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Nima Rezaei


Cancer Immunology

A Translational
Medicine Context

Cancer Immunology
Nima Rezaei

Cancer Immunology
A Translational Medicine Context
Nima Rezaei, MD, MSc, PhD
Research Center for Immunodeciencies
Childrens Medical Center
Pediatrics Center of Excellence
Tehran University of Medical Sciences
Department of Immunology
School of Medicine
and Molecular Immunology
Research Center
Tehran University of Medical Sciences

ISBN 978-3-662-44005-6 ISBN 978-3-662-44006-3 (eBook)

DOI 10.1007/978-3-662-44006-3
Springer Heidelberg New York Dordrecht London

Library of Congress Control Number: 2014952677

Springer-Verlag Berlin Heidelberg 2015

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This book would not have been possible without the continuous
encouragement by my parents and my wife Maryam.
I wish to dedicate it to my daughters Ariana and Arnika with
the hope that progress in diagnosis and treatment of these
diseases may result in improved survival and quality of life for
the next generations and at the same time that international
collaboration in research will happen without barriers.
Whatever I have learnt comes from my mentors. This book is
therefore dedicated also to all of them but most importantly to
the patients and their families, whose continuous support has
guided me during the years.

Several empirical observations suggested a long time ago that established

human tumors could melt away in response to perturbations of the immune
system such as during acute infection. Such regressions of tumors occurred
most often but not exclusively when infection occurred at the tumor site and
sparked the interest of investigators in identifying the mechanism leading to
such occurrences based on the assumption that infection acted as an adjuvant
to boost existing but insufcient immune surveillance against neoplasms.
These anecdotal observations are reected not only in the scientic literature
such as the classic reports of William Cooley in the late 1800s but even dis-
cussed by classic authors such as the doctor-writer Anton Chekhov.
It took time, however, to elevate these concepts derived from empirical
observations to a science of molecular precision. Skepticism dominated the
scene for a long time including during the late 1980s, when the introduction of
systemic IL-2 therapy for the treatment of advanced melanoma and renal cell
carcinoma provided consistent and reproducible evidence that some advanced
cancers could regress and remain in long-term remission with a treatment that

viii Foreword

had for sure no direct effect on cancer cells. Retrospectively, as too often
occurs in science, this skepticism was unwarranted, and the detractors of can-
cer immunotherapy made a disservice by slowing the progression of this bud-
ding discipline. Common criticisms were not directed against the observation
that cancers could regress but rather focused on denial about the overall effec-
tiveness of treatment, the sporadic nature of the regressions, and the relatively
high toxicity. In other words, the skeptics confused the clinical effectiveness
of a treatment with the value of a promising scientic observation.
I am emphasizing this because it is important to remember those difcult
moments now that books as sophisticated and comprehensive are presented on
a topic that was not even considered true science by most just a few decades
ago. Fortunately, several investigators did not give up, but focusing on the
value of an uncommon but reproducible observation carried the eld forward.
Thus this book! An achievement difcult to predict only two decades ago!
A book series that encompassed 77 chapters spanning biological aspects
of innate and adaptive immune responses to system biology approaches to
biomarker discovery, to portrays of clinical successes and discussion of regu-
latory processes that are about to revolutionize the development and licensing
of new investigational agents.
A signicant change occurred after the identication and molecular char-
acterization of antigens recognized by antibodies and/or T cells. Moreover,
the characterization of molecular mechanisms controlling the cross talks
between cancer and non-neoplastic somatic cells expanded the eld and
understanding of the mechanistic bases of immune-mediated tumor rejection.
These unarguable observations gave molecular precision to what was previ-
ously perceived as pointless practice. However, the true revolution came with
the clinical demonstration that some of the novel biological agents could sig-
nicantly improve the survival of patients, receiving, therefore, acceptance
and recognition as standard therapies through regulatory licensing.
Yet, challenges remain, and it is not the time to relax. Still, the benets,
though reproducible, are marginal both in terms of number of patients bene-
ting from the treatment and in the length of survival for those who benet.
Most importantly, the outcomes are capricious and unpredictable. Predictive
and surrogate biomarkers are missing in spite of novel technologies and strat-
egies that could help in the identication and stratication of patients. Still,
most clinical trials are designed to look at outcomes rather than comprehen-
sively learn in case of failures. Still, a gap exists between the potentials for
what we could do to better understand the biology of immune responsiveness
and what we actually do.
This book is written for those who want to move the eld forward both at the
clinical and the scientic level. Such a compendium can provide a contempo-
rary overlook at what has happened lately, which is remarkably logarithmic on
a time perspective. Yet, we wonder how elemental this edition may seem just
within a few years if the eld continues to evolve at the current pace. We hope
that a second edition will follow soon. Perhaps the editors should have asked
for a clairvoyants chapter. Hopefully, one of the young readers of this edition
may step forward and help dene the new frontiers of cancer immunotherapy.

Francesco M. Marincola, MD

The rapid ow of studies in the eld of cancer immunology during the last
decade has increased our understanding of the interactions between the
immune system and cancerous cells. In particular, it is now well known that
such interactions result in the induction of epigenetic changes in cancerous
cells and the selection of less immunogenic clones as well as alterations in
immune responses. Understanding the cross talk between nascent trans-
formed cells and cells of the immune system has led to the development of
combinatorial immunotherapeutic strategies to combat cancer.
Cancer Immunology Series, a three-volume book series, is intended as an
up-to-date, clinically relevant review of cancer immunology and immuno-
therapy. The book Cancer Immunology: A Translational Medicine Context, is
focused on the immunopathology of cancers. Cancer Immunology: Bench to
Bedside Immunotherapy of Cancers, is a translation text explaining novel
approaches in the immunotherapy of cancers. Finally, the book entitled

x Preface

Cancer Immunology: Cancer Immunotherapy for Organ-Specic Tumors,

thoroughly addresses the immunopathology and immunotherapy of organ-
specic cancers.
In volume I, interactions between cancerous cells and various components
of the innate and adaptive immune system are fully described. Notably, the
principal focus is very much on clinical aspects, the aim being to educate
clinicians on the clinical implications of the most recent ndings and novel
developments in the eld. To meet this purpose, this volume consists of 26
chapters. After an overview on cancer immunology in Chap. 1, the role of
innate immunity in cancers is explained in Chaps. 2 and 3, followed by the
adaptive immunity, including B cells, T cells, T regulatory and Th17 cells,
cytokines, and chemokine receptors in Chaps. 4, 5, 6, 7, and 8, respectively.
CD95/CD95L signaling pathway, MHC class I molecules, and plasmacytoid
dendritic cells are separately described in Chaps. 9, 10, and 11, respectively.
Chapter 12 focuses on cancer immunoediting, while Chaps. 13 and 14 explain
apoptosis and autophagy in cancers. Subsequently, Chap. 15 presents the
prognostic value of innate and adaptive immunity in cancers. Epigenetics and
immunogenetics are explicated in Chaps. 16 and 17, respectively. In addition,
immunodeciencies (Chap. 18), immunosenescence (Chap. 19), nutrition
(Chap. 20), allergies (Chap. 21), and transmissible cancers (Chap. 22) are
individually described in the following chapters. Chapter 23 enlightens
systems biology in cancer immunology, while immunological diagnostic
tests, including ow cytometry for cancers, are mentioned in both Chaps. 24
and 25. Finally, by allocating the nal chapter to immunohistochemistry of
different cancers, volume I comes to its end.
The Cancer Immunology Series is the result of valuable contributions of
266 scientists from 91 well-known universities/institutes worldwide. I would
like to hereby acknowledge the expertise of all contributors for generously
devoting their time and considerable effort in preparing their respective chap-
ters. I would also like to express my gratitude to the Springer publication for
providing me the opportunity to publish the book.
Finally, I hope that this translational book will be comprehensible, cogent,
and of special value for researchers and clinicians who wish to extend their
knowledge on cancer immunology.

Nima Rezaei, MD, PhD


I would like to express my gratitude to the technical editor of this book,

Maryam Ebadi, MD. With no doubt, the book would not have been com-
pleted without her contribution.

Nima Rezaei, MD, PhD


1 Introduction on Cancer Immunology

and Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Nima Rezaei, Seyed Hossein Aalaei-Andabili,
and Howard L. Kaufman
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Cancer Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 Cancer and Immune System Impairment . . . . . . . . . . . . . . . . 3
1.4 Immune System Reaction to Cancer . . . . . . . . . . . . . . . . . . . . 3
1.5 Genetic and Environmental Carcinogenesis . . . . . . . . . . . . . . 4
1.5.1 Cancer Cells Escape from Host
Immunosurveillance . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5.2 Cancer Immunodiagnosis . . . . . . . . . . . . . . . . . . . . . . 4
1.6 Cancer Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.6.1 Cancer Immunotherapy. . . . . . . . . . . . . . . . . . . . . . . . 5
1.6.2 Cancer Cell Switch . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.7 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2 Inammatory and Innate Immune Cells in Cancer

Microenvironment and Progression . . . . . . . . . . . . . . . . . . . . . . 9
Patrick Brennecke, Paola Allavena, Ilaria Laface,
Alberto Mantovani, and Barbara Bottazzi
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Heterogeneity of Myeloid Cells in the Tumor
Microenvironment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.1 Myeloid Subsets in the Tumor
Microenvironment. . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.2 Recruitment of Myeloid Cells in Tumors . . . . . . . . . . 12
2.2.3 Tumor-Derived Factors Affecting Myeloid
Differentiation and Polarized Functions . . . . . . . . . . . 13
2.3 Pro-tumoral Functions of Tumor-Associated
Myeloid Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.3.1 Tumor Proliferation and Survival . . . . . . . . . . . . . . . . 14
2.3.2 Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

xiv Contents

2.3.3 Cancer Cell Dissemination . . . . . . . . . . . . . . . . . . . . . 16

2.3.4 Suppression of Adaptive Immunity. . . . . . . . . . . . . . . 18
2.4 Selected Aspects of Therapeutic
Targeting of TAMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.5 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3 Role of Innate Immunity in Cancers and

Antitumor Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Masahisa Jinushi and Muhammad Baghdadi
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2 Role of Innate Immune Cells in Cancer and
Antitumor Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.2.1 Natural Killer (NK) Cells . . . . . . . . . . . . . . . . . . . . . . 30
3.2.2 Natural Killer T (NKT) Cells . . . . . . . . . . . . . . . . . . . 31
3.2.3 -T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2.4 Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2.5 Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.2.6 Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.3 The Role of Innate Immune Receptors on Innate
Immune Cells in Cancer and Antitumor Immunity. . . . . . . . . 32
3.3.1 Toll-Like Receptors (TLRs) . . . . . . . . . . . . . . . . . . . . 32
3.3.2 RIG-I-Like Helicases (RLHs) . . . . . . . . . . . . . . . . . . . 33
3.3.3 NOD-Like Receptors (NLRs) . . . . . . . . . . . . . . . . . . . 33
3.3.4 Phagocytosis Receptors. . . . . . . . . . . . . . . . . . . . . . . . 33
3.3.5 C-Type Lectin-Like Receptors (CLRs) . . . . . . . . . . . . 34
3.3.6 NK Cell Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3.7 B7 Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.4 The Role of Effectors Produced from Innate Immune
Cells in Cancer and Antitumor Immunity . . . . . . . . . . . . . . . . 37
3.4.1 Interferons (IFNs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.4.2 Other Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.4.3 Chemokines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.5 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

4 B Cells in Cancer Immunology: For or Against

Cancer Growth?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Qiao Li, Qin Pan, Huimin Tao, Xiao-Lian Zhang,
Shiang Huang, and Alfred E. Chang
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.2 CD40-Activated B (CD40-B) Cells . . . . . . . . . . . . . . . . . . . . 48
4.3 Tumor Killer B Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.4 Tumor-Inltrating B Cells (TIL-Bs) in Cancer. . . . . . . . . . . . 52
4.5 Resting B Cells and Regulatory B Cells in Cancer. . . . . . . . . 53
4.6 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Contents xv

5 The Role of Exhaustion in Tumor-Induced T Cell

Dysfunction in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Heriberto Prado-Garcia, Susana Romero-Garcia,
and Jose Sullivan Lopez-Gonzalez
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.2 T Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5.3 T Cell Anergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.3.1 T Cell Anergy in Cancer . . . . . . . . . . . . . . . . . . . . . . . 64
5.4 T Cell Exhaustion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
5.4.1 Mechanisms for Inducing T Cell Exhaustion . . . . . . . 65
5.4.2 Identication of Exhausted T Cells. . . . . . . . . . . . . . . 66
5.5 T Cell Exhaustion in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 67
5.5.1 A Particular Case: T Cell Exhaustion
in Lung Cancer Patients . . . . . . . . . . . . . . . . . . . . . . . 69
5.6 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

6 Regulatory T Cells and Th17 Cells in Cancer

Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Chang H. Kim
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
6.2 Diversity of Tumor Microenvironments and
Tumor Tissue Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
6.3 Generation of Tregs and Th17 Cells . . . . . . . . . . . . . . . . . . . . 80
6.4 Impact of Tumor-Derived Factors on Regulation
of T-Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
6.5 Migration of Tregs and Th17 Cells into Tumors. . . . . . . . . . . 82
6.6 Impact of Tregs and Th17 Cells on
Antitumor Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . 84
6.7 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

7 Role of Cytokines in Tumor Immunity and Immune

Tolerance to Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Murugaiyan Gopal
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.2 Cytokine Regulation of the Antitumor Immune Response . . . 94
7.2.1 IL-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
7.2.2 IL-27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
7.3 Cytokines in Immune Tolerance to Cancer . . . . . . . . . . . . . . . 101
7.3.1 TGF- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
7.3.2 IL-17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
7.3.3 IL-23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
7.3.4 IL-35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
7.3.5 IL-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
7.4 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
xvi Contents

8 Role of Chemokines and Chemokine Receptors in Cancer . . . 121

Mathieu Paul Rodero, Christophe Combadire,
and Alexandre Boissonnas
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
8.2 Chemokines and Chemokine Receptors . . . . . . . . . . . . . . . . . 123
8.3 Control of Tumor Cell Behavior . . . . . . . . . . . . . . . . . . . . . . . 125
8.3.1 Chemokines and Chemokine Receptor
Alterations During Neoplastic Transformation. . . . . . 125
8.3.2 Metastasis/Homing . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
8.3.3 Senescence, Proliferation, and Survival . . . . . . . . . . . 127
8.4 Control of Immune Cell Behaviors . . . . . . . . . . . . . . . . . . . . . 128
8.4.1 Chemokines Involved in T-Cell Antitumor
Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
8.4.2 Chemokines in Innate Immune Components . . . . . . . 130
8.4.3 Chemokine and Tumor-Induced Tolerance . . . . . . . . . 131
8.5 Alternative Tumor-Associated Physiological
Functions of Chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
8.5.1 Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
8.5.2 Fibrosis and Extracellular Matrix Remodeling . . . . . . 132
8.6 Clinical Aspect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
8.6.1 Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
8.6.2 CC Chemokines/Chemokine Receptors . . . . . . . . . . . 133
8.6.3 CXC Chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
8.6.4 CX3C Chemokine Receptors . . . . . . . . . . . . . . . . . . . 135
8.6.5 Chemokine Circulating Expression. . . . . . . . . . . . . . . 135
8.6.6 Therapeutic Strategies. . . . . . . . . . . . . . . . . . . . . . . . . 136
8.7 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

9 The CD95/CD95L Signaling Pathway: A Role

in Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Amlie Fouqu and Patrick Legembre
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
9.2 TNF Receptor Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
9.2.1 TNFR1 Signaling Pathways . . . . . . . . . . . . . . . . . . . . 144
9.2.2 TNF/TNFR: A Gold Mine for Therapeutic Tools . . . . 145
9.3 CD95: A Death Receptor?. . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
9.3.1 Structure/Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
9.3.2 Type I/II Signaling Pathways . . . . . . . . . . . . . . . . . . . 148
9.3.3 What Can We Learn from CD95 Mutations? . . . . . . . 148
9.3.4 Regulation of the Initial Steps of
CD95-Mediated Signaling . . . . . . . . . . . . . . . . . . . . . 150
9.3.5 Programmed Necrosis Also Known
as Necroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
9.3.6 CD95L, an Inammatory/Oncogenic Cytokine? . . . . 152
9.4 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Contents xvii

10 MHC Class I Molecules and Cancer Progression:

Lessons Learned from Preclinical Mouse Models. . . . . . . . . . . 161
Irene Romero, Ignacio Algarra, and Angel M. Garcia-Lora
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
10.2 MHC-I Cell Surface Expression on Tumor Cells
and Primary Tumor Growth . . . . . . . . . . . . . . . . . . . . . . . . . . 162
10.2.1 Studies in GR9 Tumor Model: H-2 Antigen
Surface Expression and Tumorigenic Capacity . . . . . 164
10.3 MHC-I Expression and Metastatic Progression . . . . . . . . . . . 166
10.3.1 MHC Class I Expression on Primary
Tumor Cells May Determine Spontaneous
Metastatic Capacity. . . . . . . . . . . . . . . . . . . . . . . . . . . 166
10.3.2 Different MHC-I Surface Expression on GR9
Tumor Clones Determines Their Spontaneous
Metastatic Capacity. . . . . . . . . . . . . . . . . . . . . . . . . . . 167
10.4 Immunotherapy as a Treatment Against Cancers
Expressing Different MHC-I Surface Expression. . . . . . . . . . 169
10.4.1 Immunotherapy as a Treatment Against
Primary Tumors with Different Levels of
MHC-I Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
10.4.2 Immunotherapy as a Treatment Against
Metastatic Progression Derived from Primary
Tumors with Different MHC-I Expression . . . . . . . . . 170
10.5 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

11 Role of Plasmacytoid Dendritic Cells in Cancer . . . . . . . . . . . . 177

Michela Terlizzi, Aldo Pinto, and Rosalinda Sorrentino
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
11.2 Localization and Trafcking Patterns of Plasmacytoid
Dendritic Cells (pDCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
11.3 Plasmacytoid Dendritic Cells (pDCs) Phenotype . . . . . . . . . . 179
11.4 Activation of pDCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
11.5 pDCs: Bridging the Gap Between Innate
and Adaptive Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
11.6 pDCs and Human Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
11.6.1 Role of pDCs in Human Infections. . . . . . . . . . . . . . . 184
11.6.2 Role of pDCs in Autoimmune Diseases . . . . . . . . . . . 185
11.6.3 Role of pDCs in Cancer . . . . . . . . . . . . . . . . . . . . . . . 186
11.7 Potential Therapies: Clinical Signicance . . . . . . . . . . . . . . . 189
11.8 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
xviii Contents

12 Cancer Immunoediting: Immunosurveillance,

Immune Equilibrium, and Immune Escape . . . . . . . . . . . . . . . 195
Alka Bhatia and Yashwant Kumar
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
12.2 Cancer Immunoediting with Its Three Es: Reection
of the Dual Role of Immunity in Cancer. . . . . . . . . . . . . . . . . 196
12.2.1 Immune Elimination: Evidences For and Against . . . 197
12.2.2 The Equilibrium Phase: The Most Controversial
and the Least Understood Phase . . . . . . . . . . . . . . . . . 200
12.2.3 Immune Escape: The Best Studied Phase . . . . . . . . . . 201
12.3 Tumor Antigens and Cancer Immunoediting . . . . . . . . . . . . . 203
12.4 The Tumor Microenvironment During
Cancer Immunoediting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
12.5 Clinical Relevance of the Immunoediting
Process in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
12.6 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

13 Apoptosis and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Mei Lan Tan, Heng Kean Tan,
and Tengku Sifzizul Tengku Muhammad
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
13.2 Mechanisms of Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
13.2.1 Extrinsic Apoptosis Pathway. . . . . . . . . . . . . . . . . . . . 212
13.2.2 Intrinsic Apoptosis Pathway . . . . . . . . . . . . . . . . . . . . 213
13.3 Apoptosis and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
13.4 Apoptosis Signaling Pathways and Therapeutic
Targets in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
13.4.1 TRAIL (TRAIL Ligands, Monoclonal
Antibodies Against TRAIL-R1 and TRAIL-R2) . . . . 220
13.4.2 Bcl-2 Family Proteins (BH3 Mimetics
and Bcl-2 Antisense) . . . . . . . . . . . . . . . . . . . . . . . . . . 225
13.4.3 Proteasome Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 227
13.4.4 Inhibitor of Apoptosis Protein (IAP) Antagonists. . . . 229
13.5 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

14 Autophagy and Necroptosis in Cancer. . . . . . . . . . . . . . . . . . . . 243

Mei Lan Tan, Heng Kean Tan, Ahmed Ismail Hassan Moad,
and Tengku Sifzizul Tengku Muhammad
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
14.2 Autophagy and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
14.3 Autophagy Signaling Pathways and Therapeutic
Strategies in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
14.3.1 mTOR Signaling Pathway Inhibitors . . . . . . . . . . . . . 249
14.3.2 Pro-autophagics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
14.3.3 Autophagy Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 251
14.4 Mechanisms of Necroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . 252
Contents xix

14.5 Necroptosis and Possible Therapeutic

Targets in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
14.6 Crosstalk in Apoptosis, Autophagy, and Necroptosis . . . . . . . 261
14.7 Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
14.8 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

15 Prognostic Value of Innate and Adaptive Immunity

in Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Fabio Grizzi, Giuseppe Di Caro, Federica Marchesi,
and Luigi Laghi
15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
15.2 Immune Inltration as a Major Player of the
Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
15.3 Cellular Players of the Innate Immunity in Cancer. . . . . . . . . 277
15.3.1 Tumor-Associated Macrophages (TAM). . . . . . . . . . . 277
15.3.2 Tumor-Associated Neutrophils (TAN) . . . . . . . . . . . . 278
15.4 Cellular Players of the Adaptive Immunity in Cancer . . . . . . 278
15.5 Prognostic Value of Innate and Adaptive Cells
of the Immune System in Cancer . . . . . . . . . . . . . . . . . . . . . . 279
15.6 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

16 Epigenetics and microRNAs in Cancer . . . . . . . . . . . . . . . . . . . 285

Petra M. Wise, Kishore B. Challagundla, and Muller Fabbri
16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
16.2 MiRNAs Regulate Effectors of the Epigenetic Machinery. . . 286
16.3 MiRNAs Are Epigenetically Regulated in
Several Types of Human Cancers . . . . . . . . . . . . . . . . . . . . . . 289
16.4 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

17 Immunogenetics of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Armin Hirbod-Mobarakeh, Ali Akbar Amirzargar,
Behrouz Nikbin, Mohammad Hossein Nicknam,
Anton Kutikhin, and Nima Rezaei
17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
17.2 Cancers: Why Are There Different Faces? . . . . . . . . . . . . . . . 296
17.3 Immune Polymorphism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
17.4 Immunogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
17.4.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
17.4.2 Immunogenetic Tools . . . . . . . . . . . . . . . . . . . . . . . . . 298
17.5 Immunogenetics: A Champion in Fighting
the Losing Battle Against Cancer . . . . . . . . . . . . . . . . . . . . . . 303
17.6 Human Leukocyte Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
17.6.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
17.6.2 Genes Behind HLA . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
17.6.3 From Polymorphisms to Clinic . . . . . . . . . . . . . . . . . . 306
xx Contents

17.6.4 HLA Typing and HLA Association Studies:

Lessons from the Past . . . . . . . . . . . . . . . . . . . . . . . . . 308
17.6.5 Typing Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
17.6.6 Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . 311
17.6.7 Linkage Disequilibrium . . . . . . . . . . . . . . . . . . . . . . . 311
17.7 The Cytokine Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
17.7.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
17.7.2 Interleukin-1 Superfamily . . . . . . . . . . . . . . . . . . . . . 313
17.7.3 Interleukin-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
17.7.4 Interleukin-6 (IL-6) . . . . . . . . . . . . . . . . . . . . . . . . . . 317
17.7.5 Interleukin-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
17.7.6 Interleukin-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
17.7.7 Interleukin-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
17.7.8 Tumor Necrosis Factor- and Lymphotoxin- . . . . . 324
17.7.9 Interferon Gamma (IFN-) . . . . . . . . . . . . . . . . . . . . 330
17.7.10 Transforming Growth Factor- (TGF-). . . . . . . . . . 330
17.8 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

18 Primary Immunodeciencies and Cancers . . . . . . . . . . . . . . . . 343

Mona Hedayat, Waleed Al-Herz, Asghar Aghamohammadi,
Kim E. Nichols, and Nima Rezaei
18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
18.2 Primary Antibody Deciencies . . . . . . . . . . . . . . . . . . . . . . . . 344
18.2.1 Common Variable Immunodeciency. . . . . . . . . . . . . 344
18.2.2 X-Linked Agammaglobulinemia. . . . . . . . . . . . . . . . . 345
18.2.3 Selective IgA Deciency. . . . . . . . . . . . . . . . . . . . . . . 346
18.3 Combined Immunodeciencies. . . . . . . . . . . . . . . . . . . . . . . . 346
18.3.1 IL-2-Inducible T-Cell Kinase Deciency . . . . . . . . . . 346
18.3.2 Purine Nucleoside Phosphorylase Deciency. . . . . . . 347
18.3.3 Dedicator of Cytokinesis 8 Deciency . . . . . . . . . . . . 348
18.3.4 RHOH Deciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
18.3.5 MAGT1 Deciency. . . . . . . . . . . . . . . . . . . . . . . . . . . 350
18.4 Phagocyte Defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
18.4.1 Severe Congenital Neutropenia
(Kostmann Syndrome) . . . . . . . . . . . . . . . . . . . . . . . . 351
18.4.2 ShwachmanDiamond Syndrome. . . . . . . . . . . . . . . . 352
18.4.3 GATA2 Deciency . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
18.5 Defects in Innate Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . 353
18.5.1 Epidermodysplasia Verruciformis . . . . . . . . . . . . . . . . 353
18.5.2 Warts, Hypogammaglobulinemia, Infections,
and Myelokathexis Syndrome . . . . . . . . . . . . . . . . . . . . . . 354
18.6 Diseases of Immune Dysregulation . . . . . . . . . . . . . . . . . . . . 354
18.6.1 X-Linked Lymphoproliferative Disease . . . . . . . . . . . 354
18.7 Syndromes with Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . 355
18.7.1 Autoimmune Lymphoproliferative Syndrome . . . . . . 355
18.7.2 Autoimmune Polyendocrinopathy with
Candidiasis and Ectodermal Dystrophy . . . . . . . . . . . 356
Contents xxi

18.8 Other Well-Dened Immunodeciencies . . . . . . . . . . . . . . . . 356

18.8.1 DNA Repair Defects . . . . . . . . . . . . . . . . . . . . . . . . . . 356
18.8.2 Signal Transducer and Activator of
Transcription 3 Deciency . . . . . . . . . . . . . . . . . . . . . 357
18.8.3 WiskottAldrich Syndrome. . . . . . . . . . . . . . . . . . . . . 360
18.8.4 Chromosome 22q11.2 Deletion Syndrome . . . . . . . . . 361
18.9 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

19 Immunosenescence, Oxidative Stress, and Cancers . . . . . . . . . 377

Tamas Fulop, Graham Pawelec, Gilles Dupuis,
Rami Kotb, Bertrand Friguet, and Anis Larbi
19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
19.2 Immune System and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 378
19.2.1 Immunosenescence or Immune Aging . . . . . . . . . . . . 378
19.2.2 Innate Immune System . . . . . . . . . . . . . . . . . . . . . . . . 379
19.2.3 Adaptive Immune System . . . . . . . . . . . . . . . . . . . . . . 383
19.2.4 Interaction Between Innate and Adaptive
Immune Responses: Effect of Aging. . . . . . . . . . . . . . 384
19.3 Inammation Aging and Oxidative Stress . . . . . . . . . . . . . . . 385
19.4 Immunosenescence and Cancer . . . . . . . . . . . . . . . . . . . . . . . 387
19.5 Modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
19.6 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

20 Nutrition, Immunity, and Cancers . . . . . . . . . . . . . . . . . . . . . . . 395

Hassan Abolhassani, Niyaz Mohammadzadeh Honarvar,
Terezie T. Mosby, and Maryam Mahmoudi
20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
20.2 Role of Nutrition in Predisposition
of Cancer from an Immunologic View . . . . . . . . . . . . . . . . . . . 396
20.2.1 Protein-Calorie Balance . . . . . . . . . . . . . . . . . . . . . . . 396
20.2.2 Essential Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.3 Antioxidants (Selenium, Vitamin E,
and Vitamin C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.4 Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.5 Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.6 Folate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.7 Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.3 Aging as a Confounder of the Triangle of Nutrition,
Immunity, and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
20.4 Role of Cancer in Predisposition to Malnutrition
from an Immunologic View. . . . . . . . . . . . . . . . . . . . . . . . . . . 398
20.5 Role of Nutritional Support in Immune
Restoration of Cancer Patients . . . . . . . . . . . . . . . . . . . . . . . . 399
20.5.1 Arginine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.2 Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.3 Branched Chain Amino Acids. . . . . . . . . . . . . . . . . . . 400
xxii Contents

20.5.4 Nucleotides, Long-Chain Omega-3 Polyunsaturated

Fatty Acids, and Eicosapentaenoic Acid . . . . . . . . . . . 400
20.5.5 Fructooligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.6 Bioactive Compounds . . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.7 Vitamins C and E . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.8 Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
20.6 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

21 Allergies and Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

Delia Rittmeyer and Axel Lorentz
21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
21.2 Molecular Mechanisms of Allergy . . . . . . . . . . . . . . . . . . . . . 408
21.3 Types of Allergic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
21.4 Molecular Basics of Carcinogenesis . . . . . . . . . . . . . . . . . . . . 409
21.5 Types of Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
21.6 Antitumor Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
21.7 Relationship Between Allergies and Cancers in General . . . . 411
21.7.1 Cancers Positively Correlated with Allergies . . . . . . . 411
21.7.2 Tumor-Promoting Effects of Allergies . . . . . . . . . . . . 412
21.7.3 Cancers Negatively Correlated with Allergies . . . . . . 413
21.8 Tumor-Protecting Effects of Allergies . . . . . . . . . . . . . . . . . . 414
21.9 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416

22 Cancer Immunology of Transmissible Cancers . . . . . . . . . . . . 419

Katrina Marie Morris and Katherine Belov
22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
22.2 Canine Transmissible Venereal Tumor . . . . . . . . . . . . . . . . . . 420
22.2.1 Prevalence and Transmission . . . . . . . . . . . . . . . . . . . 420
22.2.2 Histology and Clonality . . . . . . . . . . . . . . . . . . . . . . . 420
22.2.3 Disease Progression . . . . . . . . . . . . . . . . . . . . . . . . . . 421
22.2.4 Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
22.3 Devil Facial Tumor Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 422
22.3.1 Prevalence and Appearance. . . . . . . . . . . . . . . . . . . . . 422
22.3.2 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
22.3.3 Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
22.3.4 Do Devils Have an Impaired Immune System? . . . . . 423
22.3.5 Devils Have Low MHC Diversity . . . . . . . . . . . . . . . . 423
22.3.6 Expression of Immunosuppressive Cytokines . . . . . . 423
22.3.7 Regulation of Cell Surface MHC . . . . . . . . . . . . . . . . 423
22.4 Comparison of DFTD and CTVT . . . . . . . . . . . . . . . . . . . . . . 424
22.5 Evolution of Transmissible Cancers . . . . . . . . . . . . . . . . . . . . 424
22.6 Transmissible Tumors as a Cancer Model . . . . . . . . . . . . . . . 425
22.7 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
Contents xxiii

23 Envisioning the Application of Systems Biology

in Cancer Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Julio Vera, Shailendra K. Gupta, Olaf Wolkenhauer,
and Gerold Schuler
23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
23.1.1 The Omics Paradigm and the Use of
Statistical Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
23.1.2 Mathematical Modeling and Systems
Theory: Dissecting the Complexity
Emerging Out of the Structure
of Biochemical Networks . . . . . . . . . . . . . . . . . . . . . . 431
23.1.3 Bridging Biological Scales Through the
Integration of Biological Data in Multi-scale
Models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
23.2 One Step Further: Integrating the Different
Perspectives of Systems Biology into
a Unied Framework. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
23.3 Does Cancer Immunology Need a Systems
Biology Approach? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
23.4 A Quick View on Current Results . . . . . . . . . . . . . . . . . . . . . . 434
23.4.1 Computational Biology, Bioinformatics, and
High-Throughput Data Analysis Used in the
Design of Immune Therapies for Cancer . . . . . . . . . . 434
23.4.2 Mathematical Models Used in Basic
Oncology Research . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
23.5 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447

24 Principles of Immunological Diagnostic Tests for Cancers . . . 451

Amber C. Donahue and Yen-lin Peng
24.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
24.2 Overview of Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
24.2.1 Monoclonal vs. Polyclonal Antibodies . . . . . . . . . . . 452
24.2.2 Antibody Fragments . . . . . . . . . . . . . . . . . . . . . . . . . 453
24.2.3 Reporter Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
24.2.4 Primary and Secondary Antibodies. . . . . . . . . . . . . . 454
24.3 Immunoprecipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
24.4 Immunoblotting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
24.5 Radioimmunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
24.6 Enzymatic Immunoassays. . . . . . . . . . . . . . . . . . . . . . . . . . . 457
24.7 Immunocytochemical and Immunohistochemical Assays . . 460
24.8 Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
24.9 Bead-Based Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
24.10 Antibody Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
24.11 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
xxiv Contents

25 Flow Cytometry in Cancer Immunotherapy:

Applications, Quality Assurance, and Future . . . . . . . . . . . . . . 471
Ccile Gouttefangeas, Steffen Walter, Marij J.P. Welters,
Christian Ottensmeier, Sjoerd H. van der Burg,
Cedrik M. Britten, and Cliburn Chan
25.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
25.2 Main Flow Cytometry Assays in Cancer
Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
25.3 Panel Development and Quality Assurance . . . . . . . . . . . . . . 474
25.4 Prociency Programs Addressing Flow
Cytometry Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
25.5 Structured Reporting of Immune Assay Experiments. . . . . . . 478
25.6 Organization of Immune Monitoring in
Multicenter Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
25.7 Towards Automated Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 480
25.8 New Methods and Technologies . . . . . . . . . . . . . . . . . . . . . . . 482
25.9 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486

26 Immunohistochemistry of Cancers . . . . . . . . . . . . . . . . . . . . . . 491

Alireza Ghanadan, Issa Jahanzad, and Ata Abbasi
26.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
26.2 Immunohistochemistry of Skin Tumors . . . . . . . . . . . . . . . . . 492
26.2.1 Markers of Normal Skin . . . . . . . . . . . . . . . . . . . . . . . 492
26.2.2 Epithelial Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
26.2.3 Sweat Gland Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . 495
26.2.4 Trichogenic Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . 495
26.2.5 Sebaceous Tumors. . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
26.2.6 Melanocytic Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . 496
26.2.7 Prognostic Markers of Melanoma. . . . . . . . . . . . . . . . 497
26.2.8 Specic Mesenchymal Tumors of the Skin. . . . . . . . . 497
26.3 Immunohistochemistry of Head and Neck Tumors . . . . . . . . 499
26.3.1 Tumors of the Nasal Cavity and
Paranasal Sinuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
26.3.2 Tumors of the Larynx, Nasopharynx,
and Oropharynx. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
26.3.3 Tumors of the Salivary Glands . . . . . . . . . . . . . . . . . . 501
26.3.4 Immunohistochemistry of Salivary
Gland Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
26.3.5 Tumors of Thyroid and Parathyroid Glands . . . . . . . . 505
26.4 Immunohistochemistry of Lung Tumors. . . . . . . . . . . . . . . . . 505
26.4.1 Adenocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
26.4.2 Mesothelioma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
26.5 Immunohistochemistry of Gastrointestinal Tumors . . . . . . . . 507
26.5.1 Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
26.5.2 Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
26.5.3 Stomach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
Contents xxv

26.5.4 Small Intestine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

26.5.5 Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
26.5.6 Anal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
26.5.7 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
26.5.8 Pancreas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
26.5.9 Gastrointestinal Stromal Tumor . . . . . . . . . . . . . . . . 513
26.5.10 Neuroendocrine Carcinomas. . . . . . . . . . . . . . . . . . . 513
26.6 Immunohistochemistry of the Urinary Tract. . . . . . . . . . . . . . 513
26.6.1 Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
26.6.2 Bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
26.7 Immunohistochemistry of Female
and Male Genital Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
26.7.1 Uterine Cervix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
26.7.2 Vulva and Vagina. . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
26.7.3 Uterine Corpus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
26.7.4 Ovary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
26.7.5 Breast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
26.7.6 Prostate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
26.7.7 Testis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
26.8 Immunohistochemistry of Lymphoma . . . . . . . . . . . . . . . . . . 521
26.9 Immunohistochemistry of Soft Tissue
and Bone Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
26.9.1 Epithelial Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . 523
26.9.2 Myogenic Markers . . . . . . . . . . . . . . . . . . . . . . . . . . 526
26.9.3 Nerve and Schwann Cell Markers. . . . . . . . . . . . . . . 530
26.9.4 Endothelial Markers . . . . . . . . . . . . . . . . . . . . . . . . . 530
26.9.5 Fibrohistiocytic Markers . . . . . . . . . . . . . . . . . . . . . . 531
26.9.6 Lipocytic Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
26.9.7 Chondrocyte Markers . . . . . . . . . . . . . . . . . . . . . . . . 533
26.9.8 Osteogenic Markers . . . . . . . . . . . . . . . . . . . . . . . . . 533
26.9.9 Unknown-Origin Soft Tissue Tumors . . . . . . . . . . . . 534
26.10 Immunohistochemistry of the Nervous System . . . . . . . . . . . 534
26.10.1 Neuroepithelial Tumors. . . . . . . . . . . . . . . . . . . . . . . 535
26.10.2 Non-neuroepithelial Tumors . . . . . . . . . . . . . . . . . . . 536
26.10.3 Undifferentiated Tumors . . . . . . . . . . . . . . . . . . . . . . 538
26.10.4 Proliferative Markers. . . . . . . . . . . . . . . . . . . . . . . . . 538
26.11 Immunohistochemistry of Pediatric Tumors. . . . . . . . . . . . . . 538
26.12 Immunosurveillance, Immune Editing,
Immune Constant of Rejection, Immune Contexture,
and Immune Scoring of Cancers . . . . . . . . . . . . . . . . . . . . . . . 541
26.13 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561

Seyed Hossein Aalaei-Andabili, MD Thoracic and Cardiovascular

Surgery, Department of Surgery, College of Medicine, University of Florida,
Gainesville, Florida, USA
Research Center for Immunodeciencies, Childrens Medical Center,
Tehran University of Medical Sciences, Tehran, Iran
Ata Abbasi, MD, MPH Department of Pathology, Cancer Institute,
Imam Khomeini Complex Hospital, School of Medicine,
Tehran University of Medical Sciences, Tehran, Iran
Hassan Abolhassani, MD, MPH Research Center for Immunodeciencies,
Childrens Medical Center Hospital, Tehran University of Medical Sciences,
Tehran, Iran
Division of Clinical Immunology, Department of Laboratory Medicine,
Karolinska Institutet at Karolinska University Hospital Huddinge,
Stockholm, Sweden
Asghar Aghamohammadi, MD, PhD Research Center for
Immunodeciencies, Department of Allergy and Immunology,
Childrens Medical Center, Pediatrics Center of Excellence,
Tehran University of Medical Sciences, Tehran, Iran
Ignacio Algarra, PhD Departamento de Ciencias de la Salud, Facultad de
Ciencias Experimentales, Universidad de Jaen, Jaen, Spain
Waleed Al-Herz, MD Department of Pediatrics, Faculty of Medicine,
Kuwait University, Safat, Kuwait
Paola Allavena, MD Laboratory of Cellular Immunology, Humanitas
Clinical and Research Center, Milan, Rozzano, Italy
Ali Akbar Amirzargar, PhD Molecular Immunology Research Center,
and Department of Immunology, School of Medicine, Tehran University of
Medical Sciences, Tehran, Iran
Muhammad Baghdadi, MD, PhD Division of Immunobiology,
Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

xxviii Contributors

Katherine Belov, BSc, PhD Faculty of Veterinary Science,

University of Sydney, Sydney, NSW, Australia
Alka Bhatia, MD Department of Experimental Medicine and
Biotechnology, Post Graduate Institute of Medical Education and Research,
Chandigarh, India
Alexandre Boissonnas, PhD Centre dImmunologie et des Maladies
Infectieuses CIMI-Paris, U1135, Institut National de la Sant et de la
Recherche Mdicale (INSERM), Universit Pierre et Marie Curie Institut
Universitaire de Cancrologie (UPMC-IUC), CHU Piti-Salptrire,
Paris, France
Hopital Piti Salptrire, Universits, UPMC Univ Paris 06, CR7, INSERM,
U1135, CNRS, ERL 8255, Centre dImmunologie et des Maladies
Infectieuses (CIMI), Paris, France
Barbara Bottazzi, PhD Laboratory of Immunopharmacology,
Humanitas Clinical and Research Center, Milan, Rozzano, Italy
Patrick Brennecke, MSc, PhD Laboratory of Immunopharmacology,
Humanitas Clinical and Research Center, Milan, Rozzano, Italy
Cedrik M. Britten, MD TRON, Translational Oncology at the University
Medical Center of the Johannes-Gutenberg University gGmbH and
Association for Cancer Immunotherapy (CIMT), Mainz, Germany
Sjoerd H. van der Burg, PhD Experimental Cancer Immunology and
Therapy, Department of Clinical Oncology (K1-P), Leiden University
Medical Center, Leiden, The Netherlands
Giuseppe Di Caro, PhD Laboratory of Molecular Gastroenterology,
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
Kishore B. Challagundla, PhD Department of Pediatric Hematology/
Oncology, Childrens Center for Cancer and Blood Diseases,
Norris Comprehensive Cancer Center, University of Southern, California,
Childrens Hospital Los Angeles, Los Angeles, CA, USA
Department of Pediatric Hematology/Oncology, Childrens Hospital Los
Angeles, Los Angeles, CA, USA
Cliburn Chan, MBBS, PhD Department of Biostatistics and
Bioinformatics, Duke University Medical Center, Durham, NC, USA
Alfred E. Chang, MD Surgery Department, University of Michigan,
Ann Arbor, MI, USA
Division of Surgical Oncology, Department of Surgery, University of
Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA
Christophe Combadiere, PhD Centre dImmunologie et des Maladies
Infectieuses (CIMI), U1135, Institut National de la Sant et de la Recherche
Mdicale (INSERM), Universit Pierre et Marie Curie Institut
Universitaire de Cancrologie (UPMC-IUC), CHU Piti-Salptrire,
Paris, France
Contributors xxix

Hopital Piti Salptrire, Universits, UPMC Univ Paris 06, CR7,

INSERM, U1135, CNRS, ERL 8255, Centre dImmunologie et des
Maladies Infectieuses (CIMI), Paris, France
Amber C. Donahue, PhD Department of Hematology/Oncology
Research and Development, Quest Diagnostics-Nichols Institute,
San Juan Capistrano, CA, USA
Gilles Dupuis, PhD Biochemistry Department and Graduate Program in
Immunology, University of Sherbrooke, Sherbrooke, QC, Canada
Muller Fabbri, MD, PhD Department of Pediatric Hematology/Oncology,
Childrens Center for Cancer and Blood Diseases, Norris Comprehensive
Cancer Center, University of Southern, California, Childrens Hospital Los
Angeles, Los Angeles, CA, USA
Department of Pediatric Hematology/Oncology and Molecular
Microbiology and Immunology, Childrens Hospital Los Angeles,
Los Angeles, CA, USA
Amlie Fouqu, PhD Universit Rennes-1, Rennes, France
INSERM U1085, IRSET, Equipe Labellise Ligue Contre Le Cancer
Death Receptors and Tumor Escape, Rennes, France
Bertrand Friguet, PhD Biological Adaptation and Ageing UMR
UPMC-CNRS 8256 ERL INSERM U1164, Unit de vieillissement stress,
inammation UR 4, Universite Pierre et Marie Curie-Paris 6, Jussieu,
Paris, France
Tamas Fulop, MD, PhD Geriatrics Division, Department of Medicine,
Research Center on Aging, University of Sherbrooke, Sherbrooke, QC,
Angel M. Garcia-Lora, PhD Servicio de Analisis Clinicos and
Inmunologia, UGC Laboratorio Clinico, Hospital Universitario Virgen de
las Nieves, Granada, Spain
Alireza Ghanadan, MD Department of Pathology, Cancer Institute,
Imam Khomeini Complex Hospital, School of Medicine,
Tehran University of Medical Sciences, Tehran, Iran
Department of Dermatopathology, Razi Dermatology Hospital,
Tehran, Iran
Murugaiyan Gopal, PhD Department of Neurology, Center for
Neurologic Diseases, Brigham and Womens Hospital, Harvard Medical
School, Harvard Institutes of Medicine, Boston, MA, USA
Ccile Gouttefangeas, PhD Department of Immunology,
Institute for Cell Biology, University of Tbingen,
Tbingen, Germany
Fabio Grizzi, PhD Laboratory of Molecular Gastroenterology,
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
xxx Contributors

Shailendra K. Gupta, PhD Department of Systems Biology and

Bioinformatics, Institute of Computer Science, University of Rostock,
Rostock, Germany
Department of Bioinformatics, CSIR-Indian Institute of Toxicology
Research, Lucknow, India
Mona Hedayat, MD Division of Immunology, Boston Childrens Hospital,
Harvard Medical School, Boston, MA, USA
Armin Hirbod-Mobarakeh, MD Molecular Immunology Research Center,
School of Medicine, Childrens Medical Center, Tehran University of
Medical Sciences, Tehran, Iran
Niyaz Mohammadzadeh Honarvar, PhD School of Nutrition and
Dietetics, Tehran University of Medical Sciences, Tehran, Iran
Shiang Huang, MD Hubei Province Stem Cell Research and Appling
Center, Wuhan Union Hospital, Wuhan, China
Department of Hematology, Wuhan Union Hospital, Wuhan, Hubei, China
Issa Jahanzad, MD Department of Pathology, Cancer Institute, Imam
Khomeini Complex Hospital, School of Medicine, Tehran University of
Medical Sciences, Tehran, Iran
Masahisa Jinushi, MD, PhD Research Center for Infection-Associated
Cancer, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
Howard L. Kaufman, MD Department of General Surgery and
Immunology and Microbiology, Rush University Cancer Center,
Rush University Medical Center, Chicago, IL, USA
Chang H. Kim, PhD Laboratory of Immunology and Hematopoiesis,
Department of Comparative Pathobiology, College of Veterinary Medicine,
Weldon School of Biomedical Engineering, Center for Cancer Research,
Purdue University, West Lafayette, IN, USA
Rami Kotb, MD, FRCPC Department of Medicine,
BCCA Victoria, British Columbia Cancer Center and The University
of British Columbia, Victoria, BC, Canada
Yashwant Kumar, MD, DNB Department of Immunopathology, Post
Graduate Institute of Medical Education and Research, Chandigarh, India
Anton Kutikhin, MD, PhD Department of Epidemiology,
Kemerovo State Medical Academy, Kemerovo, Russian Federation
Ilaria Laface, PhD Laboratory of Immunopharmacology,
Humanitas Clinical and Research Center, Milan, Rozzano, Italy
Luigi Laghi, MD Department of Biotechnologies and Translational
Medicine, Humanitas Clinical and Research Center, University of Milan,
Rozzano, Milan, Italy
Anis Larbi, PhD Singapore Immunology Network (SIgN), Biopolis, Agency
for Science Technology and Research (A*STAR), Singapore, Singapore
Contributors xxxi

Patrick Legembre, PhD Universit Rennes-1, Rennes, France

INSERM U1085, IRSET, Equipe Labellise Ligue Contre Le Cancer
Death Receptors and Tumor Escape, Rennes, France
CRLCC Centre Eugne Marquis, Avenue bataille Flandres Dunkerque,
Rennes, France
Qiao Li, PhD Surgery Department, University of Michigan,
Ann Arbor, MI, USA
Jose Sullivan Lopez-Gonzalez, PhD Departamento de Enfermedades
Cronico-Degenerativas, Instituto Nacional de Enfermedades Respiratorias
Ismael Coso Villegas, Mexico City, Distrito Federal, Mexico
Axel Lorentz, PhD Department of Nutritional Medicine, University
of Hohenheim, Stuttgart, Germany
Maryam Mahmoudi, MD, PhD School of Nutrition and Dietetics,
Tehran University of Medical Sciences, Tehran, Iran
Alberto Mantovani, MD Department of Biotechnologies and
Translational Medicine, University of Milan, Milan, Italy
Humanitas Clinical and Research Center, Milan, Rozzano, Italy
Federica Marchesi, PhD Department of Immunology and Inammation,
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
Department of Biotechnologies and Translational Medicine,
Humanitas Clinical and Research Center, University of Milan,
Rozzano, Milan, Italy
Ahmed Ismail Hassan Moad, PhD Department of Medical Laboratories,
College of Medicine and Health Sciences, Hodeidah University, Hodeidah,
Katrina Marie Morris, BAnVetBioSc, PhD Faculty of Veterinary
Science, University of Sydney, Sydney, NSW, Australia
Terezie T. Mosby, MSc, RD, LDN Department of Nutrition,
St. Jude Childrens Research Hospital, Memphis, TN, USA
Tengku Sifzizul Tengku Muhammad, PhD Institute of Marine
Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu,
Terengganu, Malaysia
Kim E. Nichols, MD Department of Oncology, St. Jude Childrens
Research Hospital, Memphis, Tennessee, TN, USA
Mohammad Hossein Nicknam, MD, PhD Molecular Immunology
Research Center, and Department of Immunology, School of Medicine,
Tehran University of Medical Sciences, Tehran, Iran
Behrouz Nikbin, MD, PhD Molecular Immunology Research Center, and
Department of Immunology, School of Medicine, Tehran University of
Medical Sciences, Tehran, Iran
xxxii Contributors

Christian Ottensmeier, MD, PhD, FRCP Faculty of Medicine,

University of Southampton, Southampton General Hospital,
Southampton, UK
Qin Pan, PhD Surgery Department, University of Michigan,
Ann Arbor, MI, USA
State Key Laboratory of Virology, Department of Immunology,
Hubei Province Key Laboratory of Allergy and Immunology,
Wuhan University School of Medicine, Wuhan, Hubei, China
Department of Immunology, Wuhan University School of Medicine,
Wuhan, Hubei, China
Graham Pawelec, MA, PhD Tbingen Ageing and Tumor Immunology
Group, Second Department of Internal Medicine, Center for Medical
Research, University of Tuebingen, Tbingen, Germany
Gilles Dupuis, MA, PhD Biochemistry Department and Graduate Program
in Immunology, Faculty of Medicine and Health Sciences, University of
Sherbrooke, Sherbrooke, Canada
Yen-lin Peng, MSc Department of Hematology/Oncology Research
and Development, Quest Diagnostics-Nichols Institute,
San Juan Capistrano, CA, USA
Aldo Pinto Department of Pharmacy (DIFARMA), University of Salerno,
Fisciano, Salerno, Italy
Heriberto Prado-Garcia, PhD Departamento de Enfermedades
Cronico-Degenerativas, Instituto Nacional de Enfermedades Respiratorias
Ismael Coso Villegas, Mexico City, Distrito Federal, Mexico
Nima Rezaei, MD, MSc, PhD Research Center for Immunodeciencies,
Childrens Medical Center, Pediatrics Center of Excellence,
Tehran University of Medical Sciences, Tehran, Iran
Department of Immunology, School of Medicine, and Molecular Immunology
Research Center, Tehran University of Medical Sciences, Tehran, Iran
Delia Rittmeyer, MSc Department of Nutritional Medicine,
University of Hohenheim, Stuttgart, Germany
Matihieu P. Rodero, PhD Centre dImmunologie et des Maladies
Infectieuses (CIMI), U1135, Institut National de la Sant et de la Recherche
Mdicale (INSERM), Universit Pierre et Marie Curie Institut
Universitaire de Cancrologie (UPMC-IUC), CHU Piti-Salptrire,
Paris, France
Hopital Piti Salptrire, Universits, UPMC Univ Paris 06, CR7,
INSERM, U1135, CNRS, ERL 8255, Centre dImmunologie et des
Maladies Infectieuses (CIMI), Paris, France
Irene Romero, PhD Servicio de Analisis Clinicos and Inmunologia,
UGC Laboratorio Clinico, Hospital Universitario Virgen de las Nieves,
Granada, Spain
Contributors xxxiii

Susana Romero-Garcia, PhD Departamento de Enfermedades

Cronico-Degenerativas, Instituto Nacional de Enfermedades Respiratorias
Ismael Coso Villegas, Mexico City, Distrito Federal, Mexico
Gerold Schuler, PhD Department of Dermatology, Faculty of Medicine,
Friedrich Alexander Universitt, University of Erlangen-Nurnberg,
Erlangen, Germany
Rosalinda Sorrentino, PhD Department of Pharmacy (DIFARMA),
University of Salerno, Fisciano, Salerno, Italy
Heng Kean Tan, BSc (Hons) Malaysian Institute
of Pharmaceuticals and Nutraceuticals, Ministry of Science,
Technology and Innovation (MOSTI), Minden, Pulau Pinang, Malaysia
Mei Lan Tan, PhD Advanced Medical and Dental Institute,
Universiti Sains Malaysia, Bandar Putra Bertam, Kepala Batas,
Pulau Pinang, Malaysia
Malaysian Institute of Pharmaceuticals and Nutraceuticals, Ministry
of Science, Technology and Innovation (MOSTI), Minden,
Pulau Pinang, Malaysia
Huimin Tao, MSc Surgery Department, University of Michigan,
Ann Arbor, MI, USA
Department of Hematology, Wuhan Union Hospital, Wuhan, Hubei, China
Michela Terlizzi, PhD Department of Pharmacy (DIFARMA),
University of Salerno, Fisciano, Salerno, Italy
Julio Vera, PhD Department of Dermatology, Faculty of Medicine,
Friedrich Alexander Universitt, University of Erlangen-Nurnberg,
Erlangen, Germany
Laboratory of Systems Tumor Immunology, Department of Dermatology,
University Hospital Erlangen, Erlangen, Germany
Steffen Walter, PhD Immatics Biotechnologies GmbH,
Tbingen, Germany
Marij J. P. Welters, PhD Experimental Cancer Immunology and Therapy,
Department of Clinical Oncology (K1-P), Leiden University Medical
Center, Leiden, The Netherlands
Petra M. Wise, PhD Department of Pediatric Hematology/Oncology,
Childrens Center for Cancer and Blood Diseases, Norris Comprehensive
Cancer Center, University of Southern, California, Childrens Hospital
Los Angeles, Los Angeles, CA, USA
Department of Pediatric Hematology/Oncology, Childrens Hospital Los
Angeles, Los Angeles, CA, USA
Olaf Wolkenhauer, PhD Department of Systems Biology and
Bioinformatics, Institute of Computer Science, University of Rostock,
Rostock, Germany
xxxiv Contributors

Xiao-Lian Zhang, PhD State Key Laboratory of Virology,

Department of Immunology, Hubei Province Key Laboratory
of Allergy and Immunology, Wuhan University School
of Medicine, Wuhan, Hubei, China
Department of Immunology, Wuhan University School of Medicine,
Wuhan, Hubei, China

3-UTR 3-untranslated region

3D Three-dimensional
3-MA 3-Methyladenine
4-OHT 4-Hydroxytamoxifen
5AC 5-Azacytidine
Ab Antibody
ABC Adenosine triphosphate-binding cassette
Abs Antibodies
AC Adenocarcinoma
ACC Acinar cell carcinoma
ACC Adenoid cystic carcinoma
Ad5 Adenovirus serotype 5
ADCC Antibody-dependent cellular cytotoxicity
ADCP Antibody-dependent cellular phagocytosis
ADP Anti-adipophilin
Ag Antigen
AHR Aryl hydrocarbon receptor
AIA Ag-induced arthritis
AICD Activation-induced cell death
AIDS Acquired immune deciency syndrome
AIF Aapoptosis-inducing factor
AILT Angioimmunoblastic T-cell lymphoma
AIRC Italian Association for Cancer Research
AIRE Autoimmune regulator
ALK Anaplastic large cell lymphoma kinase
ALL Acute lymphoblastic leukemia
ALP Alkaline phosphatase
alphaGalCer Alpha-galactosylceramide
ALPS Autoimmune lymphoproliferative syndrome
AML Acute myeloid leukemia
ANCs Absolute neutrophil counts
ANN Articial neural network
ANT Adenine nucleotide translocase
APC Antigen-presenting cells
APCP Adenosine 5-(, -methylene) diphosphate
APCs Antigen-presenting cells

xxxvi Abbreviations

APECED Autoimmune polyendocrinopathy with candidiasis and ecto-

dermal dystrophy
APL Acute promyelocytic leukemia
APM Antigen presentation machinery
APS-1 Autoimmune polyendocrine syndrome type I
ARB Average relative binding
ARDS Acute respiratory distress syndrome
ASCs Adult stem cells
ASM Acid sphingomyelinase
ASPS Alveolar soft part sarcoma
ATCL Anaplastic large cell lymphoma
ATLL Adult T-cell lymphoma/leukemia
ATM Ataxia telangiectasia mutated
ATO Arsenic trioxide
ATP Adenosine triphosphate
ATR Ataxia telangiectasia/Rad3-related kinase
ATRA All-trans retinoic acid
B SLL/CLL B-cell small lymphocytic lymphoma/chronic lymphocytic
BAFF B-cell activating factor
BALs Bronchoalveolar lavage
BCA Basal cell adenocarcinoma
BCC Basal cell carcinoma
BCG Bacillus Calmette-Gurin
BCR B-cell antigen receptor
BER Base excision repair
bFGF Basic broblast growth factor
BLI Bioluminescence imaging
Bregs Regulatory B cells
BSO Buthionine sulfoximine
BTK Brutons tyrosine kinase
BTLA B- and T-lymphocyte attenuator
C/EBPb CCAT/enhancer-binding protein b
CAFs Cancer-associated broblasts
CaP Prostate cancer
CARD Caspase-recruitment domain
CBA Cytometric bead array
CBR Clinical benet response
CC Choriocarcinoma
CC Chromophobe carcinoma
CCS Clear cell sarcoma
CD Clusters of differentiation
CD40-B CD40-activated B
CD40L CD40 ligand
CDC Complement-dependent cytotoxicity
c-FLIP Cellular FLICE-inhibitory protein
CFSE Carboxyuorescein diacetate succinimidyl ester
CGN Chromogranin
Abbreviations xxxvii

CHL Classic Hodgkin lymphoma

CHS Contact hypersensitivity
CIA Collagen-induced arthritis
CIC/CRI Cancer Immunotherapy Consortium of the Cancer Research
Institute in the USA
CIHR Canadian Institutes of Health Research
CIMT Cancer Immunotherapy
CIP CIMT Immunoguiding Program
CK Cytokeratin
CLA Cutaneous lymphocyte-associated antigen
CLEC9A C-type lectin domain family 9A
CLL Chronic lymphocytic leukemia
CLRs C-type lectin and lectin-like receptors
CLRs C-type lectin receptors
CMA Chaperone-mediated autophagy
CMC Chronic mucocutaneous candidiasis
CML Chronic myeloid leukemia
CNS Central nervous system
Con Concanavalin
CP Core particle
CpG-A ODN CpG-A oligodeoxynucleotide
CpG-ODN CpG oligodeoxynucleotide
CPS Cancer Prevention Study
CQ Chloroquine
CR Complete remission
CRC Colorectal cancer
CRDs Cysteine-rich domains
CrmA Cytokine response modier A
CRP C-reactive protein
CRT Calreticulin
CS Classic seminoma
CS&T Cytometer setup and tracking
CSC Cancer stem cell
CSF-1 Colony-stimulating factor
CSF-1R CSF-1 receptor
CSF3R Colony-stimulating factor 3 receptor
CSR Class switch recombination
c-state Cytosolic state
CTC Circulating tumor cells
CTL Cytotoxic T lymphocyte
CTS Cathepsins
CTVT Canine transmissible venereal tumor
CVID Common variable immunodeciency
Cyt Cytochrome
DAMP Damage-associated molecular pattern
DC Dendritic cells
DCC Deleted in colorectal cancer
xxxviii Abbreviations

DC-SIGN Dendritic cell-specic ICAM-3 grabbing non-integrin

DD Death domain
DDP Diamindichloridoplatin
DED Death effector domain
DES Desmin
DFTD Devil facial tumor disease
DHh Desert hedgehog homolog
DISC Death-inducing signaling complex
DKO Double knockout
DLBCL Diffuse large B-cell lymphoma
DNAM DNAX-accessory molecule
DNMTs DNA methyltransferases
DNR Dominant-negative TGF- type II receptor
DNT Double-negative T
DR Death receptor
DRMs Detergent-resistant microdomains
DSB Double-strand break
DSRCT Desmoplastic small round cell tumor
DSS Dextran sulfate sodium
DT Diphtheria toxin
DTE Desmoplastic trichoepithelioma
DTH Delayed-type hypersensitivity
DTR Diphtheria toxin receptor
DUBs Deubiquitinases
EAE Experimental autoimmune encephalomyelitis
EBNA Epstein-Barr virus nuclear antigen
EBV Epstein-Barr virus
EC Embryonal carcinoma
ECL Electrochemiluminescent
ECM Extracellular matrix
ECP Eosinophil cationic protein
EGF Epidermal growth factor
EGFR EGF receptor
ELISA Enzyme-linked immunosorbent assay
EM Effector memory
EMC Epithelial-myoepithelial carcinoma
EMSA Electrophoretic mobility shift assay
EMT Epithelialmesenchymal transition
EndoG Endonuclease G
ER Endoplasmic reticulum
ER Estrogen receptor protein
ER+ Estrogen receptor-positive
ERK Extracellular signal-regulated kinase
ES Embryonic stem
ES/PNET Ewing sarcoma/peripheral neuroectodemal tumor
EV Epidermodysplasia verruciformis
FADD Fas-associating protein with a death domain
FAK Focal adhesion kinase
Abbreviations xxxix

FasL Fas ligand

FcRII Fc receptor II
FDA Food and Drug Administration
FL Follicular lymphoma
FLIP FLICE-inhibitory protein
Flt3L FMS like tyrosine kinase 3 ligand
Fluc Firey luciferase
FRB FKBP12-rapamycin-binding domain
FSC Forward scatter light
FZD Frizzled
GAP GTPase-activating protein
GBM Glioblastoma multiforme
GC Germinal center
GCLP Good clinical laboratory practice
GEFs Guanine nucleotide exchange factors
GEM Genetically engineered mouse
GEMM Genetically engineered mouse models
GFI1 Growth factor-independent 1
GFP Green uorescent protein
GI Gastrointestinal
GITR Glucocorticoid-induced tumor necrosis factor receptor-related
Gld Generalized lymphoproliferative disease
Gli Gli transcription factors
Gln Glutamine
Glu Glutamate
GLUD1 Glutamate dehydrogenase 1
GLUL Glutamate-ammonia ligase
GM-CSF Granulocyte macrophage colony-stimulating factor
G-MDSC Granulocytic MDSC
GMP Good manufacturing practice
GPU Graphical processing units
GRAFT Genetically transplantable tumor model systems
GrB Granzyme B
GSIs Gamma secretase inhibitors
GSK-3 Glycogen synthase kinase-3
GVDH Graft-versus-host-disease
GWAS Genome-wide association studies
HAX1 HS-1-associated protein X
HBE Human bronchial epithelial
HBV Hepatitis B virus
HCC Hepatocellular carcinoma
HCL Hairy cell leukemia
HCV Hepatitis C virus
HD Healthy donors
HDAC Histone deacetylase
HDACi Histone deacetylase inhibitors
HDACs Histone deacetylases
xl Abbreviations

HEV High endothelial venules

HGF Hepatocyte growth factor
HGPIN High-grade prostate intraepithelial neoplasia
HGS Human Genome Sciences
Hh Hedgehog
HIES Hyper-IgE syndrome
HIF2 Hypoxia-inducible factor 2-
HIV Human immunodeciency virus
HL Hodgkins lymphoma
HLA Human leukocyte antigen
HLH Hemophagocytic lymphohistiocytosis
HNC Head and neck cancer
HP Human papilloma
HPC Hematopoietic progenitor cells
HPV Human papilloma virus
HRG Histidine-rich glycoprotein
HRP Horseradish peroxidase
HRR Homologous recombination repair
HS Herpes simplex
HSC Hematopoietic stem cells
HSCT Hematopoietic stem-cell transplantation
HSP Heat shock proteins
HVEM Herpesvirus entry mediator
IAP Inhibitor of apoptosis protein
IB Immunoblotting
IBCC Inltrating basal cell carcinoma
ICAD Inhibitor of caspase-activated DNase
ICAM Intercellular adhesion molecule
ICAM-3 Intercellular adhesion molecule 3
ICC Immunocytochemistry
ICOS Inducible costimulator
ICOS-L Inducible costimulator ligand
ICS Intracellular cytokine staining
IDC Invasive ductal carcinoma
IDO Indoleamine 2, 3-dioxygenase
IELs Intraepithelial lymphocytes
IFN Interferon
IFN Interferon gamma
IFN- Interferon
Ig Immunoglobulin
IgAD IgA deciency
IgE Immunoglobulin E
IHC Immunohistochemistry
IHC/ICC Immunohistochemistry and immunocytochemistry
IHh Indian hedgehog
IkB Inhibitor of kB
IKK IB kinases
IL Interleukin
Abbreviations xli

IL-10 Interleukin-10
IL-1Ra Interleukin-1Ra
IL-1 Interleukin-1
IL-2R Interleukin-2 receptor-
ILC Invasive lobular carcinoma
IM Inner mitochondrial membrane
IMPT Intensity-modulated proton therapy
IMRT Intensity-modulated radiotherapy
IMS Intermembrane space
INF Interferons
iNOS inducible nitric oxide synthase
IP Immunoprecipitation
iPS Induced pluripotent stem
IRF Transcription factor
ISPC In silico planning comparative
ITAM Immunoreceptor tyrosine-based activation motif
ITIM Immunoreceptor tyrosine-based inhibition motif
ITK T-cell kinase
IVD In vitro diagnostic
JAK Janus kinase
JNK Jun N-terminal kinase
KARs Killer activation receptors
KGF Keratinocyte growth factor
KIRs Killer cell immunoglobulin-like receptors
KIRs Killer inhibitory receptors
KSHV Kaposi sarcoma-associated herpesvirus
LAT Linker of activation in T-cell
LC Luminal cells
LCA Leukocyte common antigen
LCMV Lymphocytic choriomeningitis virus
LCs Langerhans cells
LCT Leydig cell tumor
LD Linkage disequilibrium
LIR LC3 interacting region
LMP-1 Latent membrane protein-1
LNA Locked nucleic acid
LNs Lymph nodes
LOH Loss of heterozygosity
LOX Lysyl oxidase
LPL Lymphoplasmacytic lymphoma
Lpr Lymphoproliferation
LPS Lipopolysaccharide
LTA Lymphotoxin-
LUBAC Linear ubiquitin chain assembly complex
mAb Monoclonal antibody
Mac Macrophages
MAC Microcystic adnexal carcinoma
MALT Mucosa-associated lymphoid tissue
xlii Abbreviations

MAMP Microbe-associated molecular pattern

MAPK Mitogen-activated protein kinase
MC Molluscum contagiosum
MC Myoepithelial carcinoma
MCA Methylcholanthrene
MCC Merkel cell carcinoma
MCMV Mouse cytomegalovirus
M-CSF Macrophage colony-stimulating factor
mDCs Myeloid-derived dendritic cells
MDS Myelodysplasia
MDSC Myeloid-derived suppressor cells
MEC Mucoepidermoid carcinoma
MEXT Ministry of Education, Culture, Sports, Science and Technology
MF Mycosis fungoides
MFI Mean uorescence intensity
MGMT Methylguanine methyltransferase
MGUS Gammopathy of unknown signicance
MHC Major histocompatibility complex
MIACA Minimal information on reported results including reporting
information on cellular assays
MIAME Minimal information about microarray experiments
MIATA Minimal information about T-cell assays
MIBBI Minimal information on biological and biomedical
MIC-A MHC class I chain-related A
MIF Macrophage inhibitory factor
MIG Monokine induced by interferon-
miRNAs MicroRNAs
MISC Motility-inducing signaling complex
MKPs MAP kinase phosphatases
ML-IAP Melanoma inhibitor of apoptosis protein
MM Multiple myeloma
M-MDSC Monocytic MDSC
MMP Metalloproteases
MMR Mismatch repair
MnO Manganese oxide
MOMP Membrane permeabilization
MPSC Metastatic pulmonary small cell carcinoma
MSA Muscle-specic antigen
MSCs Mesenchymal stem cells
MSF Migration-stimulating factor
MSI Microsatellite instability
m-state Matrix state
mTOR Mammalian target of rapamycin
MVD Microvascular density
MYG Myogenin
MZL Marginal zone lymphoma
NADPH Nicotinamide adenine dinucleotide phosphate oxidases
Abbreviations xliii

NAIP Neuronal apoptosis inhibitory protein

NCCD Nomenclature Committee on Cell Death
NCR Natural cytotoxicity receptor
ncRNAs noncoding RNAs
NEC Neuroendocrine carcinoma
NER Nucleotide excision repair
NF Nuclear factor
NFAT Nuclear factor of activated T cells
NF-B Nuclear factor-kappa B
NHANES National Health and Nutrition Examination Survey
NHEJ Nonhomologous end-joining
NHL Non-Hodgkin lymphoma
Ni Nickel
NiS Nickel sulde
NK Natural killer
NKG2D Natural killer group two member D
NKT Natural killer T
NLPHL Nodular lymphocyte predominant Hodgkin lymphoma
NLRs NOD-like receptors
NLRs Nucleotide-binding domain and leucine-rich-repeat-containing
NMC NUT midline carcinoma
NOD Nucleotide-binding oligomerization domain
NP Normal prostate
NPC Nasopharyngeal carcinoma
NPY Neuropeptide Y
NSCLC Non-small cell lung cancer
NSCLC Non-small cell lung carcinoma
Nt Nucleotides
NTKs Neurothekeoma
NUT Nuclear protein in testis
OARs Organs at risk
OC Oncocytoma
ODEs Ordinary differential equations
ONB Olfactory neuroblastoma
OPN Osteopontin
OPRCC Oncocytic papillary RCC
PAC Prostate adenocarcinoma
PAC Pulmonary adenocarcinoma
PAGE Polyacrylamide gel, and separated by electrophoresis
PAK p21-activated kinase
PAMPs Pathogen-associated molecular patterns
PARP Poly ADP-ribose polymerase
PAX Paired box
PB Peripheral blood
PBMC Peripheral blood mononuclear cell
PBMCs Blood mononuclear cells
PCD Programmed cell death
xliv Abbreviations

PCG Protein coding gene

PD Paget disease
PDAC Pancreatic ductal adenocarcinoma
pDCs Plasmacytoid dendritic cells
PDGF Platelet-derived growth factor
PD-L1 Programmed cell death-1 ligand
PE Phosphatidylethanolamine
PE Pleural effusion
PEMCs Pleural effusion mononuclear cells
PET Positron emission tomography
PFS Progression-free survival
PH Pleckstrin homology
PHA Phytohemagglutinin
PI3K Phosphatidylinositol 3-kinase
PIDs Primary immunodeciencies
PIP3 Phosphatidylinositol-3,4,5-triphosphate
PKB Protein kinase B
PKC Protein kinase C
PLAD Pre-ligand binding assembly domain
PLGC Polymorphous low-grade adenocarcinoma
PlGF Placental growth factor
PMA Phorbol myristate acetate
PMNs Polymorphonuclear leukocytes
PMT Photomultiplier tube
PNET/ES Peripheral neuroectodermal tumor/extraskeletal Ewing sarcoma
PNP Purine nucleoside phosphorylase
PR Progesterone receptor
PRC Polycomb Repressive Complex
PRCC Papillary RCC
pre-pDCs Precursor of pDCs
PROTOR Protein observed with Rictor
PRRs Pattern recognition receptors
PS Phosphatidylserine
PSSM Position-specic scoring matrix
Ptc Patched dependence receptor
PTCH1 Patched receptor
PTM Posttranslational modication
PTPC Permeability transition pore complex
PVDF Polyvinylidene uoride
PYGL Glycogen phosphorylase
QDs Quantum dots
QoL Quality of life
RA Rheumatoid arthritis
RAGE Receptor for advanced glycation end products
Raptor Regulatory-associated protein of mTOR
Rb Retinoblastoma protein
RCC Renal cell carcinoma
RFK Riboavin kinase
RFLPs Restriction fragment length polymorphisms
Abbreviations xlv

RHIM RIP homotypic interaction motif

RHOH Ras homolog family member H
RIA Radioimmunoassay
RICD Reactivation-induced cell death
Rictor Rapamycin-insensitive companion of mTOR
RIG-1 Retinoic acid-inducible gene I
RIP Receptor interacting protein
RISC RNA-induced silencing complex
RLHs RIG-I-like helicases
RMS Rhabdomyosarcoma
ROS Reactive oxygen species
RS Reference samples
SA Sebaceous adenoma
SAP Signaling associated protein
SBDS ShwachmanBodianDiamond syndrome
SC Sebaceous carcinoma
SCC Squamous cell carcinoma
SCCHN Squamous cell carcinoma of the head and neck
SCF Stem cell factor
SCID Severe combined immune-decient
SCLCL Small cell lung cancer
SCM Small cell melanoma
SCN Severe congenital neutropenia
SCNP Single-cell network proling
SCs Stem cells
SCT Sertoli cell tumor
SDC Salivary duct carcinoma
SDS ShwachmanDiamond syndrome
SDS Sodium dodecyl sulfate
SEC Small cell eccrine carcinoma
SED Subepithelial cell dome
SFB Segmented lamentous bacteria
Shh Sonic hedgehog
SHh Sonic hedgehog homolog
SHM Somatic hypermutation
siRNA Small interfering RNA
SIRP- Signal-regulatory protein-
SLAM Signaling lymphocytic activation molecule
SLE Systemic lupus erythematosus
SMC Skeletal muscle cells
SMM Stabilized matrix method
Smo Smoothened
SNEC Small cell neuroendocrine carcinoma
SNP Single nucleotide polymorphisms
SNUC Sinonasal undifferentiated carcinoma
SOBP Spreadout Bragg peak
SOCE Store-operated Ca2+ entry
SOPs Standard operating procedures
xlvi Abbreviations

SP Side population
SP-A Surfactant protein A
SPECT Single-photon emission computed tomography
SPIO Superparamagnetic iron oxide
SPN Solid pseudopapillary neoplasm
SS Sjgren syndrome
SS Spermatocytic seminoma
SSC Side-scattered light
SSCC Small cell squamous carcinoma
SSO Sequence-specic probes
SSP Sequence-specic primers
SSPCs Salivary gland stem/progenitor cells
STAT Signal transducer activator of transcription
STAT1 Signal transducer and activator of transcription-1
STIM Stromal interaction molecule
SVZ Subventricular zone
SYN Synaptophysin
T1D Type 1 diabetes
T2 Transitional 2 immature
TAA Tumor-associated antigens
TACI Transmembrane activator and calcium modulator and
cyclophilin ligand interactor
TADC Tumor-associated dendritic cells
TAM Tumor-associated macrophages
TAMC Tumor-associated myeloid cells
TAN Tumor-associated neutrophils
TAP Transporter associated with Ag presentation
TAP Transporter associated with Ag processing
TApDCs Tumor-associated pDCs
TAPs Peptide transporters
TAS Trait-associated SNP
TAs Tumor antigens
TB Tuberculosis
TBI Total body irradiation
tBID Truncated BID
TC/HRBCL T-cell/histiocyte-rich B-cell lymphoma
TCF-4 T cell factor
TCL T-cell lymphoma
TCR T cell receptor
TDLN Tumor-draining lymph node
TEM Tie2-expressing monocytes
TEM Transmission electron microscopy
TEMRA Terminally differentiated effector memory
TFBSs Transcription factor binding sites
TFH T follicular helper
TGB Thyroglobulin
TGF- Transforming growth factor
Th T helper
TIL Tumor-inltrating lymphocytes
TIL-Bs Tumor-inltrating B cells
Abbreviations xlvii

TLR Toll-like receptor

TLT Tertiary lymphoid tissue
TME Tumor microenvironment
TNC Tenascin C
TNF Tumor necrosis factor
TNF-R Tumor necrosis factor receptor
TNF Tumor necrosis factor alpha
TNF- Tumor necrosis factor-
TNM Tumor-node-metastasis
TRADD TNF-receptor-associated death domain
TRAIL Tumor necrosis factor-related apoptosis-inducing ligand
Tregs Regulatory T cells
TSC Tuberous sclerosis complex
TSGs Tumor suppressor genes
TSH Thyroid-stimulating hormone
TSLP Thymic stromal lymphopoietin
TTP Time to progression
U1snRNP U1 small nuclear ribonucleoprotein
UADT Upper aerodigestive tract
UC Urothelial carcinoma
UCH Ubiquitin C-terminal hydrolases
ULBPs Unique long 16 binding proteins
Unfrac Unfractionated
UNPC Undifferentiated nasopharyngeal carcinoma
uPA Urokinase plasminogen activator
UPP Ubiquitin-proteasome pathway
UPS Ubiquitin-proteasome system
USP Ubiquitin-specic proteases
USPIO Ultrasmall superparamagnetic iron oxide nanoparticles
UV Ultraviolet
UVRAG Ultraviolet radiation resistance-associated gene
VEGF-A Vascular endothelial growth factor-A
VIM Vimentin
VINIII Vulvar intraepithelial neoplasia grade III
VNTR Variable number tandem repeat
VZ Varicella zoster
WAS WiskottAldrich syndrome
WASp WAS protein
WASP WiskottAldrich syndrome protein
WGS Whole genome sequencing
WHIM Warts, hypogammaglobulinemia, infections,
and myelokathexis
WM Waldenstrom macroglobulinemia
WT Wild-type
X-IAP X-linked inhibitor of apoptosis protein
XLA X-linked agammaglobulinemia
XLN X-linked neutropenia
XLP X-linked lymphoproliferative disease
XLT X-linked thrombocytopenia
YST Yolk sac tumor
Introduction on Cancer
Immunology and Immunotherapy 1
Nima Rezaei, Seyed Hossein Aalaei-Andabili,
and Howard L. Kaufman

Contents 1.5 Genetic and Environmental

Carcinogenesis............................................. 4
1.1 Introduction................................................. 1 1.5.1 Cancer Cells Escape from Host
1.2 Cancer Immunity ........................................ 2 Immunosurveillance ..................................... 4
1.5.2 Cancer Immunodiagnosis ............................. 4
1.3 Cancer and Immune
System Impairment .................................... 3 1.6 Cancer Treatment ....................................... 5
1.6.1 Cancer Immunotherapy ................................ 5
1.4 Immune System 1.6.2 Cancer Cell Switch.................................... 6
Reaction to Cancer ..................................... 3
1.7 Concluding Remarks .................................. 6
References ............................................................... 7

N. Rezaei, MD, MSc, PhD (*)

Department of Immunology, Research Center for
Immunodeciencies, Childrens Medical Center, 1.1 Introduction
Pediatrics Center of Excellence, Tehran University
of Medical Sciences, Dr Qarib St, Keshavarz Blvd,
Tehran 14194, Iran
Cancer is a life-threatening disease, which can
involve all human organs and tissues. It is the
Department of Immunology, School of Medicine, and
Molecular Immunology Research Center,
second leading cause of death and is responsible
Tehran University of Medical Sciences, for 25 % of all deaths in the United States. In
Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran 2012, more than 1.6 million new cases (848,170
e-mail: men and 790,740 women) of invasive cancers
S.H. Aalaei-Andabili, MD were diagnosed in the United States alone [1].
Thoracic and Cardiovascular Surgery, The major cancers in adults include lung, breast,
Department of Surgery, College of Medicine,
University of Florida, Gainesville,
prostate, and colorectal cancer. In addition,
Florida 100129, USA 60,824 adolescents and young adults aged
Research Center for Immunodeciencies,
1529 years old were diagnosed with invasive
Childrens Medical Center, Tehran University cancers between 1975 and 2000 [2]. Among all
of Medical Sciences, Tehran, Iran invasive cancers, lymphoma was the most com-
e-mail: mon cancer (20 %), followed by invasive skin
H.L. Kaufman, MD cancer (15 %), male genital system cancer
Department of General Surgery and Immunology (11 %), and endocrine system cancer (11 %) [2].
and Microbiology, Rush University Medical Center,
Rush University Cancer Center, Chicago,
Although cancer incidence has increased among
IL 60612, USA people younger than 45 years old during 1975
e-mail: 2000, overall cancer incidence has decreased in

N. Rezaei (ed.), Cancer Immunology: A Translational Medicine Context, 1

DOI 10.1007/978-3-662-44006-3_1, Springer-Verlag Berlin Heidelberg 2015
2 N. Rezaei et al.

men by 0.6 % per year during 20042008. patient survival. Total mortality rates vary from
Remarkably, the rate remained stable among 6 % in thyroid cancer to 97 % in pancreatic
females due to the high rate of breast cancer [3]. cancer [6].
Many cancer predisposing factors have been
recognized; it has been found that cancer inci-
dence is signicantly associated with age from 1.2 Cancer Immunity
10 to 60 years. Additionally, male gender is at
higher risk of developing cancer compared to Cancer immunology has been studied for a long
females [2]. Race is another important factor for time; however, the molecular and cellular basis of
cancer development; before 40 years of age, non- tumor immunity is not completely understood.
Hispanic whites and, after 40 years of age, Advances in understanding the basis of immuno-
African-Americans/blacks have the highest inci- surveillance and progress in the treatment of
dence [4]. Other risks factors include life style infectious disease have had a major impact on the
choices such as tobacco use, obesity, and lack of development of tumor immunotherapy. The mod-
exercise and environmental factors such as expo- ern era of tumor immunology began in the 1950s
sure to excessive sun, radiation during childhood, when the role of T cell responses in tissue
human papilloma virus (HPV), human immuno- allograft rejection was initially identied. Since
deciency virus, and Epstein-Barr virus (EBV) then, it has been conrmed that tumors occur in
infection [4]. association with impaired function of T cells,
Cancer can be a life-threatening health prob- indicating the importance of the immune system
lem, especially when the tumor has metastasized in the development and progression of cancer [7].
to other organs. It is estimated that 577,190 The identication of tumor-associated antigens,
patients (including 301,820 men and 275,370 knowledge of effector T cell responses, and the
women) died from cancer in the United States in role of regulatory and suppressor T cell popula-
2012. Four cancers lung and bronchus, prostate, tions are now shaping the use of the immune sys-
and colorectal in men and lung and bronchus, tem to treat cancer.
breast, and colorectal in women are responsible In addition to an improved understanding of the
for approximately 50 % of cancer-related deaths. immune system, signicant advances in under-
Fortunately, the overall cancer-related mortality standing the molecular basis of neoplasia have
has been decreasing in recent years. The death occurred. Precise control of cellular activity and
rate decreased by 1.8 % per year among men and metabolism is crucial for proper physiologic func-
1.6 % per year among women. The highest mor- tion. Notably, cell division is an important process
tality reduction has been found among African- that requires precise regulation. The main differ-
Americans (2.4 % per year), followed by ence between tumor cells and normal cells is lack
Hispanics (2.3 % per year); however, American of growth control during the cell division process.
Indians/Alaska natives were an exception, and the This uncontrolled cell division can originate from
rate remained unchanged in this population [1]. various factors, such as chemical agents, viral
Cancer survival signicantly impacts infections, and mutations that lead to escape of
patients quality of life. Five-year mortality cells from the checkpoints which properly control
rates depend on several factors; survival is cell division. According to the type of tumor and
worse among males over 30 years of age, and proliferation rate, cancers can be benign or malig-
the survival gets worse for patients over nant [8]. It has been found that some tumors are
45 years old in both males and females. Non- caused by oncogenic viruses that induce malig-
Hispanic whites have the best survival rate and nant transformation. These oncogenic viruses can
African-Americans have the worst survival be both RNA and DNA viruses. Also, viral infec-
with survival differences as great as 20 % at tion may lead to leukopenia and immunode-
5 years after cancer diagnosis [5]. Furthermore, ciency, increasing the risk of malignancy.
the type of cancer is another risk factor for Therefore, prophylactic immunization against
1 Introduction on Cancer Immunology and Immunotherapy 3

oncogenic viruses (such as EBV, HPV, and HBV) immunosurveillance [12, 13]. In addition, tumors
might be a logical strategy for prevention of malig- produce soluble factors which downregulate the
nancy [9]. Indeed, a vaccine against the human interleukin-2 receptor- (IL-2R), leading to sup-
papilloma virus has shown signicant impact on pression of T cell function. Furthermore, estab-
preventing cervical intraepithelial neoplasia and lished tumors may result in severe protein
may prevent development of cervical carcinoma. expenditures in hosts, contributing to impairment
of immune system function [14].

1.3 Cancer and Immune

System Impairment 1.4 Immune System
Reaction to Cancer
It has been reported that impaired immune
response can induce tumor growth and prevent A critical question is whether cancer cells are
effective antitumor suppression, possibly through sufciently different from their normal cellular
a process of sneaking through which allows counterparts, and can thus be recognized by the
improved growth of small tumors rather than immune system. The immune system also pro-
large tumors [10]. Tumors may also produce duces a group of complementary markers with
immunosuppressive factors, such as interleukin- protective effects against cancer and other immu-
10 (IL-10), transforming growth factor- nologic or inammatory stresses. These markers
(TGF-), and alpha-fetoprotein, which suppress include proteins released by T cells and are gen-
innate immune responses against cancer. This has erally classied as cytokines. Cytokines
led to investigations using neutralizing antibodies include interleukins, interferons, tumor-necrosis
against these immunosuppressive factors [7]. factors (TNF), and lymphocyte-derived growth
In contrast, tumor-specic cytotoxic T lympho- factors. The production of tumor-specic anti-
cytes (CTLs) can be genetically altered to become bodies and/or activation of tumor antigen-specic
resistant to the TGF- inhibitory effect by trans- T cells target tumor-associated antigens typically
gene expression of a mutant dominant-negative found on the cell membrane. Studies have sug-
TGF- type II receptor (DNR). In addition, spe- gested that vaccination in the presence of com-
cic T cells genetically manipulated to produce plements can lead to tumor lysis. While
IL-12 can overcome the inhibitory effects of incompletely dened, several soluble and cellular
IL-10. On the other hand, tumors may express mediators of tumor rejection have been described,
FasL and stimulate apoptosis of tumor-inltrating including complement factors, active macro-
effector T cells. Small interfering RNA (siRNA) phages, T cells, and NK cells. While T cells
can be used to knock down the Fas receptor in require antigen specicity, the soluble and cellu-
tumor-specic CTL, leading to a signicant lar mechanisms of the innate immune response
decrease in their susceptibility to Fas-/FasL- can recognize the malignant phenotype in the
mediated apoptosis [11]. absence of antigen specicity [15].
The interaction between the immune system Since most tumor-associated antigens are self-
and established cancers is complex, because in proteins, the immune response is largely weak
addition to increasing carcinogenesis by various and patients may develop immune tolerance to
carcinogens among compromised subjects, cancer tumor-associated antigens. Furthermore, the cells
cells themselves can lead to severe immunosup- of the immune system may not adequately pene-
pression. It has been reported that patients involved trate to the internal tumor microenvironment,
with primary immunodeciency syndromes have resulting in slower immune-mediated tumor
higher risk of cancer development. In a report by elimination. However, it is possible that the
Kersey et al., subjects that had an inherited abnor- immune system may be more effective in control-
mal lymphoid system were susceptible to malig- ling tumor growth rate rather than tumor regres-
nant transformation and impairment of tumor sion [10]. Recently, it has been found that
4 N. Rezaei et al.

nutrition also plays a crucial role in protection costimulatory molecules, which mediate the
against human cancer, and normal levels of zinc activation of T cells. Another strategy resulting
are required for protection against the detrimen- in failure of tumor immunosurveillance could be
tal effects of various immunosuppressive cyto- the expression of very low levels of antigens,
kines [16]. unable to stimulate an immune response. Under
some circumstances, such as failure of the
immune response to induce a rapid response,
1.5 Genetic and Environmental cancer cells may proliferate rapidly. Further
Carcinogenesis strategies for escape of tumor cells from immu-
nosurveillance are based on inhibitory tumor-
It has been found that genetic factors are as mediated signaling by CTLs, as occurs through
important as environmental carcinogens. Trials changes in cell death receptor signaling. Other
have tested carcinogenesis of retrovirus infection strategies which allow tumor cells to evade the
between different breeds of animals. A unique immune system are the secretion immunosup-
carcinogen resulted in disparate outcomes among pressive molecules dampening tumor-reactive
different breeds, indicating the importance of effector T cells and the induction of regulatory
genetic background in the progression of cancer. and/or suppressor cells [19].
Environmental factors may also suppress immune To date, most direct evidence on tumor immu-
responses and dysregulate immunosurveillance nosurveillance originates from experimental
mechanisms [17]. studies in animal models. These models have sup-
ported the potential for antitumor immunity via
vaccination, as, for example, by administration of
1.5.1 Cancer Cells Escape from Host inactivated cancer cells, or through removal of a
Immunosurveillance primary tumor. In addition, antitumor immunity
can be adoptively transferred through administra-
Antigens that distinguish tumor cells from nor- tion of tumor-reactive T lymphocytes. The com-
mal cells depend on the histologic origin of the plexities of immunotherapy are evident as nearly
tumor. Tumor-associated antigens may be viral in all immune system components can inuence
origin, represent mutated self-antigens, be tumor growth and progression. Although there is
cancer-testis antigens which are expressed only evidence for antitumor immunity in humans and
by tumor cells and normal testes, or be normal several new agents have gained regulatory
differentiation antigens. Thus, tumor cells may approval for cancer therapy, further investigation
express similar antigens to normal cells, allowing is warranted to increase the impact of tumor
tumor cells to escape immune system attack immunotherapy for more cancer patients [20].
through induction of innate and/or peripheral tol-
erance. A corollary to this is that immunotherapy
or stimulation of immune responses to some 1.5.2 Cancer Immunodiagnosis
tumor-associated antigens may lead to damage of
normal tissues and organs, as exemplied by the Nowadays, new immunomolecular diagnostic
development of autoimmunity induced by approaches have been suggested for tumor detec-
anti-CTLA-4 or anti-PD1 monoclonal antibody tion. Monoclonal antibodies marked with radio-
(mAb) treatment [18]. isotopes have been used for in vivo diagnosis of
A number of complex mechanisms have been small tumor foci. In addition, monoclonal anti-
suggested for the escape of cancer cells from bodies have been used for in vitro recognition of
host immunosurveillance. Tumors alter their the cell of origin for tumors with poor differentia-
characteristics by decreased expression of tion. Immunodiagnostics have also been used to
immunogenic tumor-associated antigens, MHC determine the extent of metastatic disease, espe-
class I molecules, beta2-microglobulin, and cially metastasis to the bone marrow [21].
1 Introduction on Cancer Immunology and Immunotherapy 5

1.6 Cancer Treatment has been injected to the tumor mass and has had
benecial effects in the treatment of melanoma
Systemic cancer treatment is based on four and head and neck squamous cell carcinoma
general therapeutic approaches: (1) chemother- [26]. Although vaccine-based therapy has not
apy, which contains a wide group of cytotoxic been effective in some types of cancer, there are
drugs that interfere with cell division and DNA studies that have shown an overall survival bene-
synthesis; (2) hormonal therapy, which contains t compared to placebo therapy [27].
drugs that interfere with growth signaling via Another immune-targeted approach is mAbs
tumor cell hormone receptors; (3) targeted ther- which block T cell checkpoints functioning to
apy, which involves a novel group of antibodies suppress T cell responses. Cytotoxic T
and small-molecule kinase suppressors that prin- lymphocyte-associated antigen 4 (CTLA4) is a
cipally target proteins crucial in cancer cell member of a large family of molecules regulating
growth signaling pathways; and (4) immunother- T cell immune responses. CTLA4 is expressed
apy, which targets the induction or expansion of on CD4+ and CD8+ T cells, as well as on FOXP3+
antitumor immune responses [22]. regulatory T cells [28]. Administration of mAbs
targeting human CTLA4 leads to the rejection of
established tumors in a small cohort of patients
1.6.1 Cancer Immunotherapy with metastatic melanoma and demonstrated
improved overall survival in patients with meta-
Tumor immunotherapy is a novel therapeutic static melanoma, resulting in US FDA approval
approach for cancer treatment, with increasing for the treatment of metastatic melanoma [29].
clinical benets. Tumor immunotherapy is based Monoclonal antibodies which block other T
on strategies which improve the cancer-related cell checkpoints, such as the programmed cell
immune response through either promoting com- death protein 1 (PDCD1/PD1), programmed cell
ponents of the immune system that mediate an death ligand 1 (PDL1/CD274), CD276 (B7H3)
effective immune response or via suppressing antigen, V-set domain-containing T cell function
components that inhibit the immune response. inhibitor 1 (B7x), and B and T lymphocyte atten-
Two current approaches commonly used for uator, have also entered clinical trials. In addi-
immunotherapy are allogeneic bone marrow tion, early phase studies have demonstrated
transplantation and mAbs targeting cancer cells signicant therapeutic activity in several types of
or T cell checkpoints [23]. Recently, various cancer, including melanoma, renal cell carci-
other approaches have been tested for cancer noma, non-small cell lung carcinoma, and ovar-
immunotherapy, and some are undergoing further ian cancer [30]. It has been reported that PDL1
clinical evaluation. expression by tumor cells is associated with poor
Initially, anticancer vaccines were considered clinical outcome and may be associated with
for prevention and treatment of various tumors clinical response to anti-PD1 and anti-PDL1
[23]. It is estimated that more than 15 % of human therapy. Also, ligation of PDL1 leads to inactiva-
cancers are caused by viral infection [24]. tion of tumor-inltrating cells [31]. On the other
Vaccine-based immunotherapy may, thus, be hand, regulatory T cells have an immunosuppres-
most useful for virus-induced cancers. Consistent sive role in the tumor microenvironment. Studies
with this hypothesis, a 50 % complete remission of anti-PD1 and anti-PDL1 are in progress.
(CR) of HPV-associated vulvar intraepithelial Moreover, the combination of these agents with
neoplasia grade III (VINIII) has been reported anti-CTLA4 and other immunotherapy strategies
[25]. An attenuated, oncolytic herpes simplex has yielded promising results.
type 1, which is genetically engineered to secrete The combination of antitumor vaccines with
granulocyte-macrophage colony-stimulating fac- agents targeting the IL-12 receptor resulted in
tor (GM-CSF), has been developed for cancer conicting results. This may be due to the
therapy. This oncolytic immunotherapeutic agent upregulation of IL-12 receptor by both activated
6 N. Rezaei et al.

T effector cells and regulatory T cells [32]. Thus, During rapid proliferation of cancer cells, pre-
new approaches focused on more specic cise orchestrated enzyme formation needed for
targeting of regulatory T cells which reduce their suitable metabolism of its different compo-
suppressive effects on the immune system are nents might get unbalanced, and products
necessary. Adoptive T cell therapy has been which are not observed in normal dividing cells
described as an effective therapeutic approach for are produced [38]. Recently, it has been
cancer immunotherapy in early phase clinical tri- reported that these biochemical switches
als. In this method, a large number of tumor- lead to uncontrolled multiplication of cancer
specic T cells derived from peripheral blood, or cells. One switch has been found for a type of
preferably from the tumor microenvironment leukemia. It has been suggested that targeting
(with or without genetic manipulation to express tumor switches can make treatment of cancers
a high-afnity antigen-specic T cell receptor very simple [20]. Nonetheless, it is unclear
(TCR)), are adoptively transferred to patients how this may be used to optimize tumor
with established tumors [33]. Recently, CD19 immunotherapy.
which is expressed by mature B cells and a Since cancer immunology is a highly com-
majority of non-Hodgkin lymphoma (NHL) cells plex process, further research is needed to more
has been used as another novel promising thera- completely understand how the immune system
peutic target [34]. Chemotherapy-mediated cell recognizes and eradicates cancer. In this book,
death leads to immune responses in a drug- we will describe a variety of novel mechanisms
induced biochemical cell death cascade- currently under investigation for mediating
dependent manner, suggesting benecial effects aspects of tumor immunology with a particular
of chemotherapy and immunotherapy in combi- focus on promising therapeutic approaches, pro-
nation [35]. It seems that future goals of tumor ducing a complete comprehensive up-to-date
immunotherapy are headed towards chemoim- textbook.
munotherapy. Potential candidates for this com-
bination approach include antitumor vaccines,
Toll-like receptor (TLR) signaling pathway ago- 1.7 Concluding Remarks
nists/antagonists, cytokines, and mAbs targeting
T cell checkpoints, such as CTLA4, PD1, or Cancer is a life-threatening health problem which
PDL1/2 [36]. Also, it seems that radiation and is related to several genetic and environmental
radiofrequency ablation are future candidates for risk factors that manipulate immune system func-
combination therapy with immunotherapy [37]. tion. Cancers themselves produce immunosup-
Although immunotherapy and its combination pressor factors to impair cells division check
with other therapeutic approaches such as radio- points, leading to uncontrolled proliferation of
immunotherapy may be benecial for tumor cancer cells. Importantly, tumor cells have learned
treatment, there are several limitations that need how to escape from immune system attack via
to be addressed; dening the optimal target presenting of similar antigens to normal cells and
patient, optimal biological dose, and schedule, expression of very low levels of antigens.
the need for better trial designs incorporating Therefore, diagnosis of tumors and their progres-
appropriate clinical endpoints, and the identica- sion is not easy. Recently, immunodiagnostic
tion and validation of predictive biomarkers are methods are shown to be helpful in the diagnosis
just a few to point to [23]. of cancers and determining the extent of metasta-
sis. On the other hand, classic treatment of cancers
led to unsatisfactory results, and intelligent immu-
1.6.2 Cancer Cell Switch nological approaches, such as regulatory T-cell
targeting, adoptive T-cell administration, and
Cancer cells can switch on genes mostly related combination of immunotherapy and chemotherapy
to the earlier embryonic stages of development. are addressed. Results of antitumor vaccines,
1 Introduction on Cancer Immunology and Immunotherapy 7

Toll-like receptor (TLR) signaling pathway ago- 13. Kersey JH, Spector BD, Good RA. Primary immuno-
deciency diseases and cancer: the immunodeciency-
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15. Schlager SI, Ohanian SH, Borsos T. Correlation
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Inammatory and Innate Immune
Cells in Cancer Microenvironment 2
and Progression

Patrick Brennecke, Paola Allavena, Ilaria Laface,

Alberto Mantovani, and Barbara Bottazzi

Contents 2.1 Introduction

2.1 Introduction ................................................ 9
Inammation is a consistent feature of the tumor
2.2 Heterogeneity of Myeloid Cells
in the Tumor Microenvironment .............. 10
microenvironment and has been considered the
2.2.1 Myeloid Subsets in the Tumor seventh hallmark of cancer [16]. As suggested
Microenvironment........................................ 10 by current estimates, 25 % of cancers are asso-
2.2.2 Recruitment of Myeloid Cells in Tumors .... 12 ciated with chronic inammation sustained by
2.2.3 Tumor-Derived Factors Affecting
Myeloid Differentiation
infections (e.g., hepatitis) or inammatory con-
and Polarized Functions ............................... 13 ditions of diverse origin (e.g., prostatitis) [6].
In addition, even tumors not directly connected
2.3 Pro-tumoral Functions
of Tumor-Associated Myeloid Cells.......... 13 to inammation are characterized by the pres-
2.3.1 Tumor Proliferation and Survival ................ 14 ence of cells and mediators of the inammatory
2.3.2 Angiogenesis................................................ 15 response [7].
2.3.3 Cancer Cell Dissemination .......................... 16
Apart from malignant cells, host cells inl-
2.3.4 Suppression of Adaptive Immunity ............. 18
trate tumors, including leukocytes, broblasts,
2.4 Selected Aspects of Therapeutic and endothelial cells. Leukocytes, and in particu-
Targeting of TAMC .................................... 19
lar myeloid cells, are the most consistent cellular
2.5 Concluding Remarks ................................. 20 component of solid tumors. Tumor-associated
References ............................................................... 21 myeloid cells (TAMC) mainly support tumor
growth and progression, thereby contrasting the
P. Brennecke, MSc, PhD P. Allavena, MD T-cell inltrate, which mainly has antitumoral
I. Laface, PhD B. Bottazzi, PhD (*) activity. TAMC all arise from hematopoietic stem
Laboratory of Immunopharmacology, Humanitas cells (HSC) within the bone marrow (Fig. 2.1) and
Clinical and Research Center,
Via Manzoni 113, Milan, Rozzano 20089, Italy further differentiate into macrophage/granulo-
e-mail:; cyte progenitors. The tumor inltrate comprising; the myeloid populations skews tumor-mediated; immunosuppression, tissue remodeling, tumor
progression and metastasis [8, 9]. TAMC dem-
A. Mantovani, MD onstrated high plasticity, resulting in two extreme
Department of Biotechnologies and Translational
Medicine, University of Milan, 20122 Milan, Italy polarized macrophage (M1 and M2) and neutro-
phil (N1 and N2) phenotypes [10, 11]. Cross talk
Humanitas Clinical and Research Center,
Via Manzoni 56, Milan, Rozzano 20089, Italy between the different cellular components was
e-mail: demonstrated, resulting in tuning of the adaptive

N. Rezaei (ed.), Cancer Immunology: A Translational Medicine Context, 9

DOI 10.1007/978-3-662-44006-3_2, Springer-Verlag Berlin Heidelberg 2015
10 P. Brennecke et al.

Bone marrow Blood/spleen Tumor



Monocyte TAM




Neutrophil TAN



Fig. 2.1 Differentiation pathways of tumor-associated and N1N2 for neutrophils. CMP common myeloid pro-
myeloid cells. Myeloid cells originate from hematopoietic genitors, IMC immature myeloid cells, TEM Tie2-
stem cells (HSC) in the bone marrow. Here the networks expressing monocytes, MDSC myeloid-derived suppressor
that give rise to the various myeloid cell lineages in diverse cells, M-MDSC myeloid MDSC, G-MDSC granulocytic
compartments (bone marrow, blood/spleen, and tumor) and MDSC, TAM tumor-associated macrophages, TAN tumor-
their precursors are illustrated. In the tumor tissue, macro- associated neutrophils, iDC immature dendritic cells,
phages and neutrophils display a gradient of differently TADC tumor-associated dendritic cells
polarized phenotypes whose extreme are M1M2 for TAM

immune response, promotion of angiogenesis, approaches based on targeting of tumor-inltrating

and tissue remodeling [8]. myeloid cells and/or soluble mediators will be
Results obtained so far clearly indicate that discussed.
TAMC are major players in the connection
between inammation and cancer. Ongoing
efforts, which led to a better understanding of 2.2 Heterogeneity of Myeloid
their biological properties, indicated that myeloid Cells in the Tumor
cell-inltrating growing tumor could have a Microenvironment
prognostic value, thus representing an attractive
target for novel biological therapies of tumors. 2.2.1 Myeloid Subsets in the Tumor
In this chapter, we will mainly focus on Microenvironment
myeloid cells inltrating tumors and mention
soluble mediators involved in their recruitment or Solid tumors are characterized by the presence
released by TAMC, which affect tumor progres- of a leukocyte inltrate including lymphocytes
sion and dissemination (cytokines, chemokines, and myeloid cells from early stages. Growing
and proteases). Furthermore, new therapeutic evidence indicated that the leukocyte inltrate
2 Inammatory and Innate Immune Cells in Cancer Microenvironment and Progression 11

has a prognostic value. For instance, it has been responses [22, 14, 23, 11, 24]. Thus, macro-
described that inltrating T lymphocytes are phages are a very heterogeneous cell population,
associated with a favorable prognosis in colorec- able to display different functions depending on
tal cancer, melanoma, ovarian cancer, and breast the context. Macrophages can be either immuno-
cancer [12, 13]. In contrast, myeloid cells are stimulatory at the beginning of the inammatory
most frequently associated with a poor progno- response or immunosuppressive which dampen
sis [14]. TAMC (Fig. 2.1) comprise ve distinct inammation [25, 18, 14, 26, 27].
myeloid populations, namely, tumor-associ- A similar dichotomy with polarization towards
ated macrophages (TAM), monocytes express- two extreme phenotypes (N1 and N2) has been
ing the angiopoietin-2 (Ang-2) receptor Tie2 also described for neutrophils [28]. Besides
(known as Tie2-expressing monocytes or TEM), exerting antibacterial functions, neutrophils can
myeloid-derived suppressor cells (MDSC), inltrate tumors playing a major role as key
tumor-associated neutrophils (TAN), and tumor- mediators in malignant transformation, tumor
associated dendritic cells (TADC). progression, and regulation of antitumor activity
Tumor-associated macrophages belong to the [29]. Tumor-associated neutrophils (TAN) have
early inltrating leukocyte populations within received interest only recently, mainly due to
tumors, thus preceding lymphocytes, and are their short life span and the observation that
usually the most abundant immune population in tumor microenvironment can sustain and prolong
the tumor microenvironment [6, 15]. They derive the survival of polymorphonuclear leukocytes
from blood monocytes actively recruited from (PMN) [30, 31].
the circulation into tumor tissues. Early studies A particular small subset of TAMC is repre-
demonstrated that appropriately stimulated mac- sented by Tie2-expressing monocytes (TEM):
rophages are able to kill tumor cells in vitro; they express several monocyte/macrophage mark-
however, TAM, conditioned by the tumor micro- ers, along with the angiopoetin-2 receptor, Tie2,
environment, loose the cytotoxic capability and and are endowed with proangiogenic properties
rather exert several pro-tumoral functions, medi- [3235]. Tie2-expressing monocytes can be dis-
ating cancer-related inammation, angiogenesis, tinguished from the majority of TAM by their sur-
immunosuppression, tissue remodeling, and face marker prole (Tie2+, CD11b+) and their
metastasis [16, 17, 6]. preferential localization to areas of angiogenesis
The heterogeneous behavior of TAM is a [33], while they are largely missing in nonneo-
hallmark of myeloid cells and is oversimplied plastic area adjacent to tumors [35]. Indeed, Tie2
in a polarization concept with two extreme M1 is constitutively expressed at low levels by a sub-
and M2 phenotypes [1820] with distinct and stantial fraction (20 %) of circulating monocytes
somehow opposite functions. M1 macrophages and is overexpressed upon monocyte homing into
are classically activated by bacterial products growing tumors or regenerating tissues [33, 36].
and Th1 cytokines (e.g., LPS/interferon-). Following Ang-2 stimulation, Tie2+ monocytes
They are potent producers of inammatory and acquire an M2-like phenotype, with increased
immunostimulating cytokines, trigger adaptive expression of IL-10, CCL17, arginase 1 (Arg-1),
responses, secrete reactive oxygen species (ROS) and scavenger and mannose receptors and low
and nitrogen intermediates, and have cytotoxic expression of proinammatory molecules such as
effect towards transformed cells. On the other IL-12 and TNF- [37, 38].
hand, M2 macrophages or alternatively activated Myeloid-derived suppressor cells (MDSC) are
macrophages differentiate in response to Th2 a heterogeneous population of immature myeloid
cytokines (e.g., interleukin (IL)-4, IL-13) [21]. cells, having the ability to suppress T-cell func-
In contrast to their M1 counterpart, M2 macro- tions [39, 40]. They are derived from myeloid
phages produce growth factors, leading to tissue progenitors in bone marrows which do not differ-
repair and angiogenesis activation, have high entiate into mature granulocytes, macrophages,
scavenging activity, and inhibit adaptive immune or dendritic cells. MDSC have been isolated from
12 P. Brennecke et al.

blood, spleen, and bone marrow of tumor-bearing immature phenotype (iDC) [5557]. The immature
mice and inltrate the tumor tissue, where local stage of TADC is responsible for the tolerogenic
tumor-associated factors promote their activa- response of adaptive immunity against tumors and
tion [41]. In tumor-bearing mice, two main strongly contributes to tumor immune evasion [58].
subsets of MDSC were identied: monocytic
MDSC (M-MDSC), characterized by CD11b+,
Ly6G, and Ly6Chigh, and granulocytic MDSC 2.2.2 Recruitment of Myeloid Cells
(G-MDSC), characterized by CD11b+, Ly6Ghigh, in Tumors
and Ly6C [42]. M-MDSC were shown to gov-
ern the ability of differentiating into monocytes TAMC derive from monocytes and granulocytes,
(macrophages) and (DC), whereas G-MDSC do extravasated from the circulation and inltrating
not possess this potential [43]. These subsets the tumor mass. Recruitment of blood cells into
are functionally different: M-MDSC-mediated tumors is mediated by chemoattractants released
immunosuppression is based on upregulation of by tumor and stromal cells. CC chemokine 2
inducible nitric oxide synthase (iNOS), expres- (CCL2), originally known as monocyte chemotac-
sion of Arg-1, and production of suppressive tic protein 1 (MCP1), was the rst relevant tumor-
cytokines, whereas G-MDSC-mediated immuno- derived chemotactic factor described [59, 60].
suppression is characterized by antigen-specic Several other chemokines attracting myeloid cells
responses (including ROS release requiring pro- have been identied, including CCL5, CCL7,
longed MDSC and T-cell contacts) [44]. Tumor- CCL8, and CXC chemokine 1 (CXCL1) and
associated MDSC generally exhibit an M2-like CXCL12 [6163]. Furthermore, urokinase plas-
phenotype, while M1 and M2 phenotypes could minogen activator (uPA); growth factors such as
coexist in some mouse tumor models [45, 46]. colony-stimulating factor (CSF)-1, transforming
Human MDSC are still poorly dened [47], growth factor (TGF-), basic broblast growth
even if they have been isolated from blood of factor (bFGF, also known as FGF-2), and vascular
patients with glioblastoma, colon cancer, breast endothelial growth factor (VEGF); and antimicro-
cancer, lung cancer, or kidney cancer [4852]. bial peptides (e.g., human beta-defensin-3) were
Recent studies have proposed that human MDSC shown to be involved in myeloid recruitment into
have a characteristic CD34+, CD33+, CD11b+, and neoplastic tissues [64, 9, 6567].
HLA-DR prole [42]. Similarly to the murine The prototypic chemoattractant for neutro-
counterpart, human MDSC are divided into two phils, CXCL8, is mainly responsible for the
main subsets: monocytic MDSC (M-MDSC), recruitment of TAN; other related chemokines of
characterized by the expression of CD14, and the CXC subfamily are also involved, including
granulocytic MDSC (G-MDSC), identied by CXCL1, CXCL2, and CXCL6 [68, 69]. Moreover,
positivity for CD15. tumor-derived TGF- can promote neutrophil
A small number of dendritic (DC) are found migration [70].
in most human and murine neoplasms. Similarly CC chemokine receptor 2 (CCR2), CCL2 recep-
to macrophages and neutrophils, plasticity is a tor, CXCL12, CXCL5, and stem cell factor (SCF,
main feature of these cells. DC are differentially also known as KIT ligand) play a pivotal role in the
localized in tumors; for example, in breast cancer recruitment of MDSC into tumors [7173]; in addi-
immature langerin+ DC are interspersed within tion, Bv8, also known as prokineticine 2 (PROK2),
the tumor mass, whereas more mature CD83+, might be essential for MDSC recruitment [74, 75].
DC-LAMP+ DC are conned to the peritumoral Finally, the proinammatory proteins S-100A9 and
area [53]. In contrast to TAM, tumor-associated S-100A8, produced by MDSC, are implicated in an
dendritic cells (TADC) were found in the invasive autocrine loop promoting accumulation of suppres-
front of papillary thyroid carcinoma [54]. Growing sor cells into tumors [76, 77].
evidences demonstrate that the majority of TADC TEM do not express CCR2 and are therefore
found within the tumor microenvironment have an recruited towards tumors by different mechanisms
2 Inammatory and Innate Immune Cells in Cancer Microenvironment and Progression 13

[35, 78, 79]. Other CC chemokines, such as is driven by cytokines and other signals released
CCL3, CCL5, and CCL8, are produced by tumor in the tumor microenvironment [92]. Among
cells and could play a role in TEM recruitment these IL-10, IL-6, CCL2, CSF-1, and prostaglan-
[80]. Ang-2, overexpressed by tumor cells and din E2 (PGE2) were reported to promote M2-like
inamed tissues, has been shown to exert a che- polarization [93, 94]. TGF- is overexpressed by
motactic effect on Tie2-expressing blood mono- tumor cells and plays a crucial role in promoting
cytes in vitro, suggesting that the Ang-2/Tie2 axis an immunosuppressive phenotype, in addition to
might be involved in recruiting TEM into tumors driving N2 polarization of TAN [31].
[81, 32, 35, 34, 82]. In addition, recent data sug- Many tumor-derived factors were implicated
gest the involvement of the CXCL12-CXCR4 in MDSC expansion such as GM-CSF, M-CSF,
homing axis for TEM inltration [82]. IL-6, IL-1, VEGF, and PGE2 [44, 95]. In addi-
In recent years, it has been shown that tumor- tion, Bronte and coworkers recently found that
derived factors such as VEGF, CXCL12, CXCL8, cytokine-mediated induction of MDSC was com-
-defensins, and hepatocyte growth factor (HGF) pletely dependent on the transcription factor
are secreted into the bloodstream and are believed CCAT/enhancer-binding protein b (C/EBPb),
to attract iDC into the tumor bed [8386]. shown to function as a master regulator in this
Moreover, CCL20, CCL7, as well as the receptors process [96]. Further it was proposed that a com-
CCR5 and CCR6 were demonstrated to be impor- bination of at least two signals is necessary for
tant for TADC recruitment towards the tumor [87]. MDSC functionality and expansion, for example,
Proliferation can also contribute to sustaining GM-CSF, inhibiting maturation of myeloid cells,
TAMC levels in solid tumors. A paracrine loop and a proinammatory molecule such as
has been evidenced for TAM, with production of interferon- (INF-) [41].
colony-stimulating factor 1 (CSF-1) by murine Soluble factors released by tumor cells (i.e.,
brosarcoma cells acting on TAM-expressing IL-10, VEGF, TGF-, etc.) contribute to keep
CSF-1 receptor (CSF-1R) [88]. A nding con- DC in an immature pro-tumorigenic phenotype.
rmed more recently by Condeelis and Pollard Furthermore, in preclinical studies of breast
[89] showed the effect of epidermal growth factor cancer, it was shown that tumor-derived factors
(EGF) produced by TAM and tumor-derived altered DC maturation by secretion of thymic stro-
CSF-1 on recruitment and survival of macro- mal lymphopoietin (TSLP), which in turn induces
phages during tumor growth. Indeed, macro- the expression and secretion of the OX40 ligand,
phage proliferation has been demonstrated to a molecule that contributes to sustain the M2-like
occur during type II inammation [90]. phenotype of TAM.

2.2.3 Tumor-Derived Factors 2.3 Pro-tumoral Functions

Affecting Myeloid of Tumor-Associated
Differentiation and Polarized Myeloid Cells
Myeloid cells exposed to the tumor microenviron-
Upon arrival in the tumor, monocytes differenti- ment most frequently promote tumor progression.
ate to macrophages primarily in response to They can secrete soluble factors which support
CSF-1 produced by tumor cells. Although coex- proliferation and invasion of tumor cells, activate
istence of diverse TAM subpopulations with dis- angiogenesis, and promote resistance to therapies
tinct functions depending on tumor stage and (Fig. 2.2). High TAM or TAN inltration generally
geographical localization within the same tumor correlates with poor patient outcome [97, 6, 16,
has been proposed, they mostly have an M2-like 11, 98101], but few exceptions to this nding are
phenotype [91]. Many different studies demon- also reported. For instance, in colorectal cancer
strated that M2 (pro-tumoral) TAM polarization (CRC) contrasting results reported that TAM
14 P. Brennecke et al.

TAM Th1; Th2; Treg

Tumor cell

Immunosuppression Matrix degradation Angiogenesis


Tumor survival Local invasion Resistance to

proliferation metastasis therapies

Fig. 2.2 Pro-tumoral functions of tumor-associated the release of soluble factors (i.e., cytokines, growth and
myeloid cells. TAMC exposed to the tumor microenviron- proangiogenic factors, proteolytic enzymes, etc.) and result
ment exert several pro-tumoral functions, including promo- in higher tumor survival and proliferation, local invasion
tion of angiogenesis, matrix degradation, and suppression and dissemination, resistance to therapies
of adaptive immunity. These effects are mediated through

density is associated with positive or negative and CD86) was associated with better patient sur-
patient outcome [102, 103105]. On the same line, vival in CRC, whereas the same cell population
TAN inltrate is associated with a favorable prog- within the tumors did not have any predictive
nosis in patients with gastric carcinomas [106], but value [109, 110]. Thus, TAMC exert complex
also with more aggressive pancreatic tumors roles on growing tumors affecting different aspects
[107]. Macrophage subsets might have distinct of tumor progression, i.e. tumor cell proliferation
roles, as observed in lung adenocarcinoma were and survival, angiogenesis, tumor dissemination,
the number of CD204+ TAM showed a strong and resistance to therapies.
association with poor patient outcome, while the
CD68+ TAM population did not [108]. The con-
cept that not only the number and the presence of 2.3.1 Tumor Proliferation
specic cell subsets but also the localization of and Survival
inltrating cells might have specic functions and
predictive values is increasingly emerging. TAM were shown to have the ability to promote
Accordingly, peritumoral TAM density with high tumor growth directly through the production of
expression of co-stimulatory molecules (CD80 trophic and activating factors for stromal and
2 Inammatory and Innate Immune Cells in Cancer Microenvironment and Progression 15

cancer cells (EGF, bFGF, VEGF, platelet-derived The prototypic myeloid cell with angiogenic
growth factor [PDGF], TGF-) [111, 112, 6, properties is the Tie2 monocyte [32, 35]. TEM
113] in response to stimuli from the tumor micro- can be found in close proximity to nascent blood
environment. For example, IL-13 and IL-4 pro- vessels within solid tumors. In addition, TEM
duced by CD4+ T-cell-inltrating tumors, such as depletion completely prevented neovasculariza-
breast cancer, led to the production and secretion tion in preclinical models (spontaneous pancre-
of EGF by TAM [114]. Moreover, production of atic adenocarcinoma, human glioma grown
proinammatory cytokines, including TNF- orthotopically in the mouse) [33]. Interestingly,
and IL-6, by TAM and other cells of the tumor TEM ablation did not affect the number of inl-
microenvironment (e.g., epithelial cells), sustains trating TAM or TAN, suggesting that TEM are an
tumor growth and inhibits apoptosis [115119]. entity on their own and not just precursors of
Several lines of evidence suggest that TAN are TAM [35]. How TEM stimulate angiogenesis has
required for the rapid growth of tumor cells and not been claried yet, but preliminary indications
their depletion inhibits tumor development [120, in murine tumor models point to the fact that
28]. Proteins stored within neutrophil granules perivascular TEM secrete bFGF. It is believed
(e.g., elastase) may have a role in tumor initiation that release of such factors in close proximity to
[121]. In addition, neutrophil-derived ROS have vessels could directly stimulate angiogenesis or
been associated with DNA damage [122]. TAN MMP9 secretion, which in turn would release
were shown to be able to produce soluble factors growth factors entrapped within the extracellular
(cytokines and chemokines, HGF, oncostatin M), matrix (ECM).
driving processes like angiogenesis, wound heal- TAM have also a profound inuence on the
ing, and hematopoiesis and thus exerting a role in regulation of tumor angiogenesis [129]. It was
tumor promotion and growth [123125, 121, demonstrated in several preclinical studies that
101]. For instance, HGF released by neutrophils TAM positively correlated with microvascular
enhances the invasiveness of human cholangio- density (MVD) [130133]. Lin and coworkers
cellular and hepatocellular carcinoma cells were the rst to describe the direct role of TAM
in vitro, and HGF levels in bronchoalveolar in driving the angiogenic switch in a spontane-
lavage uids were found to correlate with neutro- ous mammary carcinoma mouse model [134].
phil number in patients with bronchoalveolar car- Likewise, depletion of monocytes by clodronate
cinomas, which further correlates with poor treatment in a preclinical model with Lewis lung
patient prognosis [101]. carcinoma led to lower TAM inltration and
angiogenesis, further underlining the importance
and the involvement of macrophages in tumor
2.3.2 Angiogenesis angiogenesis [135].
TAM express various molecules modulating
To sustain the increased metabolic demand of angiogenesis, such as VEGF, bFGF, TNF-,
growing tumors, the development of a tumor vas- IL-1, CXCL8, cyclooxygenase 2 (COX2, also
culature is required. VEGF is the primary, but not known as PTGS2), plasminogen activator, uPA,
the only, angiogenic factor released by tumor cells PDGF-, MMP7, MMP9, and MMP12 [136].
and is involved in the angiogenic switch that Hypoxia exerts a crucial role in the upregulation
can occur at various stages of tumor progression, of gene transcription in TAM, promoting VEGF
depending on the tumor type and the microenvi- expression [137141]. Other recent studies
ronment. Other factors are involved, including showed a direct involvement of TAM in tumor
PDGF-, bFGF, angiopoietins, and CXCL12 angiogenesis and neovascularization via transdif-
(SDF-1) [126]. Tumor-associated myeloid cells ferentiation into endothelial cells when stimu-
were shown to contribute to tumor angiogenesis lated by angiogenic factors [142, 143].
by production of growth factors, cytokines, and More recent studies have shown that MDSC
proteases [80] such as VEGFA, Bv8, and metal- can contribute to tumor angiogenesis. In a pre-
loproteases (MMP) [10, 127, 65, 128]. clinical model for colon cancer, MDSC positively
16 P. Brennecke et al.

correlated with tumor growth rate and blood ves- DC maturation and further promote angiogenesis
sel density [144]. Moreover, tumor angiogenesis via this autocrine loop [153, 151].
was signicantly lowered by blocking Bv8 with a
neutralizing antibody, a treatment that signi-
cantly reduced the number of MDSC [74]. 2.3.3 Cancer Cell Dissemination
Metalloproteases, particularly MMP9, MMP2,
MMP13, and MMP14, produced by MDSC, were The major cause of death in cancer results from
shown to enhance VEGF bioavailability by mobi- therapy-resistant metastases. Stephen Pagets
lization from the ECM [144, 145]. Increased conclusion in the late nineteenth century that the
recruitment of MDSC has also been demonstrated metastatic process depends on cross talk between
in the presence of hypoxia, possibly stimulating selected cancer cells (the seeds) and a specic
tumor angiogenesis [126, 74]. Parallel to TEM, organ microenvironment (the soil) is still valid
MDSC were also observed to be localized in the and is experimentally conrmed [154, 155].
vicinity of blood vessels. Under certain condi- Tumor metastasis is a complex multistep process,
tions, some MDSC acquire endothelial cell shape, during which malignant cells spread from the pri-
start to express endothelial markers including mary tumor site to secondary distant organs. The
CD31 and VEGFR2, and are eventually incorpo- different steps of cancer cell dissemination can be
rated into the tumor endothelium [144]. subdivided into local invasion, entry into the
TAN were shown to rapidly release VEGF bloodstream (intravasation), survival in the blood-
from internal storage compartments, leading to stream, extravasation, and colonization [156].
endothelial proliferation and tubule formation Mesenchymal, endothelial, and immune cells are
[146, 147]. In addition, TNF- and GM-CSF required to form an appropriate microenviron-
secreted by tumor cells were shown to trigger the ment for tumor progression [157]. Immune cells,
release of proangiogenic chemokines by particularly macrophages, neutrophils, T lympho-
TAN. The number of TAN in myxobrosarcoma cytes, and natural killer (NK) cells, are major
positively correlated with tumor MVD [148]. sources of proteases that degrade the host tissue,
Furthermore, in a xenograft mouse model of allowing cancer cells to disseminate.
human melanoma where cancer cells were engi- The set of proteolytic enzymes found in tumor
neered to constitutively produce CXCL6, it was microenvironment comprises matrix metallopro-
found that the number of TAN as well as angio- teases, serine proteases, and cysteine proteases
genesis was markedly increased [149]. Studies in (i.e., cathepsin) [158162]. Matrix proteases exert
the RIP1-TAG2 mouse model for pancreatic car- essential functions in physiological conditions as
cinogenesis revealed formation of dysplastic, active regulators of postnatal tissue development
neutrophil-bearing, angiogenic islets upon malig- and remodeling. In addition, they are important
nant transformation. In the abovementioned for tissue repair in response to injury and regulate
model, neutrophil depletion of the islets led to cancer progression modulating the tumor micro-
dramatically lowered angiogenesis [150]. environment, particularly the leukocyte inltrate
In recent years, it has become more and more [163]. MMP were shown to activate TGF-,
apparent that iDC make a profound contribution which is an important regulator of T-cell and TAN
to tumor angiogenesis [85]. TNF- and CXCL8 functions [164]. Proteases also produce specic
produced by iDC from ovarian cancer ascites cleavage fragments of target chemokines with
triggered the release of various growth factors independent biological activity, ranging from
from EC [85, 151]. Moreover, iDC were shown anergic products (CXCL7, CXCL4, CXCL1),
to release osteopontin which promotes monocyte antagonists (CCL7), or more potent chemoattrac-
secretion of the proangiogenic IL-1 [152]. tants (CXCL8), thereby modulating the leukocyte
Finally, it was recently observed that iDC pro- composition within a tumor [165167].
duced high levels of VEGF and CXCL8 under Besides their inuence on the tumor inltrate,
hypoxic conditions, which, in turn, might inhibit proteases were shown to promote cancer cell
2 Inammatory and Innate Immune Cells in Cancer Microenvironment and Progression 17

invasion and intravasation. The cleavage of cell- It has been suggested that tumor cells can
adhesion molecules like E-cadherin induces the inuence the microenvironment of secondary
disruption of cell-cell junctions leading to loos- organs promoting the formation of a pre-
ening of cell-cell contacts which, together with metastatic niche [174, 175]. Tumor-derived fac-
ECM protein turnover, facilitated cancer cell tors and HSC are crucial components of the
migration and invasion into the surrounding tis- pre-metastatic niche. VEGF derived from tumor
sue and vasculature. Tight regulation of the sin- cells promote recruitment to the secondary organs
gle proteases within the tumor microenvironment of VEGFR1-expressing HSC that induce bro-
allows the control of tumor cell invasion [168]. nectin and MMP9 expression by resident bro-
After invasion to the surrounding tissues, can- blasts, creating favorable conditions for
cer cells enter the blood circulatory system directly settlement of future metastases [176]. Other solu-
or indirectly via the lymphatic system. Since the ble factors released by tumor cells can promote
majority of circulating tumor cells (CTC) are the formation of pre-metastatic niche. In a murine
eliminated by NK cells [169], only about 0.01 % model of breast cancer, tumor cells were found to
of CTC survive in the bloodstream [157]. Platelets induce production of CCL17 and CCL22 in the
play a key role in hematogenous metastasis and lung; both attracting CCR4+ tumor and immune
contribute to the survival of CTC in the blood- cells which establish a microenvironment for
stream by both thrombin-dependent and thrombin- metastases settlement at secondary organs [177].
independent mechanisms [170]. After a passage Moreover, it was demonstrated that the proto-
into the bloodstream, CTC adhere to vessel walls typic hypoxia-induced protein lysyl oxidase
for extravasation when they are in the vicinity of (LOX), often found in tumors, leads to cross-
secondary metastatic organs. Circulating tumor linking of collagen IV in basement membranes,
cells take advantage of the capability of neutro- in addition to recruitment of CD11b+ myeloid
phils and platelets to produce and secrete adhesion cells which adhere to the abovementioned colla-
molecules, such as integrins and selectins which gen meshwork. The captured CD11b+ myeloid
all aid the nearby CTC to adhere and ultimately cells were shown to secrete MMP2, which facili-
extravasate [170, 171]. tated invasion and recruitment of metastasizing
The arrest of cancer cells to specic organs tumor cells [178].
seems to be primarily mechanical [172]. TAMC, TAM and MDSC in particular, are
However, chemokines and chemokine receptors important players of tumor progression and met-
are also involved in organ-specic colonization, astatic colonization through the cross talk with
which nally drive cells along tissue-specic tumor cells. For instance, macrophages play a
chemokine gradients. Furthermore, a non- crucial role in conferring an invasive phenotype
chemokine pathway also exists, in which immune to epidermal keratinocytes from Snail transgenic
cells support organ-specic cancer cell dissemi- mice [179]. TAM contribute to cancer cell dis-
nation. One example is represented by the two semination by releasing enzymes involved in
inammatory mediators S100-A8 and S100-A9, degradation of the ECM (i.e., MMP and cathep-
which were shown to promote metastasis through sin) [168, 161, 180, 76], or motility factors.
serum amyloid A 3 (SAA-3) [173]. Recently we found that tumor-derived soluble
The subsequent growth of arrested tumor cells factors, particularly CSF-1, activate a transcrip-
will depend on the molecular interactions tion program in macrophages resulting in upreg-
between cancer cells and the microenvironment ulation of a series of genes, especially
of the new organ. Although cancer cells are migration-stimulating factor (MSF). MSF is a
sometimes said to home to specic organs truncated isoform of human bronectin 1, physi-
(e.g., breast tumors metastasizing to bone), it is ologically expressed during fetal life and upregu-
more likely that this organ specicity is due to lated in M2-like macrophages [181, 182]. MSF
efcient organ-specic growth rather than prefer- exerts a chemotactic effect on tumor cells, indi-
ential homing of cells to a particular organ. cating that macrophage products released in the
18 P. Brennecke et al.

tumor microenvironment can support the pro- sive phenotype, with production of high levels of
invasive phenotype of tumor cells [181]. An the immunosuppressive cytokines IL-10 and
example of the cross talk between TAM and TGF- and reduced expressions of IL-12 [19,
tumor cells involved in metastatic colonization is 186, 92, 187, 188]. In addition, they have reduced
shown in breast cancer, where EGF secreted by tumoricidal activity and are poor in antigen pre-
TAM increases migration and invasion of neigh- sentation [189]. Furthermore, TAM secret che-
boring breast cancer cells which express high mokines, such as CCL17 or CCL22, that
levels of EGF receptor (EGFR). On the other preferentially attract Th1, Th2, and T regulatory
hand, cancer cells secrete high levels of CSF-1, a (Treg) lymphocytes with defective cytotoxic
main chemoattractant for TAM which expresses functions, or such as CCL18, that recruit nave
the cognate receptor CSF-R1. Therapies aiming T cells which become anergic in contact with
at inhibiting this cross talk by blocking CSF-R1 M2 macrophages and iDC [8, 190192].
and/or EGFR were shown to be successful [183, MDSC play a prominent role in the inhibition
184]. Macrophages and their reciprocal cross talk of tumor-specic immune responses. MDSC
with tumor cells are mandatory for tumor cell localized within the tumor microenvironment has
migration, regardless of the factor inducing cell an M2-like phenotype and mediate immunosup-
invasion (i.e., SDF-1). pression through multiple pathways, that is, pro-
A myeloid cell population involved in tumor duction of Arg-1 [193], iNOS [194, 195], ROI,
progression, including invasion, is represented by and suppressive cytokines including IL-10 and
MDSC. A direct role for MDSC in tumor metasta- TGF- [196], or via the activation and recruitment
sis has not been demonstrated; however, a connec- of Treg [196, 197]. MDSC inhibit homing to
tion was suggested by the study on mice decient lymph nodes of CD4+ and CD8+ T cells and sup-
for the TGF- receptor type 2 (TGF--R2), in which press their activation [198, 199]. It was found that
MDSC were concentrated on the invasive margin. cysteine uptake by MDSC limited its availability
In addition, it is possible to reduce lung metastases for uptake by T cells, which in turn disables their
by antagonizing CXCR2 and CXCR4, two recep- activation and renders them nonfunctional.
tors involved in homing of MDSC [145]. As previ- Furthermore, it was shown that posttranslational
ously mentioned, PGE2 and the proinammatory T-cell receptor modications mediated via gener-
molecule S100A9 have been identied as main ation of peroxynitrite species led to anergy of
effectors of MDSC accumulation and function. effector CD8+ T cells [196]. MDSC can also
Accordingly, S100A9 decient mice rejected impair innate immunity through cross talk with
implantation of colorectal cancer, while administra- macrophages which led to decreased production
tion of wild-type MDSC reverted the phenotype of IL-12 by macrophages and increased produc-
and colorectal cancer cells could successfully tion of IL-10 by MDSC, thus driving a polariza-
engraft [76]. In addition, TGF- was demonstrated tion towards an M2-like phenotype [200].
to be instrumental in MDSC homing, mediated via In addition to the above described mecha-
CXCL12-CXCR4 and CXCL5-CXCR2 axis in a nisms in TAM and MDSC, TADC were found to
preclinical mammary cancer model [145]. be involved in suppression of adaptive immunity.
One mechanism leading to the induction of
tumor-specic T-cell tolerance was via upregula-
2.3.4 Suppression of Adaptive tion of inhibitory molecules such as B7-H1 [201]
Immunity or by inducing the expression of Arg-1 [202].
Moreover, it was shown that the induction of
Besides the effect on tumor growth and dissemi- oxygen-dependent pathways led to the downreg-
nation, TAMC have also the potential to suppress ulation of CD3 epsilon and T-cell apoptosis
the adaptive immune response, leading to cancer [203]. Furthermore, Muller and coworkers dem-
immune evasion [185]. onstrated that upregulation of indoleamine
M2-like polarized tumor-inltrating macro- 2,3-dioxygenase (IDO) in TADC contributed to
phages are characterized by an immunosuppres- immunosuppression [204].
2 Inammatory and Innate Immune Cells in Cancer Microenvironment and Progression 19

2.4 Selected Aspects approach to affect TAM specically is to try to

of Therapeutic Targeting reeducate them to become tumoricidal or, in
of TAMC terms of polarization, to try to repolarize them
towards an M1 phenotype. Several successful tri-
The above summarized data describing the pro- als using CpG-oligodeoxynucleotide (TLR9 ago-
tumoral role of the myeloid inltrate of tumors nists) were performed in combination with
make clear that TAMC are reasonable targets for anti-IL-10 receptor or anti-CD40 antibodies,
novel therapeutic approaches. As illustrated which reverted pro-tumoral M2-like TAM to M1
above, TAMC can directly promote tumor cell macrophages displaying antitumor activity [216
growth releasing growth factors and proangio- 218]. Rolny et al. recently demonstrated that
genic molecules, in addition to suppression of skewing of M2 TAM towards M1 leads to effec-
tumor-specic immune responses. Strategies tive antitumoral activity of host histidine-rich
explored in the last years are focused on the stop- glycoprotein (HRG), which in consequence leads
page of the mechanisms leading to suppression to inhibition of angiogenesis and promoted anti-
of lymphocyte activity and, on the other side, on tumor immune responses [219]. Gazzaniga and
the reduction of recruitment of myeloid cells and coworkers reported promising results using the
repolarization of M2-like pro-tumoral cells to molecule legumain, which targets M2 polarized
proinammatory M1 macrophages. There is a TAM specically, and was able to induce a robust
wide range of preclinical and clinical research CD8+ T-cell answer leading to reduced tumor
aimed at eliminating or reprogramming TAMC growth and inhibition of tumor angiogenesis
[39]: here we only mention some examples of the [220]. Furthermore, it was shown that zoledronic
results obtained so far in this growing eld of acid was able to revert M2 towards M1 TAM and
anticancer research. inhibit breast carcinogenesis by targeting the
Many studies have shown that targeting TAM mevalonate pathway [221]. Moreover, it was
might be a successful strategy to limit tumor demonstrated that direct reeducation of TAM
growth and metastasization and to achieve bet- using the prototypical M1 polarizing cytokine
ter therapeutic responses [32, 44, 59, 82, 189, INF- [222] is successful in promoting antitumor
205, 206, 207]. One example is represented by activity in minimal residual disease [8]. In line
bisphosphonates [208] traditionally used in the with the abovementioned results are the ndings
clinic to treat osteoporosis, which were shown to that inhibition of M2 polarization led to restora-
be very effective in depleting TAM and inhibit- tion of M1 proinammatory phenotype and inhi-
ing angiogenesis as well as metastatic spread bition of tumor growth in several preclinical
in preclinical animal models for breast cancer animal models [92, 223, 224].
[209, 210]. Furthermore, Germano and cowork- To counteract the pro-tumoral activities of
ers recently showed that specic targeting of MDSC, two general strategies can be envisaged;
macrophages with the marine antitumor agent the rst consists of transforming these immature
trabectedin was very successful in four different cells into mature cells devoid of suppressive
preclinical tumor animal models [211]. activity, and the second is focused on blocking
An alternative strategy is to target circulating MDSC suppressive functions. Depletion of
monocytes known as precursors of TAM. Two MDSC producing high levels of TGF- (in an
candidate molecules are the M-CSF receptor IL-13-dependent manner) led to the restoration
(solely expressed by monocyte-macrophages) of T-cell-mediated immunosurveillance in a pre-
and the chemokine CCL2, involved in monocyte clinical mouse model for brosarcoma [225].
recruitment within tumors. Since preclinical Several studies have shown that metabolites of
studies on prostate and colon cancer [212215] all-trans-retinoic acid are able to differentiate
identied CCR2+Ly6C+ cells as targets involved MDSC into DC and macrophages, reducing
in cancer progression and metastasis, CCL2 anti- MDSC accumulation [226, 227]. This effect was
bodies are currently investigated for therapeutic demonstrated to be benecial for patients suffer-
applications in human cancer treatment. Another ing from metastasizing renal cancer, since in
20 P. Brennecke et al.

these patient less circulating MDSC were was demonstrated that delivery of regulatory
detected in the bloodstream [228]. Furthermore, miRNA, particularly miRNA 155 in a nanoparti-
one of the benecial effects of the anticancer cle formulation, leads to reprogramming of
drug gemcitabine is its potential to eliminate immunosuppressive TADC to highly active anti-
MDSC without affecting T, B, NK cells, or mac- tumoral TADC which provoked regression of
rophages [229]. established ovarian tumors [236].
The second possibility to counteract MDSC In light of the recent results, tumor therapy
function is to block their inhibitory function, for with drugs targeting the inammatory tumor
example, by using COX2 inhibitors, phosphodi- microenvironment in combination with treatment
esterase (PDE5), and nonsteroidal anti- aimed at defeating TAM, TAN, and other myeloid
inammatory drugs releasing NO [44]. Blocking cells holds promise for the future.
of IL-1 inhibits cancer progression and metasta-
sis [230] and decreases MDSC accumulation and
suppressive activity [42]. Moreover, the proan- 2.5 Concluding Remarks
giogenic chemokine Bv8 was shown to be impor-
tant for mobilization and homing of MDSC to In recent years, it has become clear that inam-
tumor sites and therefore qualies as an interest- mation has an essential role in tumor promotion
ing therapeutic target [74]. [16]. The inammatory tumor microenviron-
Complete neutrophil depletion in already ment, mainly consisting of soluble factors and
immunocompromised patients is not desirable; host cells, has a predominant role in all aspects
therefore, the strategy of choice concerning TAN of the disease (progression, angiogenesis,
might be to disturb their tumor homing ability, in immune surveillance). In particular, a heteroge-
other words to interfere with their ability to neous group of myeloid cells is the most consis-
migrate. To this purpose, preclinical experiments tent host cell component of solid tumor [8, 9].
using anti-CXCR2 antibodies were performed TAM, TEM, MDSC, TAN, and TADC display
and were shown to be successful [231]. distinct specialized functions, as well as overlap-
Furthermore, considering the well-documented ping activities (e.g. angiogenesis). Tumor and
key role of TGF- in skewing TAN towards a N2 stromal cells release different chemoattractants
phenotype, this cytokine keeps promising poten- involved in the recruitment of myeloid cells from
tial for treatment [70, 31]. the blood into the growing tumor. Cytokines and
Some studies indicate that blocking IL-10 other soluble factors released in the tumor micro-
together with the administration of CpG oligo- environment can contribute to induce a protu-
nucleotides are able to unblock the functionally moral phenotype, promoting M2 polarization of
paralyzed TADCs and to reactivate antitumor TAM [92], N2 polarization of TAN [31], MDSC
responses [232]. Another strategy enhancing expansion [41], or preventing maturation of
immunotherapy might be targeting of soluble DCs. Thus the different TAMC populations
factors like VEGF, IL-10, TGF-, gangliosides, potentially represent a target for new therapeutic
and others, which are all tumor secreted factors approaches aimed at breaking the protumoral
leading to abnormal differentiation of DC, often networks established by cancer-associated
leaving them in an immature state [233]. Other myeloid cells.
and more recent strategies make use of siRNA
nano-complexes which lead to reprogramming of Acknowledgments The authors would like to gratefully
TADC from an immunosuppressive to an acti- acknowledge the nancial supports of the European
vated anticancer phenotype [234]. Furthermore, Research Council (ERC project HIIS), the European
it was shown that in situ stimulated CD40 and Commission (FP7-HEALTH-2011-ADITEC-280873),
the Italian Association for Cancer Research, the Italian
toll-like receptor 3 (TLR3) TADC were success-
Ministry of Health and University, Fondazione CARIPLO
fully transformed from immunosuppressive to (project 20092582), and Regione Lombardia (project
immunostimulatory cells [235]. More recently it Metadistretti SEPSIS).
2 Inammatory and Innate Immune Cells in Cancer Microenvironment and Progression 21

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rophage polarization are related processes orches- 72(7):168393.
Role of Innate Immunity in Cancers
and Antitumor Response 3
Masahisa Jinushi and Muhammad Baghdadi

Contents 3.4 The Role of Effectors Produced

from Innate Immune Cells
3.1 Introduction ................................................ 29 in Cancer and Antitumor Immunity ........ 37
3.2 Role of Innate Immune Cells 3.4.1 Interferons (IFNs)......................................... 37
in Cancer and Antitumor Immunity ........ 30 3.4.2 Other Cytokines ........................................... 38
3.2.1 Natural Killer (NK) Cells ............................. 30 3.4.3 Chemokines .................................................. 39
3.2.2 Natural Killer T (NKT) Cells ....................... 31 3.5 Concluding Remarks ................................. 40
3.2.3 -T Cells ..................................................... 31
3.2.4 Macrophages ................................................ 31 References ............................................................... 40
3.2.5 Dendritic Cells ............................................. 32
3.2.6 Granulocytes................................................. 32
3.3 The Role of Innate Immune Receptors
on Innate Immune Cells in Cancer 3.1 Introduction
and Antitumor Immunity .......................... 32
3.3.1 Toll-Like Receptors (TLRs) ......................... 32 Cellular components of the innate immune system
3.3.2 RIG-I-Like Helicases (RLHs) ...................... 33
serve as a rst line of defense against tumori-
3.3.3 NOD-Like Receptors (NLRs) ...................... 33
3.3.4 Phagocytosis Receptors ................................ 33 genic cells. Recognition of transformed cells by
3.3.5 C-Type Lectin-Like Receptors (CLRs) ........ 34 pattern-recognition receptor (PRRs) on the innate
3.3.6 NK Cell Receptors ....................................... 34 immune cells activates specialized inammatory
3.3.7 B7 Family ..................................................... 37
signaling cascades, including transcription factor
nuclear factor-kappa B (NF-B) and interferon
regulatory transcription factor (IRF), which lead
to the release of various cytokines and chemo-
kines attracting and activating effector lympho-
cytes at the tumor site. In addition, effector cells
kill transformed cells through the activation of
perforin or death receptor-mediated pathways,
M. Jinushi, MD, PhD (*)
Research Center for Infection-Associated Cancer, as well as secretion of cytokines necessary
Institute for Genetic Medicine, Hokkaido University, for the initiation of immune responses against
Kita-ku, Kita 15, Nihi 7, Sapporo 060-0815, Japan transformed cells [1, 2]. However, some tumor
e-mail: cells escape from the innate immune machinery,
M. Baghdadi, MD, PhD which leads to the dysfunction of innate immune
Division of Immunobiology, compartment, signaling pathways, and effector
Institute for Genetic Medicine, Hokkaido University,
Kita 15, Nishi 7, Sapporo 060-0815, Japan functions. This manipulation of innate immune
e-mail: systems by tumor microenvironments includes

N. Rezaei (ed.), Cancer Immunology: A Translational Medicine Context, 29

DOI 10.1007/978-3-662-44006-3_3, Springer-Verlag Berlin Heidelberg 2015
30 M. Jinushi and M. Baghdadi

impairment of antigen processing and presen- NK cells have the ability to distinguish trans-
tation by antigen-presenting cells (APCs) [3], formed cells from normal cells by recognizing a
inhibition of innate immune signaling pathways variety of cell surface receptors, including killer
[4, 5], and anti-inammatory cytokines such as activation receptors (KARs), killer inhibitory
IL-10 and transforming growth factor- (TGF- receptors (KIRs), natural killer group two mem-
) [6, 7]. Moreover, tumors manipulate innate ber D (NKG2D), DNAX-accessory molecule
immune systems to create protumorigenic envi- (DNAM), etc., which will be discussed later in
ronments, which lead to further tumor progres- this chapter. For example, KIRs on NK cells has a
sion and metastasis. Therefore, it is critical high afnity to the specic alleles in HLA class I
to clarify the molecular mechanisms through molecules, transducing an inhibitory signal to the
which the interaction between tumors and innate NK cells and preventing it from eliminating non-
immune systems is modied during different transformed cells. However, deletion of a single
phases of tumorigenesis. allele in HLA class I and/or induction of activat-
In this chapter, we describe the general func- ing receptors such as NKG2D ligands, which fre-
tions of innate immunity in cancer and antitumor quently occurs on transformed cells, triggers
host response. In addition, an overview is pro- effector functions of NK cells against tumor cells
vided on the mechanism through which coor- [10, 16]. Recent studies have focused on licens-
dinated actions of innate immune signals and ing NK cells to become functionally competent
their downstream effectors have an impact on through the interaction with self-MHC mole-
the immunosurveillance and immune subversion cules. Ly49C is an inhibitory receptor expressed
within the tumor microenvironment. on a subset of NK cells, which interact with self-
MHC molecules on target cells, and plays an
unexpected role in enabling immature NK cells to
3.2 Role of Innate Immune Cells develop into functioning, mature cells. On the
in Cancer and Antitumor other hand, Ly49C-negative NK cells are consid-
Immunity ered as non-licensed and remain at an imma-
ture stage [17]. These evolutionary processes of
3.2.1 Natural Killer (NK) Cells NK cell development and activation may help
explain why donor NK cells administrated to leu-
NK cells are important effector cells for protec- kemia patients during bone marrow transplanta-
tion against viruses and some tumors, since NK tion do not always show antitumor effects [18].
cell-depleted mice were more susceptible to The NK cell-mediated cytotoxic activities medi-
3-methylcholanthrene (MCA)-induced tumors ate the release of granule contents (perforin and
[8]. Chemokines such as CXCL12 and granzyme) onto the surface of the tumor cell [19].
CXCL3L1 are key factors for NK migration to The interaction between NK cells and dendritic
tumor sites [9], where they play an important cells (DCs) is crucial for the amplication of
role in the tumor immunosurveillance [10]. NK innate responses and the induction of potent adap-
cells recognize and eliminate transformed cells tive immunity. Immature DCs are susceptible to
by releasing perforin or death signal-associated NK cell-mediated cytolysis [20], while mature
receptors such as FAS and TRAIL (tumor DCs are activated by NK cells through cytokines
necrosis factor-related apoptosis-inducing (TNF- and IFN-) and receptor (NKp30 and
ligand) [1113]. NK cells secrete interferon NKG2D)-mediated mechanisms [21, 22]. On the
gamma (IFN-) which helps to activate T-cell- other hand, activated DCs trigger effector activi-
mediated immunity and suppress tumor angio- ties of NK cells, such as IFN- production, prolif-
genesis [14, 15]. Moreover, various innate eration, and cytotoxic activities [23]. In addition,
immune networks such as cytokines and PRR treatment with TLR3 agonist polyinosinic-poly-
recognition systems play an important role in cytidylic acid (Poly (I: C)) triggers DCs to activate
stimulating effector functions of NK cells as dis- antitumor activities of NK cells [24, 25]. Thus, the
cussed later. reciprocal interaction between NK and DC
3 Role of Innate Immunity in Cancers and Antitumor Response 31

regulates the direction and quality of antitumor 3.2.3 -T Cells

immunity, which is important for the development
of effective cancer immunotherapy. Although -T cells represent a small popula-
tion among T lymphocytes, they share several
features with innate immune cells. -T cells
3.2.2 Natural Killer T (NKT) Cells show high frequencies in intraepithelial lym-
phocytes (IELs) in the skin and gut mucosa and
NKT cells are innate lymphocytes which share possess a distinct T-cell receptor on their sur-
features of both NK cells and T cells. NKT cells face with limited diversity, which may serve as
express particular NK cell markers such as a pattern-recognition receptor [35]. Moreover,
CD161 or NKR-P1, in addition to an invariant -T cells lack CD4 and CD8 expressed by
T-cell receptor alpha chain (V14-J18 in mice -T cells and express a number of molecules
and V24-J18 in humans) [26]. The invariant shared with NK cells or APCs, such as Fc
T-cell receptor alpha chain is specic for glyco- gamma RIII/CD16 and PRRs. -T cells also
lipid antigens presented by CD1d, which is an recognize lipid-derived antigens and function
MHC class I-related molecule expressed on as professional phagocytes which recognize
antigen-presenting cells and also found in some and ingest apoptotic tumor cells and may inu-
tumor cells. NKT cells were shown to play a role ence antitumor immune responses [36, 37].
in the tumor immunosurveillance, since J18/ Mice lacking -T cells showed increased
mice showed increased susceptibility to chemi- incidence of chemically induced sarcoma and
cally induced tumors and experimentally induced spindle cell carcinoma, indicating the importance
metastases [27]. Moreover, the administration of of these cells in tumor immunosurveillance [38].
-galactosylceramide, a natural lipid isolated In addition, -T cells express NKG2D receptors
from marine sponges which efciently binds to and interact with their ligands on transformed
CD1d and thus activates NKT cells, induces anti- cells, leading to enhanced cytotoxic activities and
tumor immune responses against established effector cytokine production [39, 40]. The acti-
murine tumors [28]. The antitumor activities of vated -T cells then serve as the major early
NKT cells are mediated by IFN- production, source of IFN-, which contribute to maturation
which also activates NK and CD8+ T cells. NKT of APCs and prime -T cells, and mediate cyto-
cell activities are also important for granulocyte toxicity against tumor cells [40, 41].
macrophage colony-stimulating factor (GM-CSF)
and IL-12-based cytokine strategies [29, 30].
Recent reports have identied subpopulations of 3.2.4 Macrophages
NKT cells which secrete TH1 or TH2 cytokines
and thus play different roles in the pathogenesis Macrophages serve as a rst line of defense
of many diseases. For example, CD4 NKT cells against tumorigenesis by directly killing tumor
serve as potent effectors for triggering tumor cells and producing various antitumor mediators
rejection in various murine tumor models, while [42]. On the other hand, macrophages render
CD4+ NKT cells contribute to the pathogenesis of tumor cells with the ability to acquire invasive
allergic diseases and tumors by promoting the and metastatic activities [43]. Macrophages are
release of IL-4, IL-5, and IL-13 [31, 32]. Indeed, differentiated from immature myeloid precursors
IL-13 released from NKT cells antagonizes or circulating monocytes released from the bone
tumor immunosurveillance by promoting TGF- marrow [44]. In particular, the inammatory
secretion from Gr-1+ myeloid suppressor cells monocytes expressing Ly6C are preferentially
[33, 34]. Thus, the identication of factors inu- attracted from the circulation into the tumor site
encing the differentiation of specic NKT cell by tumor-derived chemokines, such as CCL2
subsets during tumor development is important in (MCP1-1) and CCL5 (RANTES) and CXCL12
order to optimize the therapeutic interventions (SDF1) [4547]. Immature monocytes are then
which utilize NKT cell functions against tumors. differentiated into either M1 or M2 macrophages
32 M. Jinushi and M. Baghdadi

by distinct sets of cytokines when entered into immune response cascades against tumors [60].
distinct tumor microenvironments [48]. M1 mac- Granulocytes induce tumor destruction through
rophages may induce antitumor response by pro- the release of cathepsin G, azurocidin, reactive
ducing IFN- and IL-12 and triggering cytotoxic oxygen species, and inammatory cytokines.
activities [49, 50]. In contrast, tumor microen- Moreover, granulocytes, along with macrophages
vironments adopt multiple strategies to tip a and T cells, are main effectors that elicit antitu-
balance in the favor of differentiating M2-type mor responses by DNA vaccines in murine tumor
macrophages through complex network of cyto- models [61]. In addition, dense inltration of gran-
kines, chemokines, and growth factors [43, 51]. ulocytes in tumor tissues is associated with clini-
Taken together, macrophages have a dual role cal responses of GM-CSF-secreting cancer cells
in modulating tumorigenesis and antitumor host and Bacillus Calmette-Gurin (BCG) in patients
responses. Thus, detailed characterization of with advanced melanoma and bladder carcinoma,
molecular machineries which govern macro- respectively [62, 63]. On the other hand, granu-
phage polarization in tumors seems necessary for locytes contribute to tumor angiogenesis and
a thorough understanding of pharmacological metastasis by promoting secretion of proteinases,
targeting of macrophages and their derivatives. ROS, and cytokines that may acts as antitumor
effectors in different conditions [64]. Therefore,
granulocytes have both pro- and antitumor activi-
3.2.5 Dendritic Cells ties depending on distinct environments.

DCs are professional APCs contributing to the

induction of both innate and adaptive immune 3.3 The Role of Innate Immune
responses against pathogens as well as tumors. Receptors on Innate Immune
DCs express Toll-like receptors (TLRs) and co- Cells in Cancer
stimulatory molecules necessary for the activation and Antitumor Immunity
of various effectors [52]. Due to the potent immu-
nogenicity of DC, tumor microenvironments adopt 3.3.1 Toll-Like Receptors (TLRs)
multiple tactics to subvert DC functions. In addi-
tion, tumor-inltrating DCs can both induce tumor Toll-like receptors (TLRs) are innate immune recep-
growth and metastasis by regulating angiogenesis, tors mainly expressed on APCs, such as macro-
host immunity, and tumor metastasis [5356]. phages and dendritic cells. They play an important
Moreover, indoleamine 2, 3-dioxygenase (IDO)- role in host defense against pathogens by recogniz-
producing DCs cause poor tumor immunogenicity ing pathogen-associated molecular patterns
via generating Foxp3-positive regulatory T cells (PAMPs) and damage-associated molecular pattern
[57] and interacting with other innate lymphocytes molecules (DAMPs). The recognition of PAMPs
such as -T cells [58] and NKT cells [59]. and DAMPs by PRRs activates inammatory path-
In summary, tumor-inltrating DCs represent a ways such as NF-B and IRF-mediated signals,
double-edged sword which can induce an immune leading to antitumor mediators like type I interfer-
response against tumors or tolerize the immune ons, as well as cell survival and proliferation [65].
system against tumors and contribute to tumor Various sets of TLR ligands induce the upregula-
growth and metastasis. Thus, a deep understand- tion of co-stimulatory molecules and proinamma-
ing about DC biology at tumor microenvironment tory cytokine production by APCs, thus breaking
is critical to optimize anticancer therapies and the tolerogenic status to various tumor antigens
improve the clinical output of DC vaccines. and inducing antigen-specic antitumor immune
responses [6668]. In addition, TLR4 on DCs could
interact with high mobility group box 1 (HMGB1)
3.2.6 Granulocytes and facilitate antigen cross-presentation to anti-
tumor T lymphocytes [69]. Thus, TLRs agonists
Granulocytes, the key mediators of inamma- serve as effective adjuvants in harnessing potent
tion, have a potential role in the initiation of antitumor immune response and clinical responses.
3 Role of Innate Immunity in Cancers and Antitumor Response 33

In contrast, tumor cells license TLRs on NLR-mediated innate immune systems play an
myeloid cells to acquire invasive and metastatic important role in both antitumor immunity and
activities by promoting the secretion of vari- tumorigenicity. For example, nucleotide-binding
ous protumorigenic mediators, such as TNF- oligomerization domain-containing protein 1
and S100A8 [70, 71]. Thus, the careful opti- (NOD1) has a protective role against tumors, and
mization of suitable TLRs ligands for cancer the knockdown of NOD1 promotes tumor growth
immunotherapy is critical in order to avoid pro- in breast cancer model in vivo [78, 79]. NOD-
tumorigenic inammation caused by the TLRs like receptor family pyrin domain containing 3
expressed on innate immune cells in tumor (NLRP3) serves as a sensor for activating the
microenvironments. inammasome pathway which regulates pro-cas-
pase-1 cleavage and subsequent IL-1 activation
[80]. NLRP3 is a negative regulator of chemical
3.3.2 RIG-I-Like Helicases (RLHs) colon carcinogenesis. In a dextran sulfate sodium
(DSS) and azoxymethane-induced colon cancer
RIG-I-like helicases (RLHs) are specic fami- model, NLRP3/ mice showed increased colitis
lies of pattern-recognition receptors bear- and colitis-associated cancer, which was corre-
ing caspase-recruitment domain (CARD) at lated with attenuated levels of IL-1 and IL-18
N-terminus and helicase domains, which are at the tumor site [81]. However, in other models,
responsible for detecting intracellular dou- NLRP3 may also have a role in the promotion
ble-strand RNA and inducing innate immune of tumors as in inammation-induced skin can-
responses. RLHs include retinoic acid-inducible cers through the enhancement of inammatory
gene-I (RIG-I), myeloid differentiation anti- environment [82], which suggest a dual role for
gen-5 (MDA5), and laboratory of genetics and NLLRP3 in the regulation of host immunity for
physiology-2 (LGP2 or DHX58), which are pro- or antitumor responses. ATP released by
expressed constitutively in both immune and dying tumor cells serves as a nd-me signal
nonimmune cells. RLHs recruit specic intra- and recruits phagocytes to facilitate the engulf-
cellular adaptors to initiate NF-B- and IRF- ment of apoptotic cells [83]. Thus, ATP serves as
mediated inammatory signaling pathways that an agonist for NLRP3 whose activation triggers
lead to the synthesis of type I interferons (IFNs) IL-1 production and cross-priming of antitumor
and other proinammatory cytokines [72, 73]. CD8+ T cells [84].
The utilization of RLHs ligands as adjuvants to
trigger antitumor immune responses has been
validated by several studies. Its administration 3.3.4 Phagocytosis Receptors
with retinoic acid-inducible gene-I (RIG-I)
ligand triphosphate RNA triggers antitumor Phagocytes are specialized eating cells respon-
immune response by inducing the production sible for removing apoptotic cells in the body
of IFN-/IFN- and various immunogenic cyto- through a function of ligand-receptor interac-
kines, as well as activating antitumor immune tion. Dying tumor cells attacked by immune
response cells [74, 75]. cells or targeted by cytotoxic chemothera-
Taken together, RLHs ligands may be utilized peutic reagents are subject to recognition and
as adjuvants with other immunotherapies in order removal by phagocytic myeloid cells [85, 86].
to overcome immunosuppressive tumor microen- Molecules responsible for delivering eat
vironments. me signals, including milk-fat globule-EGF
factor 8 (MFG-E8), growth arrest-specific
6 (Gas-6), T-cell immunoglobulin-mucin
3.3.3 NOD-Like Receptors (NLRs) domain protein-4 (TIM-4), and calreticulin
(CRT), recognize the phosphatidylserine (PS)
NOD-like receptors (NLRs) are especially on apoptotic cells by integrin v3 on phago-
important for the recognition of sterile inam- cytes [8790]. On the other hand, the do not
mation such as uric acids and silica [76, 77]. eat me signal serves as negative regulators
34 M. Jinushi and M. Baghdadi

for phagocytes. One example includes the (intercellular adhesion molecule 3) facilitates
interaction between CD47 and signal-regu- the cross talk between DCs and T lymphocytes,
latory protein- (SIRP-), which provides hence inuences immunogenic responses against
inhibitory signals that block phagocytosis [91] pathogens and tumors [94]. DEC-205 is highly
(Fig. 3.1a). expressed on DCs and promotes cross-presenta-
Manipulation of phagocytic systems has tion of tumor antigens to cytotoxic T lymphocytes
emerged as one of the tumor immune evasion [95]. Indeed, agonistic antibody targeting DEC-
machineries, and pharmacological targeting of 205 elicits potent antitumor immunity and durable
these pathways provides a feasible option to aug- tumor regression in various murine tumor models
ment host immune responses and eradicate [96]. In addition, C-type lectin domain family 9A
tumors. For example, blocking CD47 with a (CLEC9A) utilizes necrotic cells for uptake, anti-
monoclonal antibody triggers tumor destruction gen presentation, and immune response, hence
by inducing phagocytosis of malignant cells [90, raising the possibility that CLEC9A-mediated rec-
92], and the treatment with anti-MFG-E8 anti- ognition of immunogenic antigens may enhance
bodies elicits potent antitumor responses in com- antitumor immunity and clinical responses [97]
bination with conventional anticancer drugs [93]. (Fig. 3.1a). Therefore, CLRs serve as promising
candidates for improving therapeutic responses
to cancer immunotherapy. Moreover, deep under-
3.3.5 C-Type Lectin-Like Receptors standing of the mechanism through which CLRs
(CLRs) regulate innate immune response will lead to
improvement in cancer vaccines.
Carbohydrate-binding C-type lectin and lectin-
like receptors (CLRs) are a large family of
molecules expressed in innate immune cells 3.3.6 NK Cell Receptors
and play an important role in the regulation of
antitumor immunity. For example, the interac- NK cells possess various sets of pattern-
tion between DC-SIGN (dendritic cell-specic recognition receptors which activate or sup-
ICAM-3 grabbing non-integrin) and ICAM-3 press immune responses upon encountering

Fig. 3.1 Role of innate immune receptors in the regula- T-cell interactions. Phagocytosis receptors expressed on
tion of antitumor immunity. (a) The functions of the APCs interact with ligands on apoptotic cells and mediate
innate immune system are regulated by various receptors its removal by APCs. In some cases, ligand-phagocytosis
expressed in immune cells. C-type lectin-like receptors receptor interactions (such as CD47-SIRP-) provide an
(CLRs) regulate recognition and uptake of antigens (such inhibitory signal which blocks phagocytosis, a system uti-
as DEC-205), the interactions between immune cells lized by tumors to evade immune machineries. (b) The
(such as the interaction between DC-SIGN on APCs and balance between activating and inhibiting signals is criti-
ICAM-3 on T cells), and the recognition of dead cells, cal for NK cell activities. Upon interaction with corre-
such as CLEC-9A which recognizes necrotic cells and sponsive ligands, activating and inhibitory receptors
enhances cross-presentation of antigens derived from deliver a signal which is mediated by ITAM and ITIM in
necrotic cells to CD8+ T cells. Members of B7 family their cytoplasmic domain. Phosphorylated ITAM motifs
regulate the functions of APCs, such as B7-H1 and in activating receptors recruit adaptor proteins which acti-
B7-H4, which have immune suppressive effects, while vate downstream signaling pathways, while phosphory-
other members regulate the interaction with immune cells, lated ITIM motifs in inhibitor receptors recruit proteins
such as B7-H3, which interacts with NK cells and sup- such as SHP-1 which dephosphorylates downstream sig-
press its functions, and B7-1/B7-2 which regulates APCs- nal molecules and inhibit NK activities
3 Role of Innate Immunity in Cancers and Antitumor Response 35

36 M. Jinushi and M. Baghdadi

their target cells. The balance between activation cytotoxicity receptor (NCR) family consists of
and inhibition signals is carefully mediated by three activating receptors: NKp30, NKp44, and
signals triggered by both activation and inhibi- NKp46, which are able to induce a strong cyto-
tion receptors in combination with cytokines. toxic reaction by NK cells. Expression levels
Signals delivered from NK receptors mainly of NCRs are correlated with cytotoxic ability
mediate through immunoreceptor tyrosine- of NK cells. MHC class I molecules counteract
based activation motif (ITAM) and immunore- with NCR-mediated activation signals; in addi-
ceptor tyrosine-based inhibition motif (ITIM). tion, the loss of MHC-I molecules, frequently
ITAM and ITIM bear conserved sequences of observed in transformed cells, activates NCRs
four amino acids repeated twice in the cytoplas- on NK cells [105107].
mic tails of NK cell receptors. Phosphorylation Killer cell immunoglobulin-like receptors
of tyrosine within ITAM motifs recruits adaptor (KIRs) are a family of cell surface molecules
proteins such as DNAX-activating protein-12 expressed on NK cells. KIRs have many mem-
(DAP12) and DNAX-activating protein-10 bers divided into two groups depending on the
(DAP10) involved in activating downstream sig- number of extracellular Ig domains (2D or 3D) or
naling pathways. On the other hand, phosphory- the length of their cytoplasmic tail, long vs. short
lation of tyrosine within ITIM motifs recruits (L or S). L-forms are shown to have inhibitory
proteins such as SHP which dephosphorylates functions, while S-forms enhance cytotoxic
downstream signal molecules to inhibit NK activities of NK cells in DAP12-mediated signal
stimulation [98] (Fig. 3.1b). pathways. KIRs regulate NK cells killing func-
Tumor cells evolve multiple strategies to tion through the interaction with MHC class I
evade NK cells by modulating ligand expression, molecules [100, 108].
ligand shedding, and upregulation of MHC mol- The interaction between inhibitory KIRs
ecules, in addition to the production of immu- and normal MHC-I molecules inhibits NK cell
nosuppressive cytokines. Thus, it is important stimulation. Correspondingly, NK cell stimu-
to understand the underlying mechanism of NK lation can occur due to an interaction between
cell activation and inhibition by their receptors, activating KIRs and polymorphic self-MHC
which eventually regulate immunosurveillance. class I molecules. Inhibitory KIRs were shown
NKG2D is a homodimeric C-type lectin- to be involved in the escape mechanism of acute
activating receptor expressed on NK, NKT, and myeloid leukemia (AML) from NK cell immune
activated CD8+ T cells [16, 99]. Ligands for surveillance, mechanism of which includes a
NKG2D include stress-induced proteins, such mismatch between donor KIRs and recipient
as MHC class I chain-related A and B (MICA human leukocyte antigen ligands [109]. Thus,
and MICB) as well as unique long 16 binding the understanding of KIR-mediated recognition
proteins (ULBPs) in human [99] and RAE1, of missing self is important in the treatment of
H60, and Mult1 in mice. NKG2D ligands are AML [110].
upregulated in stress conditions, such as viral Ly49 family is a large group of receptors
infection and transformation [99102]. Several expressed in mice but not in humans [111].
signaling pathways are involved in the induction Functionally Ly49 is similar to human KIRs,
of NKG2D ligands, including HSP70-mediated containing both activating and inhibitory recep-
cellular stress [101] and ATM/ATR-mediated tors. Inhibitory Ly94 receptors possess ITIM
DNA damage pathways [103]. Importantly, motifs which recruit SHP-1 to trigger an inhibi-
blocking of NKG2D pathways increases the tory signal, while activation receptors interact
susceptibility of mice to chemically induced car- with DAP12 to activate lytic machinery in NK
cinogenesis [104], indicating the importance of cells [112]. Ly49H is an activating NK receptor
NKG2D in tumor immunosurveillance. Natural which recognizes m157 glycoprotein encoded by
3 Role of Innate Immunity in Cancers and Antitumor Response 37

mouse cytomegalovirus (MCMV). Upon interac- tory signal which suppress cytotoxic activities of
tion with m157, Ly49H associates with DAP12 NK cell. In addition, blocking of B7-H3 could
and DAP10 to stimulate NK cell-mediated cyto- restore the antitumor effects of NK cells [116].
toxic activities against infected cells [113], sug- Finally, B7-H4 promotes protumorigenic and
gesting a role for Ly49H in the protection against immunosuppressive phenotypes of macrophages;
viral infection-associated tumors [114]. for example, the blockade of B7-H4 normalized
DNAM-1 (CD226) is an adhesion molecule immunogenicity of macrophages and augmented
expressed on the surface of NK cells, monocytes, antitumor immunity in ovarian tumor tissues
and a subset of T cells. DNAM-1 belongs to the [123] (Fig. 3.1a).
immunoglobulin superfamily containing 2 Ig-like
domains of the V-set. DNAM-1 is reported to
bind to two ligands: CD112 and CD155 [115].
CD112 and CD155 are highly expressed in some 3.4 The Role of Effectors
tumors like melanoma and neuroblastoma. Produced from Innate
Importantly, neuroblastoma cells that do not Immune Cells in Cancer
express CD112 and CD155 are resistant to NK and Antitumor Immunity
cells, indicating that NK lysis of this neuroblas-
toma cells requires DNAM-1 interaction with its 3.4.1 Interferons (IFNs)
ligands on tumor cells [116].
Type I IFNs are produced by many different cells
in response to viral or bacterial infections. Type I
3.3.7 B7 Family IFNs (IFN-/IFN-) enhance proliferation and
activation of innate immune cells such as DCs,
B7 family consists of co-stimulatory and co- macrophages, and NK cells [124]. In addition,
inhibitory receptors found on activated APC and they stimulate antigen processing and presenta-
T cells, which regulate the interaction between tion to antigen-specic lymphocytes, which
APCs and T cells. B7-1 and B7-2 are expressed greatly contribute to tumor immunosurveillance
on APCs and are involved in the stimulation of [125]. The importance of type I IFNs in tumor
T-cell response. B7-1 and B7-2 on APCs serve immunosurveillance also validated enhanced
as co-stimulatory molecules and play a critical susceptibility to tumorigenesis by treatment with
role in regulating antitumor immune responses anti-IFN-/IFN- neutralizing antibodies or in
through reciprocal interaction of their receptor mice with targeted mutations of type I IFN recep-
CD28 and cytotoxic T-lymphocyte antigen-4 tor [126, 127].
(CTLA-4) on T lymphocytes [117, 118]. B7-H1 Type II IFN (IFN-) is a cytokine involved in
(PD-L1) expression in DCs is induced by IL-10 the activation of adaptive immune cells. IFN- is
and VEGF at ovarian tumors [119]. B7-H1 on primarily produced by various innate immune
DCs suppresses IL-12 and promotes IL-10 secre- lymphocytes such as NK, NKT, and -T cells
tion, creating an immunosuppressive tumor and plays a critical role in the induction of Th1
environment. Moreover, the blockade of B7-H1 immune responses and the production of NO and
enhances antitumor immunity by DC-mediated ROS by macrophages, leading to enhanced cyto-
T-cell activation [119, 120]. In addition, treat- toxic activities against transformed cells [128].
ment with PD-1 neutralizing antibodies has been IFN- has an important role in the protection
found to decrease tumor growth and metastasis against transplanted tumors or chemically
in B16 melanoma and colon cancer models [121, induced tumors by increasing intrinsic immuno-
122]. B7-H3 on APCs bind to an unidentied genicity of tumor cells [129, 130]. IFNGR/
receptor on NK cells and transduce an inhibi- mice or mice decient in IFN--downstream
38 M. Jinushi and M. Baghdadi

signaling molecule Stat-1 developed tumors immunogenic cytokines, such as IFN-, IL-2,
more rapidly and in greater frequencies com- TNF-, and GM-CSF, and also suppresses anti-
pared to wild-type mice [131, 132]. Thus, IFN-- gen presentation by APCs. On the other hand,
mediated regulation of tumor immunogenicity the carcinogen-mediated tumor incidence was
has a great impact on innate immunity and tumor increased in IL-10-knockout mice, whereas
immunosurveillance. IL-10 overexpression protects mice from aris-
ing tumors [138]. Thus, IL-10 has a complex
role in tumorigenesis, and the pro- and antitu-
3.4.2 Other Cytokines mor effects of IL-10 may depend on the differ-
ent experimental models. TGF- is a regulatory
Interleukins have an important role in regulat- cytokine which has important roles in the regu-
ing innate immune functions in tumor micro- lation of immune responses and immune toler-
environments. Several cytokines, such as IL-2, ance as well as carcinogenesis [139, 140]. TGF-
IL-12, IL-18, IL-15, and IL-21, serve as NK can inhibit the activities of NK cells through the
cell-stimulants, competent in targeting trans- suppression of IFN- production [141], as well
formed cells. Mice decient for IL-12p40 are as the downregulation of activating receptors
susceptible to carcinogen-induced tumorigen- such as NKp30 and NKG2D [142]. On the other
esis; in addition, IL-21/ mice showed reduced hand, TGF- negatively regulates recruitment
colitis-associated cancers [133], indicating the and differentiation of myeloid-derived suppres-
role of these cytokine in protecting hosts from sor cells (MDSCs) in tumor tissues derived from
arising tumors. With respect to the mechanisms mammary carcinomas, contributing to enhanced
of action, NKG2D systems are involved in the host immunity and tumor rejection [143].
enhancement of NK cell cytotoxic activities by Therefore, TGF- has different roles in antitu-
all cytokines suggested above, and perforin-gran- mor immunity and tumorigenicity, which are in
zyme pathways play an important role in exert- part dependent on the phase of tumor progres-
ing NK cell cytolysis by IL-18. Moreover, IL-21 sion and different cellular components in tumor
induces NK cell effector functions by increasing microenvironments [144]. Vascular endothelial
sensitivities to IFN-, and IL-15 regulates sur- growth factor-A (VEGF-A) also plays a critical
vival, activation, and proliferation of NK cells role in suppressing DC maturation and differ-
[134]. Cytokines produced from innate immune entiation, therefore impacting tumor immuno-
cells serve as feasible adjuvants in activating genicity and host immunosurveillance [145].
antitumor responses in patients with advanced Thus, various cytokines are responsible for
cancer. For example, the systemic administra- attenuating immunogenic potentials of innate
tion of high doses of recombinant IL-2 or the immune systems in tumors.
adaptive transfer of IL-2-stimulated NK cell can Several cytokines derived from innate lym-
trigger potent antitumor responses and medi- phocytes contribute to smoldering inammation
ate durable tumor regressions in patients with and tumor progression. IL-23-IL-17 pathway
advanced melanoma and renal cell carcinoma operated in endogenous tumor microenviron-
[135]. The clinical efcacy of IL-12 has been ments represents prototypical mediators which
evaluated as a monotherapy or in combination promote tumor-associate inammation. IL-23
with other immunotherapies in patients with can- promotes tumor cell growth and invasion through
cer; however, they did not induce durable clinical upregulation of proteins of the matrix metallo-
responses [136, 137]. proteinase-9 (MMP9), COX-2, and angiogen-
Several cytokines antagonize immuno- esis. In contrast, IL-23/ mice showed reduced
genic potential of tumors and innate lympho- inammation and thus attenuated tumor forma-
cytes. IL-10 downregulates the expression of tion [146]. IL-17 is elevated in various tumors,
3 Role of Innate Immunity in Cancers and Antitumor Response 39

where it plays an important role in tumor growth. cells which express appropriate chemokine
IL-17 can enhance tumor growth by direct effects receptors. Two types of chemokines have been
on tumor cells and tumor-associated stromal identied: CC chemokines that are chemotactic
cells by activating IL-6-Stat3 pathways [147]. for monocytes and CXC chemokines which
Furthermore, the altered composition of com- attract polymorphonuclear leukocytes (PMNs).
mensal microbes and disruption of epithelial Chemokines have a central role in tumor progres-
barrier functions facilitate differentiation of sion through the recruitment of innate immune
IL-17-producing T lymphocytes by IL-23 from cells into tumor site. Most studies have focused
myeloid cells in intestine, leading to increased on CCL2 and CCL5 as the major chemokines in
colon tumorigenesis [148, 149]. tumor microenvironment.
Granulocyte-macrophage colony-stimulating CCL2 (MCP-1) is produced by tumor cells
factor (GM-CSF) is produced in vivo by many and tumor-associated stromal cells and attracts
cells including mast cells, macrophages, T cells, CCR2+ inammatory monocytes to the tumor
broblasts, and endothelial cells in response to microenvironment, which differentiate into
immune activation and proinammatory cyto- tumor-associated macrophages and promote
kines. GM-CSF creates an immunosuppressive tumor aggressiveness, and the blockade of CCL2-
tumor microenvironment by differentiating CCR2 signaling by neutralizing antibodies sup-
immature myeloid-derived suppressor cells presses metastasis and prolongs overall survival
(MDSCs) into tumor tissues [150]. On the other of tumor-bearing mice [156]. The levels of CCL2
hand, the therapeutic administration of GM-CSF expression and macrophage inltration into
has been emerged as a potent immunogenic adju- tumors are correlated with poor prognosis and
vant to stimulate antitumor immunity by enhanc- metastases in human breast cancer, suggesting
ing APC functions [151]. signicance of CCL2-mediated immune regula-
Macrophage colony-stimulating factor M-CSF tion in cancer patients [157].
(also known as CSF-1) is a dimeric polypeptide CCL5, another important chemokine, plays an
growth factor which regulates the proliferation, important role in the recruitment of monocytes
differentiation, and survival of macrophages and into the tumor microenvironment [158]. CCL5
their bone marrow progenitors. CSF-1 expression induces expression of CCL2, CCL3 (MIP-),
is elevated in different tumors and is found to be CCL4 (MIP-), and CXCL8 (IL-8) by mono-
accompanied by high grade and poor prognosis cytes, which leads to the recruitment of myeloid
[152]. Targeting of CSF-1 has been evaluated in cells into tumor site [159]. CCL5 also induces
preclinical and clinical studies [153]. The admin- CCR1 expression on monocytes [160]. Hence,
istration of anti-CSF1R-neutralizing antibody chemokines lead to the recruitment of mono-
(AFS98) or a CSF-1R inhibitor (Ki20227) cytes, which produce more chemokines to further
resulted in reduced numbers of tumor-inltrated attract more monocytes as well as other leuko-
macrophages in an implanted osteosarcoma cytes into the tumor site. CCL5 enhances antitu-
model and reduced vascularization, angiogene- mor immune responses against tumors [161],
sis, and tumor growth [154, 155]. while it promotes tumorigenesis and metastases
in some conditions [162, 163]. These ndings
suggest dual function of CCL5 in cancer and
antitumor immunity.
3.4.3 Chemokines Taken together, the dynamic interactions
between tumor cells and innate immune cells
Chemokines are small cytokines secreted by governed by chemokine networks play a pivotal
many cell types in response to pathological con- role in the regulation of tumor immunosurveil-
ditions, in order to activate and attract effector lance and tumorigenicity (Fig. 3.2).
40 M. Jinushi and M. Baghdadi

Fig. 3.2 Role of the innate immune system in cancer and metastasis. For example, IDO+ DC induces differentiation of
antitumor immunity. Innate immune system serves as the FoxP3+Treg cells which suppress antitumor immunity, and
rst defense line against cancers. Innate immune cells such molecules released by PMNs may have protumorigenic or
as DC, NK, NKT, -T cells are attracted into the tumor site, antitumor effects. Furthermore, tumors secrete chemokines
where they recognize the transformed cells and release mul- and cytokines that attract inammatory monocytes into the
tiple factors which initiate an antitumor immune response. tumor microenvironment and induce its differentiation into
On the other hand, other innate immune cells may also M2 macrophages, which play important roles in tumor pro-
involve in the promotion of tumor growth, angiogenesis, and gression, metastases, angiogenesis, and chemoresistance

3.5 Concluding Remarks Acknowledgments We apologize to the authors whose

work could not be cited due to space constraints.
This study is partially supported by a Grant-in-Aid for
Innate immune system serves as the rst line of Scientic Research and Scientic Research for Innovative
defense against pathogens and cancers. In tumors, Areas from the Ministry of Education, Culture, Sports,
innate immune cells are attracted into the tumor Science and Technology (MEXT) and the Ministry of
Health, Labour and Welfare, The Naito Foundation, and
site. Factors released from stressed cells at the the Astellas Foundation for Research on Metabolic
tumor microenvironment, such as PAMPs and Disorders (M.J.).
DAMPs, are recognized by another set of recep-
tors, including TLRs, RLRs, and NLRs, which
trigger distinct innate signaling pathways; these References
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B Cells in Cancer Immunology:
For or Against Cancer Growth? 4
Qiao Li, Qin Pan, Huimin Tao, Xiao-Lian Zhang,
Shiang Huang, and Alfred E. Chang

Contents H. Tao, MSc

Department of Surgery, University of Michigan,
4.1 Introduction ................................................... 48 3302 Cancer Center, 1500 E,
4.2 CD40-Activated B (CD40-B) Cells............... 48 Medical Center Drive, Ann Arbor,
MI 48109, USA
4.3 Tumor Killer B Cells ..................................... 50
Department of Hematology,
4.4 Tumor-Inltrating B Cells Wuhan Union Hospital, Wuhan, Hubei, China
(TIL-Bs) in Cancer ........................................ 52 e-mail:
4.5 Resting B Cells and Regulatory X.-L. Zhang, PhD
B Cells in Cancer ........................................... 53 State Key Laboratory of Virology,
Department of Immunology,
4.6 Concluding Remarks .................................... 55 Hubei Province Key Laboratory of Allergy
References ............................................................... 57 and Immunology, Wuhan University School
of Medicine, Donghu Road 185#,
430071 Wuhan, Hubei, China
Department of Immunology,
Wuhan University School of Medicine,
Donghu Road 185#, Wuhan,
Hubei 430071, China
Q. Li, PhD (*)
Department of Surgery, University of Michigan,
3302 Cancer Center, 1500 E, Medical Center Drive, S. Huang, MD
Ann Arbor, MI 48109, USA Hubei Province Stem Cell Research & Appling
e-mail: Center, Wuhan Union Hospital, Wuhan, China
Q. Pan, PhD Department of Hematology,
Department of Surgery, University of Michigan, Wuhan Union Hospital, Wuhan, Hubei, China
3302 Cancer Center, 1500 E, Medical Center Drive, e-mail:
Ann Arbor, MI 48109, USA
A.E. Chang, MD
State Key Laboratory of Virology, Department of Surgery, University of Michigan,
Department of Immunology, Hubei Province Key 3302 Cancer Center, 1500 E,
Laboratory of Allergy and Immunology, Medical Center Drive, Ann Arbor,
Wuhan University School of Medicine, MI 48109, USA
Donghu Road 185#, 430071 Wuhan, Hubei, China
Division of Surgical Oncology,
Department of Immunology, Department of Surgery, University of Michigan
Wuhan University School of Medicine, Comprehensive Cancer Center,
Donghu Road 185#, Wuhan, Hubei 430071, China 3302 Cancer Center, Ann Arbor, MI, USA
e-mail: e-mail:

N. Rezaei (ed.), Cancer Immunology: A Translational Medicine Context, 47

DOI 10.1007/978-3-662-44006-3_4, Springer-Verlag Berlin Heidelberg 2015
48 Q. Li et al.

4.1 Introduction tolerance regarding the induction of nave T

cells. Recent studies have shown that activa-
In the 1960s, B cells were rst dened in birds tion of mouse and human B cells using CD40L
when researchers found that removal of the bursa in vitro upregulates the expression of major his-
in newly hatched chicks severely impaired the tocompatibility complex (MHC) I, MHC II, and
ability of the adult birds to produce Abs [1, 2]. costimulatory molecules on B cells [69, 13, 14,
A decade later, it was found that mammalian B 16]. These B cells present exogenous antigens by
cells are derived from bone marrow and develop MHC class I or II molecules and stimulate anti-
into plasma cells that are the source of antibodies gen-specic T cells [7, 8]. CD40-B cells induce
(Abs). Over the years, most studies on B cell T cell proliferation, interferon- (IFN-) produc-
function in immune response have focused on tion, and specic cytotoxic T lymphocyte (CTL)
antigen presentation and antibody production. responses [69, 1115]. In mouse models, it has
However, recent advances in B cell biology have been shown that CD40-B cells directly present
capitalized on old ndings and demonstrated that antigen to nave CD8+ T cells, in order to induce
B cells can also act as effector cells or as regula- the generation of potent T effectors which are
tory cells [3, 4]. able to secrete cytokines and kill target cells [16].
B cells are often overlooked in tumor immu- Moreover, CD40-B cells express the full lymph
nology, likely because of the common notion that node homing triad CD62L, CCR7/CXCR4, and
humoral and cytolytic responses work in opposi- leukocyte function antigen-1 (LFA1), suggesting
tion. The eld of tumor immunology has focused that they could co-localize with T cells in the T
on CD8+ T cells due to their ability to directly kill cell-rich areas of secondary lymphoid organs [11,
tumor cells, as well as the close association 15]. This will facilitate CD40-B cell and T cell
between tumor-inltrating CD8+ T cells and can- contact for antigen presentation.
cer patients survival [5]. To date, the role of B Using a metastatic mouse model, Li et al. pro-
cells in tumor immunity has remained largely vided direct experimental evidence that the aug-
elusive. Results from different research groups mented antitumor activity by anti-CD40
are somewhat controversial. In this chapter, we monoclonal antibody (mAb)-stimulated tumor-
review the roles of B cells in tumor immunology, draining lymph node (TDLN) cells requires the
which may either positively or negatively affect presence of APCs, e.g., B cells as well as DCs.
tumor growth and patient outcomes. They found that anti-CD40 mAb augments anti-
tumor responses of TDLN cells via ligation to
CD40 on both B cells and DCs [17].
4.2 CD40-Activated B Typically, TDLN cells are composed of
(CD40-B) Cells approximately 60 % CD3+ T cells, 30 % CD40+ B
cells, and 5 % DCs. In a murine sarcoma model,
CD40-activated B (CD40-B) cells are thought to anti-CD3-/anti-CD40-activated MCA205 TDLN
be an excellent source of professional antigen- T cells secreted signicantly higher amount of
presenting cells (APCs) for antigen-specic tumor IFN- in an antigen-specic manner (in response
immunotherapy. They have demonstrated potent to MCA205 tumor, but not to MCA 207 tumor),
effects on cellular immunotherapy of cancers in comparison with solely anti-CD3-activated
[617]. CD40-B cells induce potent expansion of TDLN T cells (Fig. 4.1a). However, when B cells
antigen-specic CD4+ and CD8+ T cells, includ- were depleted from MCA205 TDLN cells, anti-
ing nave CD8+ T cells [69, 12, 16]. One reason CD3/anti-CD40 activation could not increase the
that dendritic cells (DCs) are considered as excel- IFN- anymore. This effect is very similar to DC
lent APCs in tumor immunotherapy is that they depletion (Fig. 4.1a). In vivo, adoptive transfer of
can powerfully prime nave T cells, while rest- anti-CD3-/anti-CD40-activated MCA205 TDLN
ing B cells cannot. Resting B cells poorly express T cells mediated signicantly higher MCA205
costimulatory molecules, resulting in immune tumor regression in a pulmonary metastasis
4 B Cells in Cancer Immunology: For or Against Cancer Growth? 49

Fig. 4.1 Anti-CD40 mAb a 3,000

augmented antitumor Anti-CD3: Unfrac.
responses of anti-CD3- Anti-CD3/anti-CD40: Unfrac.
activated TDLN cells via 2,500 * Anti-CD3/anti-CD40: CD19-
ligation to CD40 on both B Anti-CD3/anti-CD40: CD11c-
cells and DCs. (a) Activated Anti-CD3/anti-CD40: CD19-/CD11c-
total unfractionated (Unfrac)
TDLN cells were co-cultured

with MCA 205 vs. MCA207 1,500
tumor cells to determine
IFN- production. B cells
were removed by CD19 1,000
depletion (CD19), and DCs
were removed by CD11c 500
depletion (CD11c).
(b) Activated total TDLN
(Unfrac) cells or B cell, 0
T alone T cells + MCA205 T cells + MCA207
DC-depleted TDLN cells
(CD19 and/or CD11c) b 300
TDLN cells adoptively
>250 >250 >250
transferred into tumor- 250
bearing mice for therapy.
Pulmonary metastases

*p < 0.05 compare with

any other group in (a, b), 200
respectively (Adapted
by permission from 150
the American Association
of Immunologists,
Inc. Copyright 2005: 100 *
Li et al. [17])

No treatment IL-2only Unfrac. Unfrac. CD19- CD11c-CD19-/CD11c-
Anti-CD3 Anti-CD3/anti-CD40

setting, compared to anti-CD3-alone-activated They found that tumor growth inhibition was
TDLN T cells (Fig. 4.1b). However, B cell signicantly diminished in the B cell-decient
removal signicantly reduced the therapeutic mice after T cell + IL-2 + IL-21 combined ther-
efcacy conferred by CD40 engagement, and so apy (Fig. 4.2).
did DC removal. Together, these studies indicate In contrast to DCs, large numbers of B cells
that B cells, as well as DCs, are required in the can be obtained from the blood of patients after
generation of potent antitumor T effector cells ex vivo expansion (up to 1,000-fold) in the
from TDLN cells via simultaneous targeting of presence of CD40L [6]. For example, only
CD3 on T cells and CD40 on B and dendritic about 106 DCs can be generated from 10 ml of
cells. blood, while 1091010 B cells can be produced
In a separate study, Iuchi et al. reported that from the same volume of the blood sample.
host B cells were required for adoptive trans- Additionally, CD40-B cells can be continu-
ferred T cells to mediate optimal antitumor ously expanded in long-term culture (>65 days)
immunity [18]. Tumor-bearing mice were treated without the loss of APC functionality [6].
with adoptive transfer of T cells accompanied Therefore, CD40-B cells have the advantage
with IL-2 and IL-21 administration in wild-type over DCs in terms of isolation, generation, and
and B cell knockout (B/) animals, respectively. long-term expansion. These characteristics
50 Q. Li et al.

Fig. 4.2 Requirement for

host B cells in T cell transfer 60
+ IL-2 and IL-21 administra- WT: No treatment
tion-elicited antitumor 50
WT: T cells+IL-2+IL-21
immunity (Adapted by
B/: No treatment

Area (mm2)
permission from the 40
American Association B/: T cells+IL-2+IL-21
for Cancer Research: 30
Iuchi et al. [18])


0 5 10 15 20 25
Days after tumor injection

p<0.0001 WT No treatment vs. WT T cells + IL-2 + IL-21

p<0.0001 B/ No treatment vs. B/ T cells + IL-2 + IL-21
p=0.0071 B/T cells + IL-2 + IL-21 vs. WT T cells + IL-2 + IL-21

make CD40-B cells a promising alternative as ligand (TRAIL), programmed death ligands 1
cell-based vaccines. and 2 (PD-L1 and PD-L2), and granzyme B
In current B cell vaccine preparations, (GrB), which are potentially involved in B cell-
activated B cells can be loaded with antigens by mediated direct cytotoxicity against tumor cells
pulsing with peptides, proteins, tumor lysates, or [2129].
by transfection with DNA or RNA, or transduc- Due to the well-known fact that B cells can
tion with viral vectors [9, 10, 19]. Coughlin et al. produce Abs which lead to CDC and ADCC, as
loaded RNA on CD40-B cells from pediatric well as the recent ndings that B cells may
patients. Vaccination using these B cells resulted kill tumor cells directly through antibody-
in simultaneous targeting of multiple antigenic independent mechanisms, it is hypothesized that
epitopes and induced CTLs [9]. Chung et al. appropriately sensitized and activated B cells can
reported that B cells stimulated with iNKT function as effector cells to mediate antitumor
(CD1d-restricted invariant T cells) ligand immunity. Indeed, Li et al. [30] proved that
alpha-galactosylceramide (alphaGalCer) could in vivo sensitized and in vitro activated B cells
directly prime CTLs and generate long-lasting could mediate tumor regression in cancer adop-
cytotoxic antitumor immunity in vivo [10]. tive immunotherapy. In vivo sensitized TDLN
Furthermore, Garbe et al. reported that semi- cells were activated and expanded in vitro with
allogeneic fusions of microsatellite instability LPS/anti-CD40, resulting in B cell proliferation
(MSI) tumor cells with B cells primed B cells to and differentiation. These activated B cells were
induce MSI-specic T cell responses [19]. then adoptively transferred into tumor-bearing
recipients for therapy. These tumor-primed and
tumor-activated B cells signicantly reduced
4.3 Tumor Killer B Cells lung metastases in an adoptive immunotherapy
model (Fig. 4.3). Furthermore, total body irradia-
B cells can directly kill tumor cells through anti- tion (TBI) could enhance the antitumor activity
body (Ab)-independent mechanisms [20]. Recent of the adoptively transferred B cells. This study
studies have shown that B cells express death- represents one of the early studies demonstrating
inducing ligands and can therefore mediate cell that effector B cells could confer antitumor
death under many circumstances. Evidence has immunity after adoptive transfer into tumor-
emerged that B cells express Fas ligand (FasL), bearing mice [30].
tumor necrosis factor-related apoptosis-inducing
4 B Cells in Cancer Immunology: For or Against Cancer Growth? 51

1,000 a 150 p < 0.05

No treatment
TBI p < 0.05
Tumor size (mm2)


Number of pulmonary
B cell transfer

metastatic nodules
TBI+ B cell transfer


0 5 10 15 20 25 30 0
T cell B cell IL-2 No
Days after tumor inoculation Treatment Treatment only treatment
*p < 0.03 vs. any other group.
p < 0.05
Fig. 4.3 TBI (total body irradiation) signicantly b
augmented the therapeutic efcacy of adoptively trans-
ferred B cells in the s.c. D5 tumor model (Adapted by

Number of pulmonary
metastatic nodules
permission from the American Association of 40
Immunologists, Inc. Copyright 2009: Li et al. [31])

Using a murine 4T1 pulmonary metastatic 20

model, it was found that adoptive transfer of

4T1-primed and LPS-/anti-CD40-activated
TDLN B cells signicantly inhibited 4T1 pulmo- 0
B cell T+B cell T+B cell No
nary metastasis in tumor-bearing mice [31] Treatment Treatment Treatment treatment
(Fig. 4.4). The efcacy mediated by B cells was (10 106) (10 106) (20 106)

comparable to that mediated by an equal number Fig. 4.4 (a) Adoptively transferred 4T1 TDLN B cells
of T cells, which served as a positive control in mediated effective inhibition of the spontaneous pulmo-
the experiment (Fig. 4.4a). Of note, adoptively nary metastasis of 4T1 breast cancer cells similarly to
transferred 4T1 TDLN T + B cells mediated inhi- equal numbers of T cells. (b) adoptively transferred 4T1
TDLN T + B cells mediated inhibition of the spontaneous
bition of the spontaneous pulmonary metastasis pulmonary metastasis of 4T1 better then B cells alone,
of 4T1 in a dose-dependent manner (Fig. 4.4b). and the efcacy was dose dependent (Adapted by permis-
This study also showed that activated 4T1 sion from the American Association for Cancer Research:
TDLN B cells caused tumor cell lysis directly Li et al. [31])
in vitro in the absence of Ab and other effector
cells and this direct cytotoxicity was tumor spe- In line with these ndings, Kemp et al. dem-
cic (Fig. 4.5). In these experiments, 4T1 mam- onstrated that CpG-A oligodeoxynucleotide
mary carcinoma murine tumor-primed TDLN B (CpG-A ODN) stimulation of human PBMCs
cells were activated with LPS and anti-CD40 leads to high levels of functional TRAIL/Apo-2L
mAb, washed thoroughly, and then co-cultured expression on B cells, and these B cells mediate
with 4T1 tumor cells or irrelevant tumor controls, TRAIL-/Apo-2L-dependent tumor cell lysis [25].
Renca (renal cell carcinoma) and TSA (sarcoma). Additional studies support the observation
The effector B cells killed 4T1 cells directly in a that B cell can function as effector cells in antitu-
dose-dependent way and were signicantly more mor responses. For example, Penafuerte et al.
effective than their killing of the control tumors. reported that B effector cells activated with a
These data support the conclusion that tumor chimeric protein consisting of IL-2 and the
antigen-primed and in vitro activated B cells are ectodomain of TGF- receptor II (also known as
able to kill tumor cells independent of Ab or FIST) induce potent antitumor immunity [32].
complement. However, the mechanism(s) by In this study, the B effector cells were
which the killer B cells lyse tumor cells directly characterized by the production of TNF and
in such a setting remains to be identied. IFN- and potent antigen presentation properties
52 Q. Li et al.

Expt 1 Expt 2
25 4T1 20 4T1
Renca **
20 *
Cytotoxicity (%)

Cytotoxicity (%)

0 0
1:1 3:1 10:1 25:1 3:1 25:1

*p < 0.01, 4T1 vs. Renca at the **p < 0.05, 4T1 vs. TSA at the
same E:T ratio same E:T ratio

Fig. 4.5 Activated 4T1 TDLN B cells mediate direct and tumor-specic cytotoxicity of 4T1 cells (Adapted by
permission from the American Association for Cancer Research: Li et al. [31])

[32]. In addition, Forte et al. found that patients [36, 37, 42, 44, 45], lower relapse rate
administration of a specic CD73 inhibitor, ade- [41], or low metastasis [42]. In a study on immune
nosine 5-(, -methylene) diphosphate (APCP), inltrates in high-grade serous ovarian cancer, it
to melanoma-bearing mice induced signicant was revealed that intraepithelial CD20+ TIL-Bs
tumor regression [33]. They observed that after are associated with increased disease-specic
APCP administration, the presence of B cells in survival [37]. Importantly, the association
the melanoma tissue was more than that observed between the immune inltrates and survival was
in control mice. This was associated with the pro- dependent on histological subtype, because
duction of IgG2b within the melanoma, implying immune inltrates were less prevalent in the
a critical role for B cells in the antitumor activity other histological subtypes compared to the high-
of APCP [33]. Together, these studies suggest grade serous cases [37]. In breast cancer, TIL-Bs
that the mechanisms underlining B cell-mediated are present in about 24 % of tumors and comprise
antitumor immunity may involve multiple cellu- up to 40 % of the lymphocytic inltrates [34].
lar and molecular events, as well as direct killing TIL-Bs have been shown to undergo antigen-
of the tumor cells. driven clonal proliferation and afnity matura-
tion in situ [35]. Very recently, in a large patient
cohort of different histological and biological
4.4 Tumor-Inltrating B Cells subtypes, Mahmoud and colleagues provided
(TIL-Bs) in Cancer evidence for a favorable outcome when high
numbers of CD20+ TIL-Bs were present [36].
Tumor-inltrating B cells (TIL-Bs) have Additionally, TIL-Bs may be involved in humoral
revealed controversial roles in antitumor immu- immune response in situ. Using recombinant Ab
nity. They have been found in breast cancer cloning techniques, Hansen et al. reported an
[3436], ovarian cancer [37], lung cancer [38], antigen-driven humoral immune response
colorectal cancer [39, 40], cervical cancer [41], directed against -actin exposed on apoptotic
cutaneous melanoma [42], and prostate cancer mammary carcinoma cells [46]. Yasuda and
(CaP) [43]. A few studies have indicated that coworkers identied TIL-Bs which produce
TIL-Bs are correlated with favorable survival of tumor-specic Abs against mutated p53 [47].
4 B Cells in Cancer Immunology: For or Against Cancer Growth? 53

Maletzki et al. also reported that TIL-Bs from enhanced T cell antitumor activity and signicant
colorectal carcinoma show an activated improvement in survival rate [4852]. It has been
immunophenotype (CD23+, CD80+) and produce reported that there are increased effector T cells
IgGs that specically bind to allogeneic target [48], increased T cell inltration of tumors [52],
tumor cells [40]. higher Th1 cytokine and antitumor CTL response
On the other hand, TIL-Bs may produce cyto- [49, 51, 52], and even reduced T regulatory cell
kines contributing to tumor development. It has (Treg) frequencies [53] in these B cell-decient
been reported that TIL-Bs in castration-resistant mice. Some studies explored the possible mecha-
CaP produce lymphotoxin by an inammation- nisms involved. B cells present in the priming
responsive IB kinase (IKK)--dependent path- phase result in disabled CD4+ T cell help for
way, which then in turn activates IKK- and CTL-mediated tumor immunity [51]. B cells
STAT3 in tumor cells to enhance hormone-free produce IL-10 which can repress antitumor
tumor survival [43]. In this study, B cell inltra- immunity [49, 54]. Similarly, Abs were shown to
tion was detected in 100 % of human CaP sam- promote primary tumor formation in a trans-
ples, while B cells were undetectable in normal genic mouse model of inammation-associated
prostate or benign prostatic hyperplasia [43]. carcinogenesis [55]. Autoantibody responses to
Castration-resistant CaP growth was delayed in self-proteins triggered by cancer vaccines may
mice reconstituted with bone marrow from JH/ inuence the efcacy of vaccination [56].
mice, which lack mature B cells [43]. It was fur- Additionally, B cells have been shown to have
ther found that these CaP allografts exhibited other pro-tumorigenic roles. For example,
IKK- nuclear translocation, which was depen- enhanced NK cell antitumor activity has been
dent on IKK- in B cells. IKK- deletion abol- reported in B cell-decient mice [48, 50, 52];
ished lymphotoxin expression by B cells. When however, the mechanisms are poorly understood.
lymphotoxin- was ablated in B cells, growth of We hypothesize that the effects of B cells on
castration-resistant CaP was delayed. Similarly, antitumor immunity depend on the presence of
another study showed that tumor-inltrating T B cell subsets mainly involved under certain
and B cells were not associated with long-term tumor conditions. In the past two decades,
survival of patients with non-small-cell lung can- investigators have identied B cell subsets
cer [38]. which are capable of suppressing the immune
The roles of TIL-Bs may be complicated, response. Suppression of an immune response
since the tumor environment is dynamic and was rst reported in 1974 where spleen B cells
changes during tumor onset and progression. were found to impair delayed-type hypersensi-
TIL-Bs need to contact other immune cells or tivity (DTH) responses in guinea pigs [57, 58].
tumor cells to be activated or regulated, so their This nding led to the conclusion that DTH
contributions to immune responses are likely to responses and T cell function can be regulated
vary in different cancers and during the course of by suppressor B cells. Subsequently, convincing
cancer. data have demonstrated that IL-10-producing B
cells, termed regulatory B cells (Bregs) by
Mizoguchi et al. [59], can suppress inamma-
4.5 Resting B Cells tory responses in experimental autoimmune
and Regulatory encephalomyelitis (EAE), collagen-induced
B Cells in Cancer arthritis (CIA), and colitis [5961]. Recently,
Bregs and their potential immunomodulatory
In contrast to activated B cells, there is abundant activities have been examined in several
evidence indicating that resting B cells can immune-related diseases. In the majority of
promote the development or progression of these studies, the function of Bregs is dependent
cancer. Resting B cells are small B cells in the G0 on IL-10 production, whereas the mechanisms
stage of cell cycle, prior to activation. Studies are still undened partly because of conicting
have shown that B cell-decient mice exhibit results regarding the phenotypic characterization
54 Q. Li et al.

of IL-10-producing cells. For example, the alongside an increase in IFN--producing CD8+

following B cells have been reported as putative T cells in the spleen [54]. In this study, Tnf/
mouse Bregs: CD1dhigh subset of B cells in mice were resistant to chemical carcinogenesis
chronic colitis in TCR-decient mice [59], of the skin. LPS-stimulated CD19+ B cells iso-
CD21highCD23low B cells in contact hypersensi- lated from Tnf/ mice produced less IL-10.
tivity (CHS) mouse model [62], CD21highCD23high These mice had a reduced absolute number of
T2-MZ precursor B cells in CIA model [63], IL-10+CD19+ B cells in their spleens, and Tnf/
CD1dhighCD5+ B cells (termed B10 cells by mice were decient for CD19+CD21high B cells.
Yanaba et al.) in CHS [64] and EAE models The authors speculated that resistance to carci-
[65], CD138+CD19+ plasmablasts in Salmonella nogenesis in Tnf/ mice may result from
typhimurium infection [66], and T cell Ig increased CD8+ IFN--producing T cells and
domain and mucin domain protein (TIM)-1+ B decreased IL-10-producing B cells. In another
cells [67]. For human, CD19+CD24hiCD38hi B study, Horikawa et al. reported that production
cells have been found as putative Bregs [68, 69]. of IL-10 by Breg inhibits lymphoma depletion
Triggering Toll-like receptors (TLRs) [7072], during CD20 immunotherapy in mice [76].
the BCR [64], CD40 [73], or combinations They found that adoptive transfer of
thereof have been shown to promote IL-10 CD1dhighCD5+ B cells (that are enriched for B10
production by B cells. BCR-mediated Ca2+ ux cells) eliminates the therapeutic benet of CD20
appears to be required for IL-10 production, since mAbs in mouse lymphoma model. The trans-
B cells decient in the calcium sensors stromal ferred B10 cells in this model downregulated
interaction molecule (STIM) 1 and STIM2 have a the expression of MHC II molecules and CD86
profound defect in IL-10 secretion and abrogated on macrophages and reduced LPS-induced
suppression abilities in vivo [74]. Nuclear factor nitric oxide and TNF- production by macro-
of activated T cells (NFAT) 1, a transcription fac- phages, indicating that B10 cells suppress the
tor, is involved in Ca2+-dependent IL-10 produc- antitumor response at least partly by downregu-
tion [74]. Therefore, their proposed model for lation of macrophage activity. Our unpublished
IL-10 production by B cells is that, after BCR data support that Bregs play a negative role in
stimulation, STIM and Orai-dependent Ca2+ antitumor immunity. In melanoma and breast
increase by store-operated Ca2+ entry (SOCE) carcinoma models, depletion of IL-10-producing
activates calmodulin/calcineurin and then B cells from TDLN cells resulted in the genera-
NFAT1, leading to IL-10 expression. In addition, tion of potent effector B cells which dramati-
the TLR signaling pathway is also required cally inhibit tumor metastasis after adoptive
for IL-10 secretion [7072]. Given that TLR transfer in two genetically distinct immune
stimulation does not induce Ca2+ mobilization in competent hosts, B6 and Balb/c mice,
B cells, crosstalk between Ca2+ and Ca2+- respectively.
independent TLR cascades may be involved in Although little is known about the mecha-
IL-10 production. nisms by which Bregs undermine effective
IL-10 is an immunomodulatory cytokine antitumor immunity, several possibilities are
and inhibits Th1 polarization, prevents Th2 suggested by studies on inammation and auto-
responses, and suppresses pro-inammatory immunity. Bregs impair Th1 immune responses.
cytokine production by monocytes and macro- The initial nding about Th1 response regulated
phages [75]. So far, the potential role of Bregs by Bregs was reported by Skok et al. [77]; they
in tumor immunology is not clear, but several found that IL-10 produced by B cells is involved
studies suggest that Bregs can negatively in the feedback regulation of Th1 development.
regulate antitumor immunity. Using a mouse It has been reported that Bregs suppress the Th1
chemical carcinogenesis model, Schioppa et al. cell-mediated immune reactions in a number of
found that resistance to papilloma development mouse models, including EAE, CIA, CHS, and
in Tnf/ mice was associated with a signicant diabetes mellitus [60, 61, 64, 65, 72, 78, 79].
reduction in IL-10-producing B regulatory cells Fillatreau et al. reported that B cell IL-10
4 B Cells in Cancer Immunology: For or Against Cancer Growth? 55

deciency correlates with enhanced type I transitional 2 immature (T2) B cells stimulated
autoreactivity; in addition, transfer of IL-10+ B with agonistic anti-CD40 (T2-like Bregs) to
cells was found to result in resolution of EAE, convert autologous effector T cells into Tr1 cells
characterized by enhanced encephalitogenic [86]. Sayi et al. also showed that B cells activated
Th1 response [60]. Later, Lampropoulou et al. by Helicobacter TLR-2 ligands produce IL-10
showed that TLR signaling in B cells suppresses and induce IL-10-producing CD4+CD25+ Tr1
inammatory T cell responses (both Th1 and cells depending on TCR signaling and a direct
Th17) and stimulates recovery from EAE [72]. T-B cell interaction through CD40/CD40L and
Similarly, using mouse model of CIA, Mauri CD80/CD28 pathways [85].
et al. showed that transfer of IL-10-producing B
cells inhibits T helper type 1 differentiation and
prevents arthritis development [61]. Yanaba 4.6 Concluding Remarks
et al. also revealed that CD1dhighCD5+ B cell
transfer normalized inammation in CHS model B cells are phenotypically and functionally het-
[64]. Using NOD mouse model of type 1 diabe- erogeneous. Characterization of B cell subpopu-
tes (T1D), Hussain et al. found that BCR- lations is shown in Table 4.1. B cells play multiple
stimulated B cells produce IL-10 and attenuate roles in tumor immunity (Fig. 4.6). On one hand,
islet inammation by polarizing CD4+ T cell accumulating literature indicate that B cells are
response toward a Th2 phenotype [79]. signicantly involved in antitumor responses. In
Bregs induce the differentiation of Tregs. this regard, B cells present tumor antigens to T
Given that MT/ B cell-decient mice display cells to generate antitumor CTLs. Upon tumor
reduced Treg frequencies in comparison with antigen stimulation, B cells can differentiate into
wild-type mice [53] and that these mice develop plasma cells to produce antibodies to target tumor
exacerbated EAE and Ag-induced arthritis (AIA) cells via ADCC and/or CDC. In addition, B cells
[60, 80], a role for Bregs in modulating Tregs was may act as killer cells to directly cause tumor cell
proposed. Several disease models have demon- lysis in the absence of antibodies. B cells migrate
strated that IL-10 produced by Bregs is important to tumor tissue and become TIL-Bs which may
for the generation and/or maintenance of Tregs. induce humoral immune response or act as killer
Sun et al. reported that after oral tolerance cells in situ. On the other hand, regulatory B cells
induction, Treg cells increase much more in WT have been described which downregulate antitu-
than in MT/ mice. However, adoptive transfer mor responses by producing immunomodulatory
of B cells before treatment normalized Treg cytokine IL-10, suppressing Th1 immune
cell development in MT/ mice [81]. In this responses, and enhancing Treg and Tr1 responses.
study, they found that sublingual tolerization Further characterization of B cell subsets respon-
with OVA/CTB (Ag conjugated to cholera toxin sible for these conicting functions demonstrated
B subunit) enhances the tolerogenic activity of B in tumor immunity and understanding of the
cells and their production of IL-10, which was molecular mechanisms involved would help
associated with the generation of Ag-specic develop novel clinical strategies for cancer
Foxp3+CD25+CD4+ Tregs [81]. This relationship immunotherapy.
between Bregs and Tregs is further supported
by the results from mouse models of airway Acknowledgments This work was supported in part by
sensitization. These results showed that Bregs the Gillson Longenbaugh Foundation, the National
Outstanding Youth Foundation of China (81025008,
prevent and reverse allergic airway inammation
Xiao-Lian Zhang), and the National Natural Science
via FoxP3+ T regulatory cells [82, 83]. Foundation of China (31270176, Qin Pan).
Additionally, Bregs can induce the differentiation
of T regulatory 1 (Tr1) cells [8486]. Gray et al.
reported that autoimmune inammation could be Competing Interests
protected by the induction of Bregs which induce The authors have declared that no competing
T cell-derived IL-10 [84]. Blair et al. used the interest exists.
56 Q. Li et al.

Table 4.1 Phenotypic characterization of B cell subpopulations

Marker Source References
Resting B cells Human CD19+CD38IgD+CD27 Tonsils [87, 88]
CD38IgM+IgD+CD27 Blood [88]
Mouse IgMlowIgDhighHSAlowCD21intCD23brightMel14 Lymph node [89]
IgMhighIgDhighCD23bright Spleen [90]
CD40 B cell Human CD19+CD38+CD80+CD86+CD71+ Tonsils [87]
CD19+CD23+CD54+CD58+CD80+ Blood [6]
Mouse B7.1highB7.2highICAM+MHCIhigh Spleen [90, 91]
Putative Breg Human CD19+CD24highCD38high Blood [68, 69]
Mouse B220+CD1dhighCD21intermediate(int) Lymph nodesa [59]
B220+CD21highCD23low Spleen in CHS model [62]
B220+CD21highCD23high IgMbrightCD1dhigh Spleen in CIA model [63]
CD1dhighCD5+ CD19+ B220+ Spleen in CHS model [64]
CD1dhighCD5+ CD19+ Spleen in EAE model [65]
CD138+CD19+ Spleen of mice infected [66]
with Salmonella
TIM-1(T cell Ig domain and mucin domain Spleen [67]
TIL-Bs Mostly unknown. Related to cancer types and progression
Human CD19+CD20+ CD23+CD80+ From colorectal [40]
Killer B Unknown
From TCR-decient mice
4 B Cells in Cancer Immunology: For or Against Cancer Growth? 57

Fig. 4.6 Potential roles Tumor cell

played by B cells in
tumor immunity. ADCC
antibody-dependent Tumor
cellular cytotoxicity, CDC antigen
cytotoxicity, TIL-Bs IL-10
tumor-inltrating B cells B cell Breg


T cell Macrophage

B cell Th17 IFN-
Lysis CDC

Lysis Tumor cell IL-17


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The Role of Exhaustion in Tumor-
Induced T Cell Dysfunction 5
in Cancer

Heriberto Prado-Garcia, Susana Romero-Garcia,

and Jose Sullivan Lopez-Gonzalez

Contents 5.1 Introduction

5.1 Introduction ................................................ 61
T cells are divided into two major functional
5.2 T Cell Activation......................................... 62
types: helper and cytotoxic T cells. Helper T cells
5.3 T Cell Anergy.............................................. 63 (CD4+) release an array of cytokines and orches-
5.3.1 T Cell Anergy in Cancer............................... 64
trate diverse immune responses, which integrate
5.4 T Cell Exhaustion ....................................... 65 both adaptive and innate effector mechanisms.
5.4.1 Mechanisms for Inducing Cytotoxic T cells (CD8+ effector T cells) are pri-
T Cell Exhaustion ......................................... 65
5.4.2 Identication of Exhausted T Cells .............. 66 marily involved in the recognition and elimina-
tion of body cells compromised by intracellular
5.5 T Cell Exhaustion in Cancer ..................... 67
pathogens or oncogenic transformation.
5.5.1 A Particular Case: T Cell Exhaustion
in Lung Cancer Patients ............................... 69 Thus, T cells are essential components of the
immune system, which have been the major
5.6 Concluding Remarks ................................. 72
focus of immunotherapeutic strategies to boost
References ............................................................... 72 endogenous antitumor immunity. However,
despite homing to tumor sites, inltrating T cells
seldom control tumor growth, as a consequence
of the tumor microenvironment, which contains a
wide array of suppressive mechanisms that allow
tumors to escape T cell effector functions.
Even when T cell anergy has been considered
responsible for T cell hyporesponsiveness in can-
cer patients, cancer is also regarded as a chronic
disease, similar to chronic viral infections, where
H. Prado-Garcia, PhD (*) S. Romero-Garcia, PhD T cells are continuously stimulated. Thus, with
J.S. Lopez-Gonzalez, PhD
Departamento de Enfermedades Cronico-Degenerativas, chronic stimulation, tumor-specic T cells grad-
Instituto Nacional de Enfermedades Respiratorias ually become less functional until they undergo
Ismael Coso Villegas, Calzada de Tlalpan 4502, cell death, a phenomenon known as T cell
Col. Seccion XVI, Mexico City, Distrito Federal exhaustion. This chapter will focus on the latter
14080, Mexico
e-mail:;; mechanism and its participation in cancer- induced T cell dysfunction.

N. Rezaei (ed.), Cancer Immunology: A Translational Medicine Context, 61

DOI 10.1007/978-3-662-44006-3_5, Springer-Verlag Berlin Heidelberg 2015
62 H. Prado-Garcia et al.

5.2 T Cell Activation which interact with CD28 expressed on the

T cell membrane [1] (Fig. 5.1). CD28 is the
T cell activation requires two signals delivered primary costimulatory molecule for nave T
by antigen-presenting cells (APCs). The rst cells; this molecule is essential for initiating
signal involves the presentation of antigen by T cell responses. The interaction of CD80 and
APCs, in the form of peptides bound to MHC CD86 with CD28, together with TCR signal-
class I or class II molecules, to the T cell recep- ing, promotes the expansion and differentiation
tor (TCR), expressed on the surface of the T cell. of antigen-stimulated T cells into effector and
The second signal, or costimulatory signal, stim- memory cells. The interaction of CD28 with its
ulates T cells in conjunction with the antigen. ligands (1) enhances the production of interleu-
The molecules expressed on APCs engage their kin-2 (IL-2) and other cytokines, (2) promotes
corresponding costimulatory receptors on the energetic metabolism, (3) induces the cell cycle
surface of T cells generating costimulatory sig- progression, (4) promotes T cell survival, and
nals. CD80 (B7-1) and CD86 (B7-2) are well- (5) maintains T cell responsiveness upon subse-
characterized costimulatory signal molecules, quent restimulation [1].

Fig. 5.1 T cell activation

requires recognition of the
antigen and costimulatory
signals. Inammation
generated by tissue damage
or infections activates
antigen-presenting cells
(APCs) and stimulates the
expression of costimulatory
molecules, such as CD80/
CD86. Presentation of the
antigen to the T cell receptor
(TCR), in the context of major
histocompatibility complex
(MHC) molecules and CD80/
CD86 that interact with
CD28, stimulates the
expansion and differentiation
of nave T cells (top panel).
Resting APCs express few or
no costimulatory molecules
and fail to activate T cells,
this leads to anergy (middle
panel). CTLA-4 is a
coregulatory molecule that
binds CD80 and CD86 and is
upregulated on activated T
cells. CD80/CD86-CTLA-4
interactions inhibit T cell
responses and mediate
5 The Role of Exhaustion in Tumor-Induced T Cell Dysfunction in Cancer 63

Although costimulatory molecules were 5.3 T Cell Anergy

initially identied as stimulators of T cell
responses, some costimulatory (coregula- T cell anergy induces peripheral tolerance; this
tory) receptors inhibit T cells [1]. Cytotoxic mechanism protects the host from autoimmune dis-
T-lymphocyte-associated antigen-4 (CTLA-4) eases. In addition, anergy has been suggested to
is a CD28 homolog that also binds CD80 and play an important role in the induction of T cell dys-
CD86. Nevertheless, CTLA-4 expression is function in cancer patients. T cell anergy is a toler-
inducible after T cell activation, and is involved ance mechanism in which, after antigen encounter,
in the induction and maintenance of tolerance, as the T cell is intrinsically and functionally inacti-
its ligation inhibits IL-2 production and blocks vated [5]. The cell remains alive in this hyporespon-
cell cycle progression [1]. sive state for an extended period of time. Anergic T
After the discovery of homologs of CD28/ cells neither produce nor respond to proliferative
CTLA-4 and their ligands, many other coregu- signals and are unable to exert effector functions,
latory molecules have been identied, some of such as cytolysis or cytokine secretion. A character-
which include the inducible T cell costimula- istic of anergy is that it must be cell autonomous,
tor (ICOS or CD278) with its ligand CD275 which distinguishes this process from immunoregu-
(ICOS-L, B7h, B7-RP), the inhibitory pro- lation mediated through other regulatory cells, such
grammed death-1 (PD-1, CD279) with its as regulatory T cells (Tregs) [5, 6].
ligands PD-L1 (B7-H1, CD274) and PD-L2 There are at least ve distinct sets of circum-
(B7-DC, CD273), and the B- and T-lymphocyte stances that lead to T cell anergy [5, 7]: (1) TCR
attenuator (BTLA, CD272) which binds the ligation in the absence of full costimulation; (2)
herpesvirus entry mediator (HVEM). BTLA exposure to partial agonists, peptides with minor
is an additional receptor of the immunoglobu- sequence differences from native agonist antigenic
lin superfamily that negatively regulates T cell peptides that exhibit reduced avidity for TCR liga-
activation. In addition, HVEM interacts with tion; (3) full signaling without IL-2 receptor-
another negative regulator of T cells, CD160. driven cell division; (4) TCR ligation in the
Recent studies of the lymphocyte activation presence of IL-10 or transforming growth factor-
gene-3 (LAG-3, CD223) suggest that this mol- (TGF-); and (5) anergy induced through CTLA-4
ecule also plays an important role in the regula- or other coregulatory molecules (Fig. 5.1).
tion of T cell responses. Moreover, the T cell Thus, anergy is the consequence of factors
immunoglobulin domain and mucin domain-3 that negatively regulate proximal TCR-coupled
(TIM-3), with its ligand galectin-9, are involved signal transduction, together with active tran-
in terminating Th1 cell responses and establish- scriptional silencing, which is reinforced through
ing tolerance [2, 3]. epigenetic modications [8]. This state of nonre-
T cells that recognize antigen in the absence sponsiveness is molecularly distinct from T cell
of costimulation either fail to respond and exhaustion. T cell anergy is induced upon the rst
undergo cell death or enter a state of unrespon- encounter with the antigen and is quickly initi-
siveness, a phenomenon known as anergy. Thus, ated, in contrast with T cell exhaustion, which
costimulation is a key factor in the outcome of T is progressive. Gene expression proles show
cell interactions with the antigen. Signicant that anergy and exhaustion are partially distinct.
efforts have been undertaken to characterize Genes, such as Rnf128 (Grail), Egr2, and Egr3,
costimulatory molecules in order to augment are upregulated in anergic but not in exhausted
antitumor responses; recent evidence has demon- T cells, whereas NFAT is upregulated under
strated the importance of coregulatory molecules both conditions [9]. The detailed characteriza-
in the inhibition of immune responses. Thus, tion of the differences between anergy and T cell
interfering with these regulatory pathways has exhaustion will have important implications for
gained interest as a potential strategy for cancer therapeutic interventions in immune-mediated
therapy [4]. diseases and chronic infections.
64 H. Prado-Garcia et al.

Fig. 5.2 T cell exhaustion during chronic inammation. stages of dysfunction, losing effector functions (cytolysis
In an acute inammatory process, nave T cells are primed and secretion of cytokines) and proliferative potential in a
by an antigen, costimulatory molecules, and cytokines that hierarchical manner. Finally, deletion of T cells by apop-
promote differentiation into effector T cells. After clear- tosis occurs. As antigen load increases or CD4+ T helper
ance of the antigen and once inammation is resolved, a subpopulation decreases, T cells become more exhausted.
subset of effector T cells differentiates to become mem- Expression of coregulatory receptors is correlated with the
ory cells. During chronic processes, such as viral infec- level of exhaustion. The scale of each activity is presented
tions, the antigen persists, and T cells go through several from high (+++) to low ()

5.3.1 T Cell Anergy in Cancer coregulatory molecules, such as PD-L1, PD-L2,

ICOS-L (B7-H2, CD275), and B7-H3 (CD276),
Anergy has been proposed to play a role in the indicating a poor costimulatory and a high inhibi-
impairment of T cell function in human cancers. tory anergy-promoting environment. Evidence
Tumor cells are poor APCs, as these cells express that cancer induces T cell anergy comes from
antigens on MHC class I molecules but do not studies where the transfection of CD80 in tumor
express costimulatory molecules to provide a cells or the blockage of the B7 family coregula-
second signal for full T cell activation; thus, tory molecules results in reduced tumor growth
tumor-inltrating lymphocytes (TILs) are ren- or tumor rejection in mice models [2, 1114].
dered anergic [10]. In addition, immature Analysis of the functional state of TILs has
myeloid-derived dendritic cells (mDCs), demonstrated that these cells are characterized by
plasmocytoid dendritic cells (pDCs), myeloid- impairment of cytolytic activity, decreased cyto-
derived suppressor cells (MDSCs), and tumor- kine secretion, reduced expression of IL-2R
associated macrophages (TAMs) potentially (CD25), and diminished activation of extracellu-
induce anergy in TILs [8, 11, 12]. Several studies lar signal-regulated kinase (ERK) after TCR acti-
have shown that human tumor cells, mDCs, vation. Thus, T cell anergy occurs in the tumor
pDCs, MDSCs, and TAMs express high levels of microenvironment of some human tumors [1416].
5 The Role of Exhaustion in Tumor-Induced T Cell Dysfunction in Cancer 65

Nevertheless, direct evidence that T cell anergy late stages of exhaustion. Finally, during the most
occurs in cancer has been difcult to obtain due extreme stages of exhaustion, deletion of T cells
to the lack of surface markers to identify anergic occurs through apoptosis [19, 24] (Fig. 5.2). Like
T cells [8]. CD8+ T cells, CD4+ T cells also lose function
Based on mouse tumor models, the induction during chronic infections; however, there is little
of antigen-specic T cell anergy has been sug- information about the mechanisms of exhaustion
gested to be an early event in the progression of in this T cell subpopulation [19].
tumors, which occurs in the equilibrium phase of Exhausted T cells possess a molecular prole
immunoediting, before immunosuppression that is distinct from those of memory, effector,
takes place in advanced tumors (escape phase) and anergic T cells [9]. First, many membrane
[10, 17]. However, Klein et al. showed that highly inhibitory receptors are upregulated, for instance,
immunogenic tumors evade immunosurveillance PD-1, LAG-3, and TIM-3. Second, transcription
due to antigen overload and an insufcient num- of genes encoding molecules involved in TCR
ber of tumor-specic T cells, resulting in the signaling (such as Lck and NFATc) and cytokine
exhaustion of the immune cells [18]. Thus, from receptors (IL7 and IL-15 receptors) are down-
a temporal perspective, T cell anergy predomi- regulated. Third, the pattern of genes involved in
nantly occurs during the early stages of tumor chemotaxis, migration, and adhesion is changed.
progression, whereas T cell exhaustion might Fourth, there is an altered pattern of differentia-
play a crucial role in T cell dysfunction during tion compared with memory or effector T cells.
the late stages of cancer [10]. Finally, exhausted T cells present deciencies in
translational, metabolic, and bioenergetic pro-
cesses, such as the Krebs cycle [9].
5.4 T Cell Exhaustion

T cell exhaustion has been dened as a stage of T 5.4.1 Mechanisms for Inducing
cell differentiation where T cells have poor effec- T Cell Exhaustion
tor functions, sustained coregulatory receptor
expression, and a transcriptional state distinct Immunoregulation is critical in T cell exhaustion.
from that of functional effector or memory T Coregulatory receptors play a key role in many
cells [19]. Originally, this phenomenon was iden- aspects of adaptive immunity, including self-
tied in chronic viral infections in mice and later tolerance, prevention of autoimmunity, and can-
in chronic viral infections in humans, e.g., human cer. The mechanisms of regulation through
immunodeciency virus (HIV), hepatitis B virus coregulatory receptors have not been character-
(HBV), and hepatitis C virus (HCV) [1922]. ized in detail; nevertheless, several studies sug-
Chronic bacterial and parasitic infections have gest that these receptors attenuate T cell responses
been demonstrated to induce T cell exhaustion; in many ways. Accumulating evidence highlights
also, cancer has been suggested to induce a simi- the pivotal role of the PD-1/PD-L1 pathway in
lar phenomenon [20, 23]. maintaining an immunosuppressive tumor micro-
During chronic infections, antigen-specic environment. This pathway has been proposed to
CD8+ T cells initially acquire effector functions, be the most important coregulatory pathway
but gradually become less functional as the infec- involved in T cell exhaustion [25, 26].
tion progresses. The dysfunction of exhausted T A transmembrane receptor of the Ig superfam-
cells is hierarchical, showing the initial loss of ily, PD-1 (CD279), is upregulated in mice chroni-
properties, such as cytotoxic activity, prolifera- cally infected with lymphocytic choriomeningitis
tive potential, and interleukin 2 (IL-2) synthesis; virus (LCMV) [25]. PD-1 interacts with its
followed by diminished tumor necrosis factor- ligands PD-L1 (B7-H1, CD274) or PD-L2 (B7-
alpha (TNF-) secretion and subsequent loss of DC, CD273), which are members of the B7 fam-
interferon-gamma (IFN-) production during the ily [26]. PD-1 is rapidly upregulated on activated
66 H. Prado-Garcia et al.

T cells; then, after antigen clearance, the expres- secretion of effector cytokines, such as IFN-,
sion of this receptor is reduced on effector T TNF-, and IL-2 [19]. Interestingly, CD8+ T cells
cells. Upon subsequent antigen stimulation, expressing both coregulatory receptors also pro-
effector T cells show upregulated PD-1 expres- duce the suppressive cytokine IL-10 [37].
sion. Thus, the continuous stimulation of T cells Remarkably, functional effector T cells express
(nave or effector) during chronic infections coregulatory receptors during activation; however,
induces the accumulation of PD-1+ T cells [19]. as indicated above, the prolonged and increased
High levels of PD-L1 expression on APCs (or expression of multiple coregulatory receptors is
tumor cells) might sustain PD-1 expression on T a key feature of CD4+ and CD8+ T cell exhaus-
cells, impair T cell effector maturation, and allow tion. However, exhausted T cells do not necessar-
the progression of chronic infection [2729]. ily coexpress all of the coregulatory molecules.
Studies in mouse tumor models demon- The pattern as well as the level of expression of
strated that the inhibition of PD-L1 or PD-1 coregulatory receptors simultaneously expressed
using blocking monoclonal antibodies (mAbs) in the same CD8+ T cell might considerably inu-
increases the cytolytic activity of CD8+ T ence the severity of dysfunction [38].
cells and reverses T cell dysfunction [30, 31]. Several factors, such as duration of the infec-
Subsequently, Barber et al. showed that the inhi- tion, level of antigen exposure, availability of
bition of PD-1 using anti-PD-1 mAbs in chroni- CD4+ T cell help, and the type of APCs that
cally infected mice enhances the proliferation present the antigen, have been implicated in the
and effector functions of exhausted T cells [25]. severity of T cell exhaustion. Ligand availability
Since the publication of these seminal reports, for coregulatory receptors could also inuence
many other studies have shown that PD-1 with the degree of exhaustion, as well as environmen-
its ligand (PD-L1) is crucially involved in T cell tal factors such as the presence of immunoregu-
exhaustion in chronic human pathogen infec- latory cytokines [19]. In chronic viral infections,
tions and cancer [2124, 3234]. IL-10 expression is associated with T cell dys-
In addition to PD-1, many other cell surface function [38, 39]. In addition, TGF- has also
inhibitory receptors also participate in T cell been linked to exhaustion in chronic infections
exhaustion. These coregulatory receptors regu- in humans [40, 41]; nevertheless, the mecha-
late distinct cellular functions. For instance, PD-1 nisms underlying IL-10 and TGF--mediated T
pathway affects T cell survival and proliferation, cell exhaustion are unclear. Interestingly, both
whereas LAG-3 affects cell cycle progression, cytokines are secreted by several human tumors
but has less inuence on apoptosis [19]. Several [42, 43].
receptors belonging to the tumor necrosis recep-
tor family are upregulated in exhausted T cells,
such as Fas, TNF-R, and tumor necrosis factor- 5.4.2 Identication of Exhausted
related apoptosis-inducing ligand (TRAIL) T Cells
receptors; hence, these death receptors have been
implicated in the induction of exhaustion, as T Exhausted T cells show a poorly differentiated
cells might become prone to activation-induced phenotype (CD27hiCD28loCD57loCD127loCCR7-
cell death (AICD) [19, 35, 36]. CD45RA+ or CD27+CD45RO+) correlated with T
TIM-3 is an inhibitory molecule that down- cell dysfunction. Although PD-1 upregulation in
regulates effector Th1 responses; upregulation of T cells was initially considered as a hallmark of T
this molecule has been found in HIV-specic and cell exhaustion, this molecule is upregulated
HCV-specic CD8+ T cells in patients with pro- along with activation markers, such as CD38 or
gressive HIV and HCV infections, respectively. HLA-DR [44]. In healthy adults, the percentage
Importantly, the coexpression of TIM-3 and PD-1 of PD-1+ cells varies from 40 to 80 % of
has been associated with severe CD8+ T cell (CCR7+/CD45RA) memory T cells; remark-
exhaustion in terms of the proliferation as well as ably, these cells do not exhibit characteristics of
5 The Role of Exhaustion in Tumor-Induced T Cell Dysfunction in Cancer 67

exhaustion [45]. Thus, PD-1 is also associated tumor-specic T cells, virus-specic T cells are
with T cell activation and differentiation. more frequent and easily detectable, facilitating
Many cell surface coregulatory receptors are the ease in identication, phenotypic character-
expressed in exhausted T cells. LAG-3, TIM-3, ization, and isolation of T cells [10]. However, in
CD244 (2B4), CD160, CTLA-4, and the recently the tumor microenvironment, inltrating T cells
described B- and T-lymphocyte attenuator become dysfunctional and show reduced effector
(BTLA) are coexpressed in antigen-specic CD8+ functions. Several reports suggest that PD-L1
T cells during chronic infection. The pattern and expression on tumor cells plays an important role
level of coregulatory receptors simultaneously in tumor-induced T cell dysfunction. PD-L1
expressed in the same CD8+ T cell consider- membrane expression has been observed using
ably inuence the severity of dysfunction [38]. immunohistochemistry on many human tumors,
However, depending on the chronic infections or such as melanoma, lung, larynx, colon, breast,
cancer, exhausted T cells might express a differ- cervix, and stomach [26]. In breast, esophageal,
ent pattern of coregulatory molecules. gastric, and renal carcinomas, the increased
Genomic strategies have provided a molecular expression of PD-L1 on the surface of tumor
prole for exhausted T cells. These genomic cells is strongly associated with poor prognosis
studies support the notion that T cell exhaustion [26, 47]. Thus, T cell exhaustion has been pro-
represents a particular state of differentiation, posed as a mechanism for inducing T cell dys-
different from that of effector or memory T cells function through the PDL-1/PD-1 pathway.
[9, 19]. However, as previously indicated, PD-1 expres-
Several transcriptional pathways have been sion cannot be considered as the sole marker of T
associated with T cell exhaustion. The increased cell exhaustion in chronic diseases and cancer;
expression of transcriptional repressor Blimp-1 is hence, other markers must be considered.
associated with the upregulation of many coregu- In human metastatic-melanoma lesions, TILs
latory receptors (PD-1, LAG-3, CD160, and show upregulation of PD-1 expression, accompa-
CD244). In addition, the transcription factor nied with reduced production of IFN- TNF-, and
NFATc1 (NFAT2) is also upregulated but shows a IL-2. Both tumor-inltrating CD8+ T cells, par-
dysregulated function [9]. The transcription fac- ticularly MART-1-specic, and tumor-inltrating
tor T-bet also plays a role in protection against T CD4+ T cells show signicantly higher levels of
cell exhaustion, as T-bet promotes terminal dif- PD-1 expression than CD8+ and CD4+ T cells
ferentiation after acute infection, and the from peripheral blood and normal tissues from
increased expression of this transcription factor cancer patients. In addition, a large proportion
inhibits the expression of coregulatory receptors of CD8+ T cells from TILs were PD-1+CTLA-4+
during chronic viral infection. T-bet expression is cells compared with normal tissues and blood.
downregulated through persistent antigenic stim- Furthermore, PD-1+CD8+ cells from TILs lacked
ulation, resulting in T cell exhaustion [46]. CD25 as well as IL-7RA expression, suggesting
that these cells were unable to proliferate, pro-
duce effector cytokines, and differentiate into
5.5 T Cell Exhaustion in Cancer memory cells [48]. PD-1+NY-ESO-1-specic
CD8+ T cells, from patients with advanced mela-
Cancer and chronic viral infections have been noma, upregulate TIM3 expression and are more
thought to share similar mechanisms in establish- dysfunctional than TIM3-PD-1+ and TIM3-PD-
ing high antigen load and an immunosuppressive 1NY-ESO-1-specic CD8+ T cells, producing
environment. However, there is a fundamental less IFN-, TNF-, and IL-2 [49].
difference between both diseases: viral antigens Derr et al. showed that tumor antigen
are exogenous and extremely immunogenic, (Melan-A/Mart-1)-specic CD8+ T cells express
whereas tumor antigens are self-molecules that high levels of BTLA and are susceptible to func-
are weakly immunogenic. Thus, compared with tional inhibition through its ligand HVEM [50].
68 H. Prado-Garcia et al.

In addition, Baitsch et al. recently showed that in defects in proliferation and cytotoxicity, but with
melanoma, tumor antigen-specic CD8+ T cells increased production of IFN- and TNF-, nor-
with effector phenotypes simultaneously express mal production of IL-2, and increased expression
four or more of the inhibitory receptors BTLA, of T-bet. Thus, although CD8+ T cells show fea-
TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1, or tures of T cell exhaustion, these cells retain the
CTLA-4 [51]. Moreover, tumor antigen-specic ability to produce cytokines [57]. However, head
CD8+ T cells present a large variety of genes with and neck cancers that are positive for human pap-
a similar genetic prole as that of exhausted T illomavirus (HPV) present a high inltration of
cells from chronic viral infections [52]. Taken PD-1+ T cells, and the numbers of PD-1+ cells are
together, these reports show that tumor antigen- positively associated with a favorable clinical
specic CD8+ T cells are exhausted in melanoma outcome. Nevertheless, these PD-1+ T cells
patients. express activation markers, 50 % of this popula-
Additional evidence for T cell exhaustion in tion lack TIM-3 expression, and are functional
other cancers comes from studies in patients with after the blockade of the PD-1/PD-L1 pathway,
ovarian cancer. Matsusaki et al. reported that suggesting that PD-1+ T cells are activated rather
NY-ESO-1-specic CD8+ T cells, from the than exhausted [58].
peripheral blood of patients with ovarian cancer, Interestingly, Haymaker et al. proposed that
show impaired effector functions along with PD-1highCD8+ T cells in cancer patients are not
coexpression of the inhibitory molecules LAG-3 exhausted [59]. This hypothesis is based on the
and PD-1. The expression of LAG-3 and PD-1 on observation that CD8+ T cells from the TILs of
the surface of CD8+ T cells is upregulated through melanoma patients recover their proliferative
IL-10, IL-6, and tumor-derived APCs. In addi- potential ex vivo, despite expressing high levels
tion, LAG-3+PD-1+CD8+ T cells are decient in of PD-1. These TILs mediate antitumor responses
IFN-/TNF- secretion compared with LAG- upon adoptive transfer into patients [60, 61].
3+PD-1 or LAG-3PD-1 subsets [53]. Under this hypothesis, inltrating and peripheral
In hepatocellular carcinoma, PD-1+CD8+ T blood CD8+ T cells, expressing PD-1, BTLA,
cells are higher in tumor tissues than in non- along with other coregulatory receptors, are not
tumor tissues, presenting decreased proliferative exhausted. Instead, these cells are highly acti-
abilities as well as effector functions, as demon- vated effector-memory cells T cells that can be
strated by reduced granule and cytokine expres- stimulated through immunotherapy [59].
sion compared with PD-1CD8+ T cells [54]. Nevertheless, these observations have been pri-
Nevertheless, no other marker of T cell exhaus- marily achieved in melanomas. In other cancers,
tion was analyzed. the reduced proliferative and effector capacities
PD-L1 expression is upregulated in Hodgkin persist, even after stimulation, and immunothera-
lymphoma (HL) and several T cell lymphomas, peutic strategies have failed to induce potent anti-
but not in B cell lymphomas. In addition, PD-1 is tumoral responses [53, 57, 62, 63].
upregulated in TILs as well as peripheral blood Some of the phenotypic, functional, and
T cells from HL patients and the blockade of the molecular changes that occur in T cells during
PD-1 pathway restores IFN- production in chronic infections are exhibited in TILs as well
T cells [55]. Moreover, LAG-3 is expressed on as peripheral blood T cells from several cancer
TILs from patients with this malignancy [56]. types. The initial aim of tumor immunotherapy
Taken together, these reports suggest that TILs was to prevent anergy and tolerance towards
from patients with HL are exhausted. tumor antigens. However, the efcacy of this
In patients with chronic lymphocytic leuke- strategy is potentially limited by T cell exhaus-
mia (CLL), CD8+ and CD4+ effector T cells show tion [10]. Interestingly, Hailemichael et al.
the increased expression of CD244, CD160, and showed that in mice vaccinated with gp100 mela-
PD-1, with the expansion of the PD-1+ BlimpH1 noma peptide, the persisting tumor antigen at
subset. CD8+ T cells from CCL patients show vaccination sites induces the sequestration of
5 The Role of Exhaustion in Tumor-Induced T Cell Dysfunction in Cancer 69

CD8+ T cells, resulting in the dysfunction and from chronic viral infections [24], which sug-
death of these cells [63]. gests that CD8+ T cells are exhausted.
PD-1 blockage results in the recovery of T cell Recently, pleural effusion CD8+ T cells,
effector functions in vitro and in animal models derived from lung cancer patients, have been
in several tumors, thus signicantly enhancing shown to be susceptible to AICD. This phenome-
antitumor immunity [30, 31, 49, 64]. This knowl- non is preferentially observed in memory as well
edge has been translated into several clinical tri- as terminally differentiated CD8+ T cells. AICD
als [34, 65]. Recently, Brahmer et al. showed that is associated with upregulation of FasL and
the antibody-mediated blockade of PD-L1 TRAIL molecules. Interestingly, CD4+ T cells
induced durable tumor regression along with pro- from malignant pleural effusions are not prone
longed disease stabilization in patients with to AICD [75]. Thus, chronic stimulation by the
selected advanced cancers, including non-small lung tumor mass may sensitize CD8+ T cells to
cell lung cancer [65]. Thus, understanding T cell AICD, as it has been shown in TILs from vari-
exhaustion in cancer will contribute to the ous types of human cancers [75]. Nevertheless,
advancement of tumor immunotherapy. evaluation of exhaustion in lung tumor-specic
CD8+ T cells has not been possible, since lung
tumor-associated antigens are not shared among
5.5.1 A Particular Case: T Cell all lung cancer patients [62].
Exhaustion in Lung Cancer Here, it is shown PD-1 expression on CD8+
Patients and CD4+ T cells from pleural effusions and
peripheral blood of lung cancer patients who
Lung cancer is the leading cause of cancer-related were admitted to the National Institute of
mortality in developed countries and the second Respiratory Diseases Ismael Coso Villegas.
leading cause of death in countries with emerging Pleural uid was obtained by thoracocentesis
economies. Lung cancer is one of the most com- used for routine diagnostic procedures. Diagnosis
monly diagnosed cancers worldwide, represent- was established by histological examination of
ing 13 % of all cancer cases and approximately pleural biopsy or cytological observation of
18 % of all cancer deaths [66]. Some reports malignant cells in pleural effusion. None of the
show that the presence of TILs with memory patients received any type of anticancer therapy
phenotype in lung cancer is predictive of a favor- before the study or had clinical signs of acute or
able clinical outcome [6769]. Also, it has been chronic infection, which might interfere with the
shown that CD8+ T cell subpopulation is PD-1 analysis. For comparison, two groups of
decreased in the pleural compartment with patients with acute (pneumonias) and chronic
respect to peripheral blood from lung cancer (tuberculosis) infections that presented pleural
patients, whereas the CD4+ T cell subpopulation effusion were included. In lung cancer patients,
is increased [70, 71]. PD-1 was expressed on average at about 40 % of
Both in TIL and in the pleural compartment, pleural effusion CD8+ T cells, which was signi-
CD8+ T cells are functionally impaired and are cantly higher compared to percentages of
poorly responsive or unresponsive to several T PD-1+CD8+ T cells from pleural effusions sec-
cell-activating stimuli, even though memory cells ondary to acute or chronic tuberculosis infec-
inltrate lung tumors. CD8+ T cells present low tions. Also, PD-1 was expressed in more than
proliferation rate, diminished production of some 30 % of peripheral blood CD8+ T cells from lung
Th1 cytokines, and reduced cytotoxic potential cancer patients; in contrast, approximately 23 %
[70, 7274]. Pleural effusion CD8+ T cells from of peripheral blood CD8+ T cells from healthy
lung cancer patients express cell markers associ- subjects expressed PD-1 (Fig. 5.3). With respect
ated with a memory phenotype (CD45RA- to CD4+ T cells, the percentages of PD-1+ cells
CD45RO+CD27+Granzyme AlowPerforin), were signicantly higher in malignant effusions
similar to those markers found in CD8+ T cells compared to tuberculosis and acute effusions
70 H. Prado-Garcia et al.

Fig. 5.3 Determination of PD-1 on CD4+ and CD8+ T FITC-conjugated anti-PD-1 or isotype control mAb. Cells
cells. Pleural effusion (PE) and peripheral blood (PB) of were washed, xed with 1 % paraformaldehyde, and ana-
lung cancer patients (n = 23), patients with acute diseases lyzed using ow cytometry. FSC vs. SSC dot-plot graphs
(non-chronic n = 8), patients with tuberculosis (TB, n = 9), were done for cellular debris and necrotic cell exclusion.
and PB from healthy donors (HD, n = 9) were evaluated. From a lymphocyte gate containing 50,000 lymphocytes,
For PD-1 membrane staining, peripheral blood mononu- CD4+ or CD8+ cells were gated from a CD4+ or CD8+ vs.
clear cells (PBMCs) or pleural effusion mononuclear cells SSC dot-plot graph. For the analysis of PD-1 expressions,
(PEMCs) were incubated with anti-CD4 PE or anti-CD8- and to rule out nonspecic antibody binding and autouo-
PECy5 monoclonal antibodies (mAbs), in addition to rescence, quadrants were set according to isotype control

(Fig. 5.3). Similar percentages of PD-1+CD4+ T peripheral blood. However, TIM-3 expression on
cells were found in peripheral blood, between CD8+ T cells was not associated with any clinical
lung cancer patients and healthy donors. Thus, a pathological parameter in lung cancer patients
greater percentage of CD8+ and CD4+ T cells (e.g., tumor size, lymph node metastasis, and
from the pleural compartment are PD-1+, which tumor stage) [77].
is a consequence of the underlying pathology, In this chapter, TIM-3 expression on
rather than the anatomical compartment. CD4+ and CD8+ T cells derived from pleu-
Recently, Zhang et al. reported that tumor- ral effusion of lung cancer patients is shown
inltrating CD8+ T cells derived from patients (Fig. 5.4). Percentages of TIM-3+ cells were
with non-small cell lung carcinoma express signicantly higher in pleural effusion CD8+
increased levels of PD-1 [76]. These CD8+ T T cells in comparison with CD4+ T cells from
cells are impaired in cytokine production as well the same cancer patients (Figs. 5.4 and 5.5).
as proliferative potential. Blockade of the PD-1/ Interestingly, pleural effusion CD8+ T cells
PD-L1 pathway by anti-PD-L1 antibody partially from cancer patients showed higher percent-
restores cytokine production and cell prolifera- ages of TIM-3+ cells compared to those from
tion. However, PD-1 expression cannot be con- the nonmalignant group (tuberculosis). Hence,
sidered as the sole marker of T cell exhaustion; TIM-3 is likely to be upregulated in response
additionally, TIM-3 has been shown to mark to tumor-derived environmental factors absent
exhausted CD8+ T cells [38]. In a study by Gao in pleural effusions from patients with tuber-
et al., TIM-3 was found to be highly upregulated culosis. Nevertheless, in contrast with results
on both CD4+ and CD8+ T cells from lung tumor reported by Gao et al., who found that in lung
tissues, but almost undetectable on T cells from tumor tissues the majority of TIM-3+TILs are
5 The Role of Exhaustion in Tumor-Induced T Cell Dysfunction in Cancer 71

Cancer Tuberculosis
4 7.2 4.5 4 0.22 0.29
200K 10 10

91 Gated on
100K 10
3 CD8+ cells
CD3+ cells
0 10 3 32.2 3 51.4
19 10 10
0 50K 100K 150K 200K 250K 4
SSC 3 4 3 4
0 10 10 0 10 10
250K 73 10
0.34 0.43 10
0.18 0.30

200K 3
3 3 4 5
150K 10 0 10 10 10 3 3
10 10 CD4+ cells
100K CD4

50K 3 35.1 103 56.9


3 3 4 5 3 4 3 4
10 0 10 10 10 0 10 10 0 10 10
CD3 PD-1

Fig. 5.4 Gating strategy for the analysis of PD-1 and were plotted on PD-1 (FITC) vs. TIM-3 (APC) 5 % con-
TIM-3 expression on T cells. PEMCs were plotted rst on tour outlier plots; quadrants were set according to isotype
SSC vs. FSC with selection of the lymphocyte population. controls. Immunostaining was done as indicated in
Gated cells were then plotted on CD3 (PE-Texas Red) vs. Fig. 5.3. Representative data from patients with lung can-
FSC and further gated on CD4 (Alexa 700) vs. CD8 cells cer or tuberculosis are shown
(APC-Cy7). CD4+ (lower row) or CD8+ (upper row) cells

Fig. 5.5 (a) Percentage of

TIM-3+ cells on CD4+ and
CD8+ T cells from lung
cancer (n = 9) and tuberculo-
sis patients (n = 5). (b)
Percentage of cells express-
ing PD-1 and TIM-3 in
pleural effusion CD8+ T
cells. Determination of
TIM-3 and PD-1 was done
as indicated in Fig. 5.4.
* p = 0.015, ** p = 0.023.
Bars depict mean standard

PD-1+ [77], in the pleural compartment, most tion of exhausted CD8+ T cells. Nevertheless,
PD-1+CD8+ T cells did not coexpress TIM-3 TIM-3 expression is likely responsible for
(Figs. 5.4 and 5.5). Thus, PD-1+CD8+ T cells the absence of CD8+ T cell responses in lung
might be activated in a microenvironment that cancer patients.
does not provide the sufcient signals to fully Interestingly, the administration of PD-1 anti-
differentiate into effector T cells. Because body as a blocking agent against PD-1 pathway
PD-1 was not coexpressed in TIM-3+ CD8+ T has shown durable partial tumor regression in
cells (Fig. 5.5), further studies are required to patients with lung cancer, which was long thought
dene whether this subset belongs to a popula- to be a non-immunogenic tumor [65]. Thus,
72 H. Prado-Garcia et al.

reactivation of immune responses in lung cancer [52]. Nevertheless, it is not clear whether
patients, via blocking PD-1, TIM-3, or other reg- exhausted T cells share similar molecular and
ulatory pathways, in combination with other ther- genetic patterns in patients with chronic infec-
apeutic modalities, such as radiotherapy or tions and other types of cancer.
chemotherapy, will provide major clinical bene- Understanding the mechanisms of tumor-
ts to lung cancer patients. induced T cell exhaustion will conduce to the
development of vaccine-induced T cells aimed at
promoting tumor rejection. Preliminary clinical
5.6 Concluding Remarks ndings with blockers of immune-regulatory
pathways, such as the PD-1/PD-L1 pathway, sug-
T cell exhaustion is a stage of T cell differentia- gest that this strategy is promising for enhancing
tion where T cells show poor effector functions, antitumor immunity with the potential to produce
sustained coregulatory receptor expression, as long-lasting clinical responses.
well as a transcriptional state distinct from mem-
ory, effector, and even anergic T cells. From a
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Regulatory T Cells and Th17 Cells
in Cancer Microenvironment 6
Chang H. Kim

Contents 6.1 Introduction

6.1 Introduction ................................................... 77
Organs and tissues in the body are highly
6.2 Diversity of Tumor Microenvironments
and Tumor Tissue Factors ............................ 79
heterogeneous in producing tissue factors that
affect the development and maintenance of
6.3 Generation of Tregs and Th17 Cells ............ 80
immune cells. In general, organs and tissues in
6.4 Impact of Tumor-Derived Factors the body maintain highly tolerogenic conditions.
on Regulation of T-Cell Differentiation....... 81 This is important to prevent unwanted autoim-
6.5 Migration of Tregs and Th17 Cells mune or inammatory responses to harmless
into Tumors .................................................... 82 antigens and immune stimulants. Tumors, formed
6.6 Impact of Tregs and Th17 Cells in tolerogenic tissue environments, are naturally
on Antitumor Immune Responses ............... 84 hypoimmunogenic and utilize a number of mech-
6.7 Concluding Remarks .................................... 85 anisms to actively suppress the generation of
effector T cells [1, 2]. Tumors maintain tolero-
References ............................................................... 86
genic environments to avoid antitumor immune
responses. Tumors harbor high numbers of
FoxP3+ T cells (commonly called Tregs). Despite
the tolerogenic nature of the tumor microenviron-
ment, tumors variably produce many factors that
affect T-cell differentiation and maintenance. The
numbers of effector T-cell populations in tumors
are relatively more variable. Certain cancers are
linked to chronic inammation [3]. Cancers
formed in certain tissues, such as the intestine
and in patients with chronic infection, are
exposed to microbes, which can form inamma-
tory conditions in tumors. Cancers formed in
C.H. Kim, PhD (*) these tissues would be inuenced by inamma-
Laboratory of Immunology and Hematopoiesis, tory conditions. Necrotic tumor cells also induce
Department of Comparative Pathobiology, inammation through damage-associated molec-
College of Veterinary Medicine, Weldon School of
Biomedical Engineering, Center for Cancer Research,
ular pattern (DAMP) receptors such as TLR2,
Purdue University, West Lafayette, IN, USA TLR4, and the receptor for advanced glycation
e-mail: end products (RAGE) [4]. Inammatory tumors

N. Rezaei (ed.), Cancer Immunology: A Translational Medicine Context, 77

DOI 10.1007/978-3-662-44006-3_6, Springer-Verlag Berlin Heidelberg 2015
78 C.H. Kim

a b Non-inflammatory c Inflammatory


FoxP3 Th17 Th17 FoxP3

Naive Th17


Naive Th17 Th1/CD8/

FoxP3 FoxP3 FoxP3 Th17/Th1/CD8/

Fig. 6.1 Potential roles of FoxP3+ T cells and Th17 cells priming conditions. (b) FoxP3+ T cells can promote tumor
in tumors. (a) FoxP3+ T cells are made in the thymus as growth by suppressing antitumor immune responses at
nave-type FoxP3+ T cells, which migrate to lymphoid tis- early and late stages. On the other hand, Th17 cells can
sues. These FoxP3+ T cells can become the memory type induce immune responses that lead to eradication of
after activation in secondary lymphoid tissues. Induced tumor cells in a manner similar to other effector, CD8+,
FoxP3+ T cells with memory-type FoxP3+ T cells and and T cells. (c) In inammatory conditions, FoxP3+ T
Th17 cells are made from nave CD4+ T cells. FoxP3+ T cells and Th17 cells have the potential to play different
cells suppress effector T cells and other immune cells and roles. Th17 cells cause inammation in tissues; hence,
decrease tissue inammation. Th17 cells produce IL-17 to inammatory tumors are formed and stimulated to grow.
induce inammatory responses. FoxP3+ T cells and Th17 FoxP3+ T cells suppress the function of Th17 cells and
cells can trans-differentiate into other T-cell subsets such other inammatory T cells, leading to suppression of the
as Th1 and T-FH cells in appropriate cytokine and antigen tumorigenic process in inamed tissues

harbor FoxP3+ T cells and effector T cells, includ- cells [79]. In certain cancers, FoxP3+ T cells
ing Th17 cells and Th1 cells [5, 6]. FoxP3+ T increase, whereas Th17 cells decrease in number
cells can suppress the function of antitumor as cancers advance to more aggressive stages [9].
effector T cells and other immune cells to pro- The presence of FoxP3+ T cells and Th17 cells in
mote tumorigenesis (Fig. 6.1). On the other hand, tumors and associated tissues not only reects
FoxP3+ T cells can suppress tissue inammation the nature of tumor microenvironments but also
to prevent the emergence of tumor cells follow- indicates the types of active T-cell-mediated
ing chronic tissue inammation. Effector T cells immune responses in tumors. In this chapter, we
produce inammatory cytokines that promote will discuss tumor factors that regulate T-cell dif-
tumorigenesis by increasing tissue inammation ferentiation into Tregs and Th17 cells, migration
and angiogenesis, but they can also promote anti- of the T-cell subsets into tumors and associated
tumor immunity. An inverse correlation was lymphoid tissues, and the functions of Tregs and
observed between frequencies of FoxP3+ T cells Th17 cells in regulating antitumor immune
and effector T cells such as Th17 cells and Th1 responses.
6 Regulatory T Cells and Th17 Cells in Cancer Microenvironment 79

6.2 Diversity of Tumor cytotoxicity and cytokine secretion by CD8+ T

Microenvironments cells are impaired at the low pH range [19].
and Tumor Tissue Factors Cells in the tumor microenvironment produce
various cytokines and growth factors [20]. Some
Tumor microenvironment is highly heterogeneous, of these factors are drained into lymphatic ves-
depending on tumor types and sites of formation. sels and form tumor-associated microenviron-
Together with tumor cells, broblasts, myobro- mental milieu in lymph nodes. If tumors have
blasts, endothelial cells, mast cells, and other tissue tumor-specic or tumor-associated antigens,
cells make up tumors. Moreover, immune cells are these antigens are drained or transported into
an important component of tumors and are mainly lymph nodes and presented to T cells via antigen-
composed of T cells, B cells, innate lymphoid cells, presenting cells (APCs). Effector and regulatory
and myeloid cells. Tumor-associated myeloid cells T cells can be made during this antigen priming
are heterogeneous as well and contain immature process. The cytokine milieu is critical in deter-
and mature myeloid cells. mining the fate of differentiating T cells in tumor-
Myeloid-derived suppressor cells (MDSC) are draining lymph nodes. Again, the type and
immature myeloid cells and highly enriched in amount of cytokines and other factors produced
tumors [10]. MDSC are composed of heteroge- in tumors are highly diverse among tumor types.
neous myeloid cells at various different stages. Expression of IL-1, IL-1, IL-6, IL-11, and
Compared to mature myelocytes such as dendritic TNF- was observed in colon carcinoma, colon
cells (DCs) and macrophages, MDSC do not adenoma, ovarian cancer, and gastric cancer [21
highly express cytokines, co-stimulatory mole- 27]. IL-2 and IL-15 are expressed in melanoma.
cules, and MHC class molecules. Therefore, they IL-10 and TGF- are expressed in myeloma,
poorly support antitumor effector T-cell responses. colon cancer, lung cancer, and mammary carci-
Moreover, MDSC express various molecules that noma [28, 29]. Expression of IL-17, IFN-, and
dampen immune responses. MDSC produce per- IL-4 has been observed in certain tumor types
oxynitrite for nitration and nitrosylation of many [3032]. Expression of M-CSF, GM-CSF, and
proteins in the tumor environment [11, 12]. A IL-3 has been observed as well [3335]. These
major target protein for nitration and nitrosylation tumor-derived hematopoietic cytokines regulate
is TCR, which becomes ineffective at activating myeloid cell-mediated inammation and affect
T cells after the modications [13]. They also T-cell activity in tumors. Chemokines such as
express Arg1, inducible nitric oxide synthase CXCL chemokines (CXCL1, 3, 6, 8, 10, and 12)
(iNOS), and TGF-1, among others [14]. Tumors and CCL chemokines (CCL1, 2, 5, 17, 25, and
also harbor many macrophages, which can be 28) are expressed in various tumor types [3639].
made from MDSC or myeloid progenitor cells Growth and angiogenic factors such as VEGF,
[15]. Dendritic cells express indoleamine EGF, and HGF are broadly expressed in a num-
2,3-dioxygenase (IDO) to regulate available tryp- ber of cancer types [40, 41]. The cell types pro-
tophan [16]. Other immune cells such as mast ducing these factors are not limited to tumor cells
cells, NK cells, CD8+ T cells, and B cells are fre- but can be from various cell types in tumors. For
quently found in many tumor types. example, tumor-associated macrophages produce
The tumor environment is low in both oxygen both inammatory and immunosuppressive cyto-
and pH. Tumor cells rapidly divide and therefore kines such as IL-1, IL-6, IL-10, and TGF- [42].
vigorously consume oxygen supplied via blood T-cell receptor (TCR) activation signals are
vessels. Tumor cells mainly utilize the aerobic gly- modied by the signals from co-stimulatory and
colysis pathway to generate energy [17]. This can co-inhibitory molecules, which are expressed by
accumulate lactic acid and protons, leading to low tumor cells and tumor-associated APC [43]. These
extracellular pH [18]. The most common pH range molecules include B7-1, B7-2, programmed
in tumors is 66.5. The low acidic tumor environ- cell death-1 ligand (PD-L1), PD-L2, ICOS-L,
ment leads to immune cell anergy. For example, B7-H2, B7-H3, B7-H4, and B7-H6. Among these,
80 C.H. Kim

PD-L1-PD and B7-1/2-CTLA-4 play important RALDH2 expression is induced during immune
roles in the formation of Tregs in tumor micro- responses to increase the concentration of RA
environments [4446]. Moreover, TNF receptor available in local tissue environments. Inamed
family members such as OX40, GITR, 4-1BB, and tissues or tumors are low in expression of
CD40 are expressed in tumors and regulate antitu- RA-producing RALDH but are high in expres-
mor immune responses [47, 48]. sion of RA-catabolizing CYP26 [58, 59]. In sum,
Inammatory mediators are produced in the tumor microenvironment is made of highly
tumors. Cyclooxygenase-2 (COX-2) is highly diverse factors. Some are from tumor cells, while
expressed in malignant tumors [49, 50]. COX-2 others are from tissue cells and immune cells.
expression is induced in hypoxic conditions or by These factors have profound effects on T cells in
cytokines and growth factors [51]. COX-2 gener- tumors and associated lymphoid tissues as dis-
ates prostaglandin H2 from arachidonic acid, cussed in detail later in this chapter.
which is processed to generate major inamma-
tory mediators such as PGD2, PGE2, PGI2, and
TXA2. These mediators regulate angiogenesis 6.3 Generation of Tregs
and various aspects of inammatory responses in and Th17 Cells
tumors [49].
Some tumor types are under the inuence of FoxP3+ Tregs are made in the thymus as natural
microbe-associated molecular pattern (MAMP) FoxP3+ T cells. They are also induced in the periph-
receptor ligands if tumors are formed in barrier tis- ery from nave CD4+ T cells. In addition, IL-10-
sues such as the intestine or in patients infected producing Tregs (Tr1 cells) are made from nave
with pathogens. In mucosal tissues, decreased bar- CD4+ T cells. Tregs produce suppressive cytokines
rier functions due to tumorigenesis or preexisting such as IL-10, IL-35, and TGF- [6062]. These
inammation can lead to bacterial invasion and Tregs play critical roles in preventing autoimmune
induction of inammatory responses. Furthermore, diseases. Tregs are generally made whenever effec-
tumors that are associated with infection by papil- tor T cells are formed during immune responses.
lomavirus (uterine cervical carcinoma), hepatitis This is important to limit the potentially inamma-
B virus (hepatocellular carcinoma), Epstein-Barr tory activities of effector T cells.
virus (Burkitts lymphoma), human T-cell leuke- Induction of effector T cells and Tregs occurs
mia virus (adult T-cell leukemia), or herpes virus mainly in secondary lymphoid tissues. One reason
(Kaposis sarcoma) would be inuenced by viral for this is that naive CD4+ T cells that become effec-
MAMPs. MAMPs and DAMPs activate Toll-like tor T cells and Tregs migrate mainly to secondary
receptors (TLRs) [52]. TLR activation can induce lymphoid tissues. However, memory/effector T
tissue inammation that promotes cancer [53]. cells can trans-differentiate into each other at any
MYD88 signaling is also required for activation of tissue sites upon antigen priming (Fig. 6.1a). Th1
dendritic cells for proper formation of effector T cells are the most readily made effector T cells from
cells. Without proper MYD88 signaling, Th2 cells nave CD4+ T cells. IL-12, a cytokine produced
ineffective in antitumor immunity can be made from DCs, promotes the generation of Th1 cells.
[54]. TLR signaling can work together with STAT3 Th2 cells are made when IL-4 is abundant. Th17
and Notch pathways to activate MAPK and NFkB, cells are generated when IL-6, TGF-, and other
which promote the survival and proliferation of inammatory cytokines are present during T-cell
tumor cells [55]. priming. MAMPs and TLR activation in tissues
Retinoic acid is an anticancer agent. Retinoic promote the generation of Th17 cells. Th1 cells are
acids such as all-trans retinoic acid (ATRA) and efcient in the promotion of cell-mediated immu-
9-cis RA are produced from retinol (vitamin A) nity through production of IFN- Th17 cells that
by retinol metabolizing enzymes such as ADH are effective at inducing inammatory conditions
and RALDH [56]. Epithelial cells and APCs in through producing IL-17. A number of inamma-
the intestine highly express these enzymes [57]. tory cytokines, neutrophil-attracting chemokines, and
6 Regulatory T Cells and Th17 Cells in Cancer Microenvironment 81

inammatory mediators are induced by IL-17 [63]. molecules such as B7-1 and B7-2 at high levels.
IL-2 is required for the induction of T-cell prolifera- In tumors, the signals to maturate DCs are diverse
tion. IL-7 and IL-15 drive proliferation of T cells and not as apparent as those in infection. Thus,
in an antigen-independent manner in lymphopenic APCs maturated in tumor microenvironment do
conditions [64, 65]. IL-2 suppresses the formation not highly express the co-stimulatory molecules
of Th17 cells [66]. IL-4, while inducing Th2 cells, [75]. Moreover, tumor-associated APCs express
suppresses the formation of induced FoxP3+ T cells co-inhibitory receptor ligands such as PD-L1 and
and Th1 cells [67, 68]. IL-27 promotes the genera- PD-L2 [76, 77]. This affects T-cell activation and
tion of Tr1 cells [69, 70]. Expression or activation differentiation. Therefore, DCs in or from tumors
of specic transcription factors is required for the have low activation potentials for T cells. This con-
generation of specialized effector T cells and Tregs. dition typically generates induced FoxP3+ T cells
For example, RORt, STAT3, and AHR are impor- but not effector T cells. Other APCs in tumors,
tant for Th17 cells. FoxP3 and STAT5 are important such as macrophages and MDSC, are also ineffec-
for the formation of induced Tregs. c-Maf and aryl tive in generating effector T cells but are prone to
hydrocarbon receptor (AHR) are important for for- induce Tregs [78].
mation of Tr1 cells [61, 60, 71]. Beyond cytokines, As mentioned, the hypoxic condition in the
many other factors can modulate the generation of tumors is another regulatory factor for T cells [79].
Tregs and Th17 cells. This subject will not be dis- It is expected that draining lymph nodes or tertiary
cussed in detail, as the generation of Tregs and Th17 lymphoid tissues within tumors have low oxygen
cells during basic immune responses is exhaustively levels. T cells become FoxP3+ T cells when they are
discussed elsewhere. activated in hypoxia [80]. This is in part mediated
by a transcription factor called HIF-1. The high
glycolytic activity in tumors leads to accumulation
6.4 Impact of Tumor-Derived of lactic acid [8183]. This promotes the generation
Factors on Regulation of FoxP3+ T cells. TGF-1 is a characteristic cyto-
of T-Cell Differentiation kine produced in the tumor environment [8486].
TGF-1 is the most efcient cytokine that induces
Most T cells in tumors are memory T cells [72]. FoxP3+ T cells in the periphery. Along with TGF-
Both antigen-specic and nonspecic bystander T 1, IL-10 acts to suppress antitumor immune
cells would be present in tumors. In general, the responses and the promotion of Tregs [87, 88].
presence of memory T cells and CD8+ T cells is IL-10 is produced by various cell types, including T
linked to positive prognosis in cancer patients. This cells, myeloid cells, B cells, and tumor cells.
indicates that it is benecial to have these T cells in PGE2 is highly produced in the tumor envi-
tumors. About 3050 % of CD4+ T cells in various ronment. PGE2 induces FoxP3+ T cells. This
tumors formed in animals are FoxP3+ T cells [72]. induction is mediated by EP4 and EP2 receptors
Th17 cells are also found in tumors, particularly [89, 90]. In this regard, inhibition of cyclooxy-
tumors formed in mucosal tissues [73, 7, 74]. In genase-2 (COX-2) decreased FoxP3 expression
contrast, Th17 cells are hard to nd in transplanted in tumors and reduced tumor burden [91].
tumors in animal models at ectopic sites [72]. Interestingly, FoxP3+ Tregs express COX-2 and
Many factors of the tumor microenvironment can produce PGE2 [92]. The PGE2 produced by
promote the generation of FoxP3+ T cells. First, Tregs suppresses effector T cells. In addition,
APCs in tumor environments are prone to generate prostaglandin D2 (PGD2) acts on DCs to induce
FoxP3+ T cells. During infection, DCs uptake anti- FoxP3+ T cells [93]. This effect is mediated
gens and undergo maturation in response to TLR through the D prostanoid receptor and cyclic
activation. Activated DCs emigrate tissue sites of AMP-dependent protein kinase A. In this regard,
infection and migrate into secondary lymphoid enforced expression of COX-2 in head and neck
tissues through lymphatic vessels. Only mature squamous cell carcinoma led to expansion of
DCs express MHC molecules and co-stimulatory IL-10+ FoxP3+ T cells [94].
82 C.H. Kim

Commensal bacterial products that activate and integrins mediate rolling and rm adhesion
TLR2 are implicated in selectively promoting of leukocytes on endothelial cell vessels [106,
FoxP3+ T cells and Th17 cells. Segmented la- 107]. Chemokines induce integrin activation
mentous bacteria (SFB) promote Th17 cells in the between rolling and rm adhesion of immune
small intestine [95]. Certain bacterial groups such cells on endothelial cells. Chemokines also
as Clostridium and Bacteroides fragilis promote induce chemotaxis for migration of immune cells
the generation of FoxP3+ T cells in the intestine within tissues. Organs and tissues express distinct
[96, 97]. Tumors, formed in the intestine, female and overlapping chemokines and adhesion mole-
reproductive tract, and skin, are expected to be cules for regulation of immune cell migration
heavily inuenced by commensal bacteria. In [108]. Since tumors are formed within special-
these tumors, bacterial MAMPs would activate ized organs and tissues, there are similarities in
APC and T cells to regulate the generation of expression of trafcking signals between normal
FoxP3+ T cells and Th17 cells. Thus, depending tissues and tumors formed within the tissues.
on the bacterial group that is dominant in the Compared to normal tissues, however, tumors
tumor environment, FoxP3+ T cells and Th17 have altered expression of chemokines and adhe-
cells can be differentially generated. sion molecules [109]. The trafcking signals and
As mentioned, retinoic acid is an important receptors required for T-cell migration into the
tumor factor. Retinoic acid affects T cells and intestine are well established. In the intestine,
tumor cells. Retinoic acid promotes the generation CCL20 and CCL25 are, respectively, expressed
of FoxP3+ T cells but suppresses that of Th17 cells in the subepithelial cell dome (SED) of Peyers
[98, 99]. Retinoic acid affects the development of patches and by small intestinal epithelial cells
DCs and generates tolerogenic DCs expressing [110113]. Endothelial cells in the intestine,
Arg1 [100]. These DCs promote the generation of Peyers patches, and mesenteric lymph nodes
FoxP3+ T cells but suppress the formation of Th17 express mucosal addressin cell adhesion mole-
cells. This function seems to be mediated through cule-1 (MAdCAM-1) [114]. T cells migrating to
RAR-. It is also reported that retinoic acid at low the small intestine express CCR9 and 47 [115
concentrations (i.e., 0.55 nM) is required for nor- 117]. Memory T cells migrating to the Peyers
mal function of effector T cells [101, 102]. Low patches express CCR6 [118, 119]. Nave T cells
concentrations of RA are found in bodily uids in migrating to Peyers patches, MLN, and PLN
most tissues. In vitamin A deciency, the migration express CCR7, 47, and CD62L [120]. Memory
and function of effector T cells are severely T cells migrating to other tissues or inamed tis-
impaired. As mentioned, tumor cells express sues variably express CCR1-6, CCR8, CCR9,
CYP26 and can decrease retinoic acid concentra- CCR10, CXCR3, CXCR5, and CXCR6 [108].
tion in tumors and associated tissues [58]. This Effector T cells frequently express P-selectin gly-
hyporetinoic acid condition would signicantly coprotein ligand-1 (PSGL-1), E-selectin ligand-1
affect the T-cell prole in tumors and associated (ESL-1), CXCR3, CCR5, and CCR4 [105, 120].
lymphoid tissues. Moreover, retinoic acid can pro- The trafcking receptors of Tregs and Th17
mote differentiation of tumor-associated MDSC cells have been determined. FoxP3+ T cells that
into dendritic cells and macrophages [103]. are freshly made in the thymus express CCR7,
CXCR4, and CD62L [121, 122]. FoxP3+ T cells
activated or induced in the periphery express
6.5 Migration of Tregs and Th17 memory-type trafcking receptors that are fre-
Cells into Tumors quently expressed by Th1 or Th2 cells. Th17 cells
express most memory-type chemokine receptors
Migration of T cells, including Tregs and Th17 [123, 124]. CCR6 is a characteristic chemokine
cells, is regulated by trafcking receptors such as receptor expressed by most Th17 cells. In general,
chemokine receptors and adhesion molecules FoxP3+ Tregs and Th17 cells follow the trafck-
[104, 105]. Adhesion molecules such as selectins ing pattern of conventional nave and memory/
6 Regulatory T Cells and Th17 Cells in Cancer Microenvironment 83

effector T cells. Conventional nave CD4+ T cells nd universal trafcking signals which govern
expressing CCR7 and CD62L lose these receptors T-cell trafcking in most tumors.
upon T-cell activation in the secondary lymphoid Our group investigated the trafcking recep-
tissues and migrate into nonlymphoid or inamed tors expressed by tumor-inltrating FoxP3+ T
tissues. Various tissue factors inuence the cells [72]. FoxP3+ T cells account for 2550 %
expression of trafcking receptors on FoxP3+ T of CD4+ T cells inltrating A20, CT26, 4T1, and
cells and Th17 cells [125, 126]. For example, reti- B16 tumors. Most of these FoxP3+ T cells are
noic acid acts on T cells undergoing activation to memory CD44+ CD62- T cells, which are down-
induce gut-homing receptors such as CCR9 and regulated for CD62L and CCR7. Downregulation
47. FoxP3+ T cells and Th17 cells express these of CCR7 was critical for the migration of FoxP3+
gut-homing receptors and migrate to the intestine T cells into tumors, as CCR7high FoxP3+ T cells
[98, 127]. In vitamin A deciency, the number of were not efcient at migrating into tumors [72].
FoxP3+ T cells and Th17 cells in the gut is signi- Downregulation of CCR7 and CD62L occurs
cantly decreased in part because most T cells do in tumor-draining lymph nodes during antigen
not migrate to the small intestine [128]. In addi- priming. Therefore, migration of T cells into sec-
tion, TGF-1 is a major cytokine that induces the ondary lymphoid tissues is required to acquire a
expression of CCR6 on FoxP3+ T cells and Th17 proper trafcking receptor phenotype for migra-
cells [123]. Moreover, IL-2 is a cytokine that tion into tumors. While downregulated for CCR7
effectively downregulates CCR6 expression and CD62L, tumor-inltrating FoxP3+ T cells
induced by TGF- 1. Thus, cytokines and tissue express CCR8 and CXCR4 at high levels [72].
factors can co-regulate the expression of trafck- This trafcking receptor phenotype reects the
ing receptors on T cells. differentiation status of the tumor-inltrating T
Researchers have been searching for che- cells and/or the trafcking receptor requirement
mokines that regulate immune cell trafcking for FoxP3+ T-cell migration into the tumors.
and antitumor immune responses [129133]. Induction of FoxP3+ T cells from FoxP3- T cells
Chemokines such as CCL3-5, CCL20, and in tumors was assessed, and the results indicate
CXCL10, often expressed in inamed tissues, are that this induction is inefcient [72]. Thus, the
also expressed in tumors [134139]. Chemokines tumor-inltrating FoxP3+ T cell in these tumors
induce chemotaxis of immune cells and tumor is largely from the FoxP3+ T cells made in the
cells. They can co-stimulate T cells and pro- thymus or secondary lymphoid tissues rather
mote angiogenesis [140, 141]. CCR2-10 and than FoxP3+ T cells induced directly in tumors.
CXCR3-5 regulate T-cell trafcking in various However, this can be quite different in other types
tumors [132]. Most of these receptors are highly of tumors where the tumor microenvironment is
expressed by FoxP3+ T cells and Th17 cells more conducive in priming T cells for differ-
in mice and humans [105, 123, 124, 121, 122, entiation into Tregs. In tumors, FoxP3+ T cells
142]. CCL17 and CCL22 are highly expressed in appear highly stable in maintaining their FoxP3
gastric cancer with CCR4-expressing FoxP3+ T expression. While detailed information on Th17
cells [131]. CCR7 is expressed by some T cells cell migration into tumors is not available, Th17
in colorectal cancers and is predictive of positive cells would probably utilize the same tissue- or
prognosis [143]. CXCR4+ T cells are increased inammation-associated trafcking signals uti-
in lung adenocarcinoma [144]. Chemokines lized by Th17 cells for regulation of general
expressed in tumors also attract hematopoietic immune responses. Th17 cells are prevalent in
progenitors, myeloid cells, NK cells, and CD8+ the gastrointestinal (GI) tract and other muco-
T cells [136, 145, 10]. An important point is sal tissues. High numbers of Th17 cells were
that chemokine signals in cancer patients are observed in aggressive forms of GI cancers [73,
highly diverse among different tumors. They are 7, 74]. Thus, these tumors would have trafcking
also affected by tissue sites and inammatory and cytokine signals appropriate for recruitment
responses in tumors. Therefore, it is difcult to and maintenance of Th17 cells or their progenitors.
84 C.H. Kim


Blood FoxP3 Tumor

Back to circulation

Th17 Th1


Naive Humoral Mac


FoxP3 LN
Memory type
trafficking receptors DC

Fig. 6.2 Migration of FoxP3+ T cells and Th17 cells into CCR5, CCR8, CCR10, and/or CXCR4. Dendritic cells
tumors. Natural FoxP3+ T cells made in the thymus can (DCs) transport and present tumor tissue antigens and
migrate into lymph nodes, but cannot migrate directly into play important roles in the generation of FoxP3+ T cells
tumors unless tumors are formed in lymphoid tissues. and Th17 cells in lymph nodes. Soluble tumor tissue fac-
FoxP3+ T cells can migrate into tumors after they are anti- tors are collected in tumor-draining lymph nodes, and
gen primed in secondary lymphoid tissues and gain the some affect T-cell priming and differentiation. In tumors,
memory/effector-type trafcking receptors. Loss of CCR7 macrophages (Mac), DCs, and MDSC suboptimally acti-
and CD62L occurs during antigen priming and is required vate T cells in tumors. These APCs play potentially
for migration of antigen-primed FoxP3+ T cells into important roles in maintaining the phenotype of FoxP3+ T
tumors. Induced FoxP3+ T cells in the tumor-draining cells and Th17 cells in tumors. There is no such thing as
lymph nodes can migrate into tumors, as they are down- tumor-specic trafcking receptors. Instead, T cells vari-
regulated for CCR7 and CD62L but upregulated for mem- ably use conventional trafcking receptors to migrate into
ory/effector-type trafcking receptors such as CCR4, different tumors

Migration of FoxP3+ T cells and Th17 cells into frequencies of memory CD4+ T cells and CD8+ T
tumors and draining lymph nodes is summarized cells in many cancer types. Tumorigenesis is
in Fig. 6.2. increased in pan-T-cell- or -T-cell-decient
animals or humans [148]. Strikingly, T cells
have a small negative effect on tumor numbers,
6.6 Impact of Tregs and Th17 but a greater positive effect on tumor size. This
Cells on Antitumor Immune implies that T cells are composed of heteroge-
Responses neous subsets with different functions, and some
of these T cells may even promote tumor growth.
The presence of T cells in tumors is a highly reli- FoxP3+ T cells and other regulatory T cells are
able prognostic factor for survival of cancer likely the T cells that suppress antitumor immune
patients [146, 147]. There is a strong posi- responses. FoxP3+ T cells can inhibit antitumor
tive correlation between patient survival and immune responses and promote tumor growth
6 Regulatory T Cells and Th17 Cells in Cancer Microenvironment 85

[149]. Many FoxP3+ T cells are self-reactive and inammatory tumors with high expression of
effective in preventing autoimmune diseases. The inammatory cytokines would harbor high
same function can be used to promote tumor numbers of Th17 cells. Tumors are heteroge-
growth. This is because tumor cells basically neous in the tumor microenvironment even
express self-antigens, and FoxP3+ T cells can within the same group of cancers, and not all
effectively suppress immune responses to self- tumors t into the inammatory vs. noninam-
antigens [150]. In the same line, the frequencies matory tumor model. While there is an inverse
of FoxP3+ T cells in many tumor types are correlation between FoxP3+ T cells and Th17
inversely correlated with patient survival rates cells, both T-cell subsets can be increased or
[151, 147]. However, lack of correlation or posi- decreased depending on the balance of cytokines
tive correlation has been noticed as well [152, and other tissue factors. An example for this situ-
153]. A good example is colorectal carcinoma, in ation is invasive ductal breast carcinoma [157].
which high frequencies of FoxP3+ T cells are
associated with a favorable prognosis [5]. It is
expected that FoxP3+ T cells can even prevent the 6.7 Concluding Remarks
formation of some tumors by suppressing tissue
inammation at early stages of tumorigenesis. As discussed throughout this chapter, FoxP3+ T
Therefore, FoxP3+ T cells have the potential to cells and Th17 cells play both positive and nega-
either promote or suppress tumorigenesis depend- tive roles in regulating antitumor immune
ing on tumor type, tissue site, and immune responses (Fig. 6.1). Despite the presence of
response. The potentially complex functions of these T cells, some tumors still develop and grow.
Tregs in tumorigenesis are depicted in Fig. 6.1. Thus, these T cells by themselves are not suf-
It has been observed that Th17 cells can pro- cient to effectively mount antitumor immune
mote CD8+ T-cell-mediated antitumor immune responses. More detailed studies on FoxP3+ T
responses in a mouse model [154]. Moreover, cells and Th17 cells in various tumors can pro-
polarization of CD8+ T cells into Tc17 cells vide systematic information regarding the tumor
increased their antitumor immunity [155]. Th17 microenvironment and therapeutic interventions.
cells may become Th1 cells or activate CD8+ T It is important to develop novel strategies to boost
cells to increase antitumor immunity. the benecial effects of the T-cell subsets and to
Paradoxically, Th17 cells can cause inammation suppress their tumor-promoting effects. The key
to initiate development of inammatory tumors is to alter tumor microenvironment to regulate
at early stages of tumorigenesis. In colorectal these T-cell subsets. This is expected to be
cancer, Th17 cells are linked to poor prognosis, achieved through control of antigen-presenting
whereas Th1 cells are positively linked to patient cells, metabolism, cytokines, chemokines, co-
survival [156]. The major cytokine product of stimulatory/inhibitory receptors, inammatory
Th17 cells, IL-17, can induce tissue inamma- mediators, and nuclear hormone receptor ligands
tion and the expression of certain angiogenic fac- such as retinoic acid. Regulation of multiple fac-
tors, including CXCL8, MMP-2, MMP-9, and tors at the same time would provide more effec-
VEGF [157]. The function of Th17 cells in can- tive strategies in tipping the T-cell balance toward
cer can be complex and appears to be determined tumor-eradicating immune responses. A one-
again by cancer type, stage, and site. The poten- size-ts-all approach is not likely to be effective
tially complex functions of Th17 cells in tumori- in changing the microenvironment and T-cell
genesis are depicted in Fig. 6.1. activity in all tumors. In this regard, another point
Apart from their effector functions, the fre- is that antitumor therapy strategies should be
quencies of FoxP3+ T cells and Th17 cells reect tailor-made based on cancer type, tissue site, and
the context of the tumor microenvironment. tumor microenvironment. It is expected that
Noninammatory tumors with low expression of application of wrong immunotherapy strategies
IL-6 and other inammatory cytokines would to regulate the T-cell subsets could even worsen
have high numbers of FoxP3+ T cells, whereas the prognosis of cancer patients. More research
86 C.H. Kim

into classication of cancer types based on tumor 12. Bentz BG, Haines 3rd GK, Radosevich JA. Increased
protein nitrosylation in head and neck squamous cell
microenvironment and immunological milieu
carcinogenesis. Head Neck. 2000;22(1):6470.
would be highly useful. 13. Nagaraj S, Gupta K, Pisarev V, Kinarsky L, Sherman
S, Kang L, et al. Altered recognition of antigen is a
Acknowledgments The author thanks Kim Lab mem- mechanism of CD8+ T cell tolerance in cancer. Nat
bers and F. Chu (Purdue University) for their inputs and Med. 2007;13(7):82835.
assistance in preparation of this chapter. This study was 14. Peranzoni E, Zilio S, Marigo I, Dolcetti L, Zanovello
supported, in part, by grants from the NIH (R01AI074745, P, Mandruzzato S, et al. Myeloid-derived suppressor
R01DK076616, 1R01AI080769, and 1S10RR028293), cell heterogeneity and subset denition. Curr Opin
the Crohns and Colitis Foundation of America, and the Immunol. 2010;22(2):23844.
National Multiple Sclerosis Society to CHK. 15. Kusmartsev S, Gabrilovich DI. STAT1 signaling regu-
lates tumor-associated macrophage-mediated T cell
deletion. J Immunol. 2005;174(8):488091.
16. Ikemoto T, Shimada M, Komatsu M, Yamada S, Saito
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Role of Cytokines in Tumor
Immunity and Immune Tolerance 7
to Cancer

Murugaiyan Gopal

Contents 7.1 Introduction

7.1 Introduction ................................................. 93
Strong evidence has been accumulated
7.2 Cytokine Regulation
of the Antitumor Immune Response .......... 94
demonstrating that cancer cells in humans and
7.2.1 IL-12 .............................................................. 95 animals are recognized in general as nonself
7.2.2 IL-27 .............................................................. 100 by the immune system [1, 2]. Both innate and
7.3 Cytokines in Immune Tolerance adaptive immune reactions to cancer have been
to Cancer ...................................................... 101 described. Many cases of spontaneous tumor
7.3.1 TGF- ............................................................ 101 regression in patients with cancer have been
7.3.2 IL-17 .............................................................. 105
reported. In addition, such spontaneous regres-
7.3.3 IL-23 .............................................................. 108
7.3.4 IL-35 .............................................................. 108 sions normally occur following an infection.
7.3.5 IL-10 .............................................................. 109 Moreover, immunosuppressed patients are at
7.4 Concluding Remarks .................................. 111 increased risk for virally induced tumors [3]. In
fact, the presence of highly adaptive immune cell
References ................................................................ 111
inltrates within the tumor can be a positive prog-
nostic indicator of patient survival [4]. Murine
models of spontaneously arising or chemically
induced tumors have been useful in demonstrat-
ing that the immune system naturally surveys for
aberrant cells and has an important role in pre-
venting tumor formation [2].
An antitumor immune response is initiated
when the cells of the innate immune system
become alerted to the presence of a growing
tumor, at least in part owing to the local tissue
damage that occurs as a result of stromal remod-
eling process integral to the basic physiology of
M. Gopal, PhD (*) solid tumor development [2, 5]. Once solid tumors
Department of Neurology, Center for Neurologic reach a certain size, they begin to grow invasively
Diseases, Brigham and Womens Hospital, and require an enhanced blood supply that arises
Harvard Medical School, Harvard Institutes
as a consequence of the production of angiogenic
of Medicine, 77 Avenue Louis Pasteur, NRB-641,
Boston, MA, 02115, USA proteins [6]. Invasive growth causes minor disrup-
e-mail: tions within the surrounding tissue that induces

N. Rezaei (ed.), Cancer Immunology: A Translational Medicine Context, 93

DOI 10.1007/978-3-662-44006-3_7, Springer-Verlag Berlin Heidelberg 2015
94 M. Gopal

inammatory cytokines and chemokines leading Tumors use several mechanisms that facilitate
to recruitment of cells of the innate immune system immune escape and prevent tumor elimination
[7]. The innate response includes several cellular including impairment of antigen presentation,
factors, such as natural killer (NK) cells, natural activation of negative co-stimulatory signals, and
killer T (NKT) cells, T cells, macrophages, elaboration of immunosuppressive factors [12].
dendritic cells (DCs), and neutrophils [8]. These In addition, tumor cells may promote the expan-
cells can reject tumors either by direct killing of sion and/or recruitment of regulatory immune
the tumor cells or by inhibition of angiogenesis. cell populations which can contribute to the
The components of innate immunity use pattern immunosuppressive network; these populations
recognition receptors and other cell surface mol- include regulatory T cells (Tregs), myeloid-
ecules to detect tumor cells. Cancer cells express derived suppressor cells (MDSCs), and distinct
families of stress-related genes such as MHC subsets of immature and mature regulatory DCs
class I-related stress-inducible surface glycopro- [12]. All these host-derived immune cell popula-
tein A and B (MICA and MICB), which function tions can impair antitumor effector cell responses,
as ligands for NKG2D receptors expressed on NK both locally at the tumor microenvironment and
cells [9]. In addition, NK cells can be triggered for systemically in the lymphoid organs. In fact, both
cytolytic activity by DCs depending on direct cell tumor-promoting and tumor-inhibitory immune
contact through their expression of cell surface cell populations have been found in patients with
molecules such as CD48 and CD70 which are various cancers. Several recent studies have
ligands for NK cell-activating receptors 2B4 and found correlations between particular immune
CD27, respectively [9]. The DCs that have been cell inltrates in tumors and patient prognosis.
recruited to the tumor site become activated either Inltration of CD8+ T cells and mature DC is
by exposure to the cytokine milieu created during associated with a favorable prognosis in patients
the ongoing attack by the innate immune system with cancer. However, an extensive macrophage
or by interacting with NK cells. The activated inltration correlates with poor patient prognosis
DC can acquire tumor antigens directly by inges- in most of the cancers analyzed. Thus, the com-
tion of tumor cell debris or potentially through plexity of the immune cell populations inltrat-
indirect mechanisms involving transfer of tumor ing tumors with their synergistic or opposing
cell-derived heat shock protein/tumor antigen effects may inuence tumor growth differently,
complexes [10]. The activated antigen-bearing depending on their cytokine secretion. A number
DCs then migrate to the draining lymph nodes, of immune-enhancing cytokines have been
where they trigger the activation of tumor antigen- shown to promote or inhibit antitumor immunity
specic CD4+ Th1 cells. In addition, DCs activate in multiple experimental models and in patients
CD8+ cytotoxic T lymphocytes (CTL) via cross- with cancer. This chapter reviews the role of anti-
presentation of tumor antigenic peptides on MHC tumor cytokines IL-12 and IL-27 in tumor immu-
class I molecules [11]. Activated tumor-specic nity and immunotherapy while discussing the
CD4+ and CD8+ T cells home to the tumor site role of pro-tumor cytokines (TGF-, IL-17,
where they kill tumor cells. Mice lacking adaptive IL-23, IL-35, and IL-10) that have pathogenic
immunity (RAG-2 gene-decient mice lacking signicance in cancer progression.
T cells) were more susceptible to carcinogen-
induced and spontaneous primary tumor forma-
tion. Thus, the development of adaptive immunity 7.2 Cytokine Regulation
may provide the host with the capacity to com- of the Antitumor Immune
pletely eliminate the developing tumor. However, Response
the development of clinically evident cancers
indicates that these innate and adaptive immune Cytokines comprise a large family of intracellu-
responses are not always enough to prevent dis- lar communicating molecules that play important
ease progression as cancer cells manage to escape roles in immunity, inammation, and repair, as
host-tumor immunity. well as general tissue homeostasis. In addition,
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 95

cytokines functions extend to many other aspects boosts antitumor immunity by contributing to
of biology, including cancer [13]. In the tumor the development of NK cells and CTLs without
microenvironment, cytokines are produced by any toxic side effects.
host stromal and immune cells, in response to
molecules secreted by the cancer cells. In addi- IL-12: Linking Innate
tion, cancer cells also produce cytokines in the and Adaptive Antitumor
same environment. Increased levels of circulating Immunity
cytokines and their receptors have been found in IL-12 plays an essential role in the interaction
patients with various types of cancer, both at between the innate and adaptive arms of antitu-
diagnosis of the primary disease and in those mor immunity [17] (Fig. 7.1). It induces IFN-
with metastases, compared with healthy people production by NK cells and T cells. In fact, NK
[14, 15]. The cytokine repertoire present at the cells and T cells were rst shown to express high-
tumor site determines the type of host response afnity receptors for IL-12 [18]. Tumor eradica-
directed against the tumor. Immunosuppressive tion after vaccinations supported by IL-12 is
cytokines secreted by tumor cells or tumor- dependent on NK cells in several animal models
inltrating immune cells can impair the host anti- [1921]. IL-12 enhances in vitro lysis of both NK
tumor response, whereas cytokines promoting cell-sensitive and NK cell-resistant tumor cells.
the development of T-cell-mediated immunity Consistent with animal studies, in patients with
can induce or enhance antitumor immunity. cancer, IL-12 enhances the cytolytic activity of
Studies of cytokine-decient mice have revealed NK cells and increases the expression of CD2,
dual role for the immune system in suppressing LFA-1, and CD56 molecules which mediate NK
and promoting tumor growth. cell migration [22]. Moreover, IL-12 was shown
to enhance the cytotoxicity mediated by NK cells
from healthy donors against cancer cells derived
7.2.1 IL-12 from patients with cancer.
In addition to its effect on NK cell cytotoxic- Overview ity, IL-12 enhances T-cell-mediated cytotoxicity
IL-12 is a heterodimeric cytokine containing a and has an enhancing effect on CD8+ T cells [23].
35 kD and a 40 kD subunit that signals through DCs play a crucial role in facilitating the interac-
a receptor of the type I family of cytokine recep- tion between CD4+ T cells and antigen-specic
tors. The principal sources of IL-12 are APCs CD8+ T cells. Priming of CTL is enabled by the
such as DCs and macrophages. Secretion of ligation of CD40 on DC and its ligand CD154 on
IL-12 is generally activated via the physiologi- activated CD4+ T cells [24, 25]. The induction of
cal stimuli of CD40 and toll-like receptors IL-12 synthesis that occurs as a result of CD40
which recognize structurally conserved mole- ligation suggests an important role for IL-12 in
cules derived from microbes [16]. IL-12 plays a the molecular mechanisms responsible for CTL
major role in the development of antitumor priming [26]. It was then shown that IL-12, in the
immune responses [17]. Numerous studies presence of antigen, acts directly on naive CD8+
report that IL-12 promotes an effective destruc- T cells to promote clonal expansion and differen-
tion of cancer cells through the induction of the tiation [27]. In addition, priming of CD8+ T cells
innate and adaptive arms of antitumor immu- in the absence of IL-12 rendered them unrespon-
nity. In addition, IL-12 has potent antiangio- sive to the same antigen [28]. Agonistic CD40
genic activity. Due to these features, IL-12 has antibodies (Abs) were shown to substitute the
been used as a systemic cancer therapeutic function of CD4+ T cells in murine models of
agent, but the clinical development of IL-12 has T-cell-mediated immunity, resulting in rapid
been hindered by its signicant toxicity and dis- expansion of CTLs that cleared established lym-
appointing antitumor effects seen in cancer phomas and provided long-term protection
patients. However, emerging studies suggest against tumor rechallenge [29, 30]. These obser-
that IL-12 in combination with other cytokines vations provided an explanation for the impaired
96 M. Gopal

Innate immunity


Tumor Macrophage

Tumor Ag
in NK
s IL-12 IL-12

Endothelial cells

Perforin CTL
IFN- IL-12


B cell IL-2,IFN-

Th1 CD4+T


Adaptive immunity Antibodies Plasma cell

Fig. 7.1 IL-12 links innate and adaptive antitumor immu- addition, IL-12 has a direct toxic effect on the some tumor
nity. IL-12 utilizes several mechanisms to induce antitu- cells. Furthermore, IL-12 secretion by DCs can induce
mor effects. IL-27 activates innate effectors, such as NK adaptive arms of antitumor immunity. IL-12 can augment
cells, NKT cells, and T cells and promotes their cyto- Th1 response necessary for cellular immune response.
lytic activity and cytokine production. IL-12 induces Il-12 stimulates the differentiation and lytic capacity of
IFN- production in macrophages that can have a cyto- CTL and promotes immune memory. Finally, IL-12 can
toxic effect on tumor cells. IL-12 induces the production mediate antibody-mediated tumor clearance via B-cell
of antiangiogenic molecules from endothelial cells. In activation

tumor antigen-specic CTL activation in CD40- ducted by different groups, indicated that the
decient mice and conrmed the key role of the injection of IL-12 directly into subcutaneous
CD40-IL-12 pathway in the regulation of antitu- tumors results in CTL response against the tumor
mor immunity. A series of experiments, con- in mice [3133].
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 97

The rejection of tumors requires CD8+ T cells Favorable antitumor responses were related to the
whose activation and maintenance depends on synthesis of Abs against tumor antigens inducing
CD4+ T cells. Upon stimulation, nave CD4+ T tumor cell lysis in a complement-dependent cyto-
cells differentiate into different lineages of T toxicity assay [42].
helper subsets including Th1, Th2, Th17, and The ability of IL-12 to facilitate cell-mediated
Tregs. These distinct CD4+ T-cell subsets have immune responses, including enhancement of
varied impact on tumor growth. While Th1 cells NK cytotoxicity, generation of CTL, and DC
promote CD8+ T-cell-mediated immunity to activation, suggests its role in both the innate and
tumors, the other CD4+ T-cell subsets Th2 and adaptive immunity resistance mechanisms
Tregs negatively regulate CD8+ T-cell function. against tumors. Experimental studies of systemic
In the presence of IL-12, nave CD4+ T cells dif- administration of the cytokine have indicated that
ferentiate into IFN--secreting Th1 cells [34]. IL-12 exerts potent antitumor activity against a
Th1 cytokines, IL-2, and IFN-, stimulate the variety of metastatic tumors and can even prevent
cytolytic activity of NK cells. High production spontaneous tumor development in HER-2/neu
of IFN- by CD8+ T cells and a Th2 to Th1 shift transgenic mice. In addition, models based on
in the cytokine secretion prole of CD4+ T cells intra-tumor cytokine delivery or in vivo transfer
were also seen in the IL-12-treated mice [35]. By of cytokine-secreting tumors have indicated that
altering the balance between Th1 and Th2 cyto- IL-12 has signicant dose-dependent antitumor
kines, IL-12 plays a critically important role in activity against a wide spectrum of murine tumors
antitumor immune responses. A shift from Th1 including melanoma and breast, ovarian, and
to Th2 cytokine production has been reported bladder tumors [17, 43, 44]. All these studies
in progressive cancer patients, and a vaccine have demonstrated that IL-12 can inhibit tumor
inducing Th2 to Th1 shift in a murine model of growth and improve the survival of tumor-bearing
tumor was shown to induce tumor rejection [36]. animals that are dependent on not only its ability
In addition, Th2 cytokines have been shown to to activate the innate and adaptive arms of antitu-
accelerate tumor growth in multiple experimental mor immunity but also through its antiangiogenic
models [37]. In fact, CD4+ T cells can directly activity.
interact with CD8+ T cells via CD40-CD154
interactions [38], which directly contrast with IL-12 and Angiogenesis
the early notion that CD4+ and CD8+ T cells are Inhibition
brought together on the same antigen-presenting Accumulating evidence indicates that the antitu-
cell for the effective delivery of IL-2 to neighbor- mor effects of IL-12 are mediated, at least in part,
ing CD8+ T cells. Moreover, a full CD8+ T-cell through mechanisms involving angiogenesis and
response is elicited by a temporal release of IL-2 its direct effects on tumors. Angiogenesis is an
from CD4+ T cells, which is consistent with the essential process for tumor growth and metasta-
ndings that neutralization of IL-2 signicantly ses. In addition, it is the result of a complex bal-
limits CD8+ T-cell growth [39]. IL-12 also plays ance between angiogenic and antiangiogenic
an important role in the establishment of mem- factors. The balance between angiogenic and
ory CD8+ T cells [40]. A strong specic CTL angiostatic molecules in the tumor microenviron-
response was observed in patients with advanced ment can determine tumor growth and survival.
melanoma after administration of IL-12. The The antiangiogenic properties of IL-12 were rst
number of tumor-specic CTL increased in the observed by Voest et al. who demonstrated that
circulation, and inux of specic memory CD8+ IL-12 treatment almost completely inhibited neo-
T cells into metastasized lesions was docu- vascularization in immunocompetent mice,
mented [41]. Additionally, IL-12 was shown to severe combined immunodecient mice, and
stimulate humoral immunity. In a model of colon T-cell-decient nude mice [45]. However, sup-
carcinoma, vaccination with IL-12-transduced pression of angiogenesis by IL-12 was dependent
tumor cells cured 40 % of tumor-bearing mice. on its ability to induce IFN- expression.
98 M. Gopal

Accordingly, administration of IFN- reproduced age, and NK cell inltration surrounding small
the antiangiogenic effects promoted by IL-12. vessels [54]. These results documented that
Moreover, it was shown that inhibition of tumor NK cell cytotoxicity of endothelial cells is a
growth by IL-12 or IFN- required an intact sig- potential mechanism by which IL-12 can sup-
naling from IFN- receptors expressed in neo- press neovascularization. The antiangiogenic
plastic cells. This indicated that IL-12 could program activated in lymphocytes by IL-12 can
inhibit tumor growth by inducing neoplastic cells also directly affect gene expression in neoplastic
to produce antiangiogenic factors. Two of the cells. In fact, upregulation of signal transducers
most relevant factors were identied as the IFN-- and activators of transcription-1 (STAT-1) and
inducible genes, IFN-inducible protein 10 (IP- angiopoietin 2 together with down-modulation
10) and monokine induced by interferon- (MIG) of vascular endothelial growth factor (VEGF)
[46, 47]. Local and systemic treatment with IL-12 has been observed in neoplastic cells exposed
was associated with the expression of IFN-, to soluble factors released by IL-12-stimulated
IP-10, and MIG in the tumor; in addition, intra- lymphocytes [55]. In addition, IL-12 treat-
tumor delivery of MIG into subcutaneously ment reduced the production of metallopro-
growing tumor in nude mice led to tumor necro- teases, playing a role in matrix remodeling, a
sis associated with vascular damage. process required during neoangiogenesis [56].
Administration of neutralizing Abs to IP-10 and Moreover, the activation of integrin V3 on
MIG substantially reduced the antitumor effects endothelial cells is reduced by the IL-2-induced
of IL-12 [48]. IP-10 and MIG interact with their IFN-, which leads to decreased endothelial cell
receptor CXCR3 to mediate their angiostatic adhesion and survival [57]. IL-12-induced secre-
activity. These results support the notion that tion of IFN- leads to an increase in p53 activity,
these chemokines, both ligands of the receptor which subsequently results in tumor suppression
CXCR3, contribute to the antitumor effects of due to the induction of apoptosis in cancer cells
IL-12 through their inhibitory effect on tumor [58]. Furthermore, IL-12 dramatically decreased
vasculature. In addition to IFN- stimulation, tumor-supportive activities of tumor-associated
IL-12 promotes the expression of interferon regu- macrophages (TAMs), which are involved in
latory factors 1 (IRF-1) and 4 (IRF-4), which are tumor angiogenesis and metastasis. The anti-
necessary for Th1 cell differentiation [49]. IRF-1 angiogenic mechanisms mediated by IL-12 are
has tumor suppressor activities in cancer cells complex and dependent not only on the direct
in vitro and decreases the tumorigenicity of cells effect on endothelial cells of the proinamma-
inoculated into athymic nude mice [50, 51]. tory cytokine/chemokines induced by IL-12 but
Similarly, IRF-4 suppresses c-Myc-induced leu- also on the recruitment of immune effector cells
kemia in animal models and inhibits BCR/ABL- such as NK and T cells.
induced B-cell acute lymphoblastic leukemia
[52, 53]. Regulation of IL-12 in Tumor
Emerging evidence indicates the involve- Microenvironment
ment of lymphocyte-endothelial cell crosstalk Although controlled Th1 and CTL responses can
at the beginning of the process of angiogen- exert a signicant antitumor immunity, the same
esis inhibition by IL-12. It has been shown that responses, if exaggerated, may result in host-
neutralization of NK cell function reversed tissue destruction and autoimmunity. Therefore,
IL-12 inhibition of angiogenesis in athymic as a part of immune homeostasis, the inamma-
nude mice. Immunohistochemistry analysis tory responses need to be counter-regulated.
revealed that neovascularization inhibited by Tregs play a major role in controlling unwanted
IL-12 displayed accumulation of NK cells and immune response to self-antigens [59]. Studies
IP-10-positive cells. In addition, experimental have revealed a signicant role for Tregs in defec-
Burkitt lymphomas treated locally with IL-12 tive immune responses to tumor antigens. Treg
displayed tumor tissue necrosis, vascular dam- functions are mediated in part through secre-
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 99

tion of immunosuppressive cytokines IL-10 and only one partial response (renal cell carcinoma)
TGF-. Both TGF- and IL-10 can inhibit DC and one transient complete response (mela-
antigen presentation, IL-12 secretion, and effec- noma), among the 40 enrolled patients. However,
tor functions of both CD4+ and CD8+ T cells common signs and symptoms of toxicity such as
[12]. Thus, it is possible that as an immunosup- fever/chills, nausea, vomiting, fatigue, and head-
pressive environment develops in the growing ache were observed [64]. Administration of IL-12
tumor, DCs secreting IL-12 become scarce. This resulted in stabilization of the disease in several
might be due to an absence of DC activation sig- renal cancer patients and partial regression of a
nals, CD40, or inhibition of activated CD4+ T metastatic lesion, but has not proceeded further
cells which could themselves activate in clinical development due to signs and symp-
DC. Moreover, the CD40-CD40L interaction toms of toxicity, including fever, vomiting, and
between DCs and T cells leads to the induction elevation of hepatic enzymes [65]. Clinical trials
of not only IL-12 but also IL-10, a pro-tumor of IL-12 treatment in combination with rituximab
cytokine that may act in an autocrine or a para- in patients with B-cell non-Hodgkin lymphoma
crine manner to downregulate IL-12 secretion (NHL) did not result in clinical response [66].
from DCs [60]. Indeed, reduced CD40 expres- However, several clinical studies revealed posi-
sion on DC or CD40-L on T cells from tumor- tive results with IL-12 administration. During
bearing hosts may explain the reason for reduced IL-12 treatment in patients with NHL, 21 % of
levels of IL-12 observed in patients with cancer the patients had a partial or complete response
[61]. In accordance with this, reduced expres- without major side effects [67]. Similarly, sub-
sion of IL-12 was observed in patients with cutaneous IL-12 treatment resulted in complete
advanced cancer types including glioblastoma, response in 56 % of the treated patients with T-cell
renal cell carcinoma, head and neck squamous lymphoma with minor toxicity [68]. Furthermore,
cell carcinoma, gastric cancer, melanoma, clinical trials on metastatic melanoma revealed
colorectal cancer, hepatocellular carcinoma, and that IL-12 administration induces tumor shrink-
gastric cancer [15]. Moreover, IL-12 production age in patients accompanied with increased fre-
by stimulated peripheral blood mononuclear quency of circulating antitumor CTLs [41]. The
cells decreased signicantly in patients with gas- low efcacy of IL-12 in the abovementioned
tric and colorectal cancer with advanced disease. clinical trials may be due to an immunosuppres-
In addition to the immunosuppressive cytokines sive microenvironment in advanced tumors. In
TGF- and IL-10, other factors present in the addition, IL-12 may self-limit its own therapeutic
tumor microenvironment can downregulate efcacy by inducing IL-10 and other suppressive
IL-12 production, for example, prostaglandin E2 factors. For example, IFN- induced by IL-12
(PGE2) produced by tumor cells or tumor- can activate immunoregulatory molecules such as
associated host cells (macrophages, endothelial programmed death ligand-1 (PD-L1) and indole-
cells, and stromal cells) known to inhibit IL-12 amine 2,3 dioxygenase (IDO) on a variety of cells
production [62]. (DC, T cells, and endothelial cells) [69]. Both
PD-L1 and IDO can abrogate antitumor immu- Clinical Studies with IL-12 nity through various mechanisms. Furthermore,
Based on the provocative preclinical studies, other factors such as environment and diet may
IL-12 was evaluated in patients with differ- alter the effectiveness of IL-12-mediated antican-
ent malignancies. However, clinical experience cer immunity. Although systemic administration
with IL-12 in humans is limited. Several phase of IL-12 in patients is limited by its signicant
I clinical trials of IL-12 in patients with solid toxicity, emerging studies in animal models indi-
tumors and hematological malignancies have cate that IL-12 in combination with other cyto-
been reported [63]. IL-12 administration in kines boosts antitumor immunity without any
patients with advanced colorectal cancer (CRC), toxic side effects [44]. Thus, selective targeted
melanoma, and renal cell carcinoma resulted in delivery of IL-12 to tumors and/or reducing the
100 M. Gopal

dose of IL-12 while combining it with other ther- with these observations, IL-27R/ mice failed
apeutics may yield better outcome. to regulate tumor growth in vivo, reiterating the
importance of IL-27 signaling in the genera-
tion of antitumor immunity [82]. Most recently,
7.2.2 IL-27 DC-derived IL-27 has been shown to induce
NK and NKT cell-dependent antitumor immu- Overview nity against methylcholanthrene-induced bro-
IL-27 is a member of the IL-12 family cytokine sarcoma and transplanted B16 melanoma [83].
that plays potent antitumor effects against vari- Moreover, IL-27 in combination with other cyto-
ous tumor models via different mechanisms, kines such as IL-2 and IL-12 boosts antitumor
depending on the characteristics of each tumor immunity by contributing to the development of
[70]. Unlike IL-12, IL-27-mediated antitumor CTLs and NK cells [84].
functions are independent of IFN-, and IL-27- In addition to the direct effect of IL-27 on
treated mice do not manifest any toxic side CD8+ T-cell activation, the inuence of IL-27 on
effects. IL-27 is mainly produced by activated CD4+ T-cell responses might provide further
APCs including DCs and macrophages. DCs therapeutic opportunities. Initial studies have
secrete IL-27 on exposure to physiological indicated that IL-27 leads to the differentiation of
stimuli such as type I and type II interferons Th1 cells [85]. IL-27 synergizes with IL-12 to
(INF) and CD40 [7173]. In addition, IL-27 enhance IFN- production [86]. Moreover, it has
expression is induced in APCs upon stimula- been shown that IL-27 inhibits Th2 polarization
tion by various TLR ligands such as poly(I:C), of nave CD4+ T cells and suppresses Th2 cyto-
lipopolysaccharide (LPS), and CpG-DNA, kine production from in vitro polarized Th2 cells
which are agonists of toll-like receptors [8789]. By altering the balance between Th1
(TLR)3, TLR4, and TLR9, respectively and Th2 cytokines, IL-27 plays a critically impor-
[7476]. tant role in antitumor immune responses. In line
with this, a recent study conrmed IL-27s capa- IL-27 in Antitumor Immunity bility in the reversion of the Th2 polarization of
IL-27 has a wide array of functions necessary in vivo primed lymphocytes from pancreatic can-
for the induction of antitumor immune response. cer patients [90]. IL-27-dependent enhancement
IL-27 has been shown to act on NK cells to of preexisting antigen-specic Th1 responses has
enhance their cytotoxic activity both in vitro also been demonstrated [36]. IL-27 may promote
and in vivo; in addition, therapeutic administra- tumor regression through the inhibition of Tregs.
tion of IL-27 increased NK cell susceptibility of IL-27 inhibits the generation of Foxp3+ Tregs
tumors [77]. By activating NK cells, IL-27 might both in vitro and in vivo. IL-27 blocks Treg dif-
enhance adaptive immunity to tumors in part; the ferentiation through a mechanism that is at least
killing of tumor target by NK cells could provide partially dependent on STAT-3 [91, 92]. In addi-
DCs with increased access to tumor antigens; tion, IL-27 can limit Treg cell expansion by
thus, IL-27 serves as a link between innate and inhibiting IL-2, a cytokine necessary for Treg
adaptive antitumor immunity. In addition to NK development [93]. In a murine model of neuro-
cell activation, IL-27 acts on CD8+ T cells and blastoma, IL-27 has been shown to inhibit IL-2-
induces the generation of CTL through enhanc- induced Treg expansion in the tumor, promoting
ing the expression of effector molecules such as antitumor immune responses [84]. IL-27 also
granzyme B and perforin [78]. Similar to mice, induces tumor-specic Ab response which coop-
IL-27 promotes IFN- and granzyme B produc- eratively elicits ADCC activity [94].
tion from human CD8+ T cells [79]. The over-
expression of IL-27 in highly immunogenic Direct Effect of IL-27 on Tumors
murine tumor cells facilitated CTL development IL-27 possesses multiple antitumor effects medi-
with enhanced IFN- production [80, 81]. In line ated by mechanisms involving angiogenesis
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 101

and its direct effects on tumors. IL-27 has been ne balance with other myeloid cell populations
shown to have antiproliferative activities which especially tumor-inltrating macrophages and
inhibit tumor growth and metastasis in murine DCs expressing OPN and IL-27.
melanoma [95]. The major antitumor role of
IL-27 relies on its antiangiogenic property of sur- Advantages of IL-27 Over IL-12
rounding endothelial cells and broblasts. IL-27 in Tumor Immunity
signicantly inhibited tumor growth in SCID IL-27-mediated antitumor mechanisms are com-
mice through the induction of antiangiogenic plex. Similar to IL-12, IL-27 utilizes effector
factors such as IP-10 and MIG from endothelial mechanisms of innate and adaptive immunity
cells [96]. Consistent with these results, IL-27 to mediate antitumor immunity. IL-27 promotes
has been shown to directly act on human umbili- tumor immunity through the induction of Th1
cal cord endothelial cells and induce production and CTL responses while inhibiting immu-
of the antiangiogenic chemokines such as IP-10 nosuppressive Th2 and Tregs. Unlike IL-12,
and MIG [97]. IL-27 strongly inhibited tumor IL-27-mediated antiangiogenic functions are
growth of primary multiple myeloma (MM) independent of IFN-. Thus, IL-27-treated mice
cells through inhibition of angiogenesis [98]. In are not observed with any toxic side effects [104].
addition, IL-27 downregulated a wide panel of The central role of IL-27 in orchestrating both the
proangiogenic genes, including matrix metallo- innate and adaptive arms of immunity together
proteinase-9 (MMP-9), TGF-, and VEGF with a with multiple antiangiogenic functions explains
concomitant upregulation of the angiostatic che- the essential contribution of this molecule to
mokines IP-10 and MIG. the development of antitumor immunity against
IL-27 may further promote tumor regression both high and poor immunogenic tumors. These
through the inhibition of a proangiogenic cyto- observations together with the lack of toxicity
kine IL-17. IL-27 suppresses the Th17 key tran- observed in vivo in preclinical trials with IL-27
scription factor RORt and thus inhibits treatment highlight the enormous therapeutic
expression of IL-17 by T cells both in humans potential of this approach.
and mice [99, 100]. Accordingly, mice decient
in either the IL-27 EBI3 subunit or IL-27R have
increased levels of IL-17 [101]. Among the Th17 7.3 Cytokines in Immune
suppressive molecules found in the tumor micro- Tolerance to Cancer
environment, IL-27 is one of the most potent
inhibitors of Th17 differentiation. IL-27 can be Although certain cytokines produced in the tumor
induced in tumor-inltrating DCs by galactin-1, microenvironment can function to inhibit tumor
IFN-, and apoptotic tumor cells in the tumor growth, others can promote tumor growth and
microenvironment [71, 102, 103]. However, the progression. Several cytokines were found to
proangiogenic molecules which dominate the serve as growth and survival factors that act on
microenvironment in advanced tumors can limit premalignant cells, stimulate angiogenesis and
the availability of IL-27. Osteopontin (OPN), a metastasis, and maintain tumor-promoting
proinammatory cytokine, inhibits the expres- immunosuppression and inammation.
sion of IL-27 in DCs while inducing Th17 dif-
ferentiation [72]. OPN promotes tumor growth
through mechanisms involving angiogenesis, 7.3.1 TGF-
tumor migration, and metastasis, suggesting that
OPN may release the brake on Th17 cell Overview
responses by suppressing IL-27 in DCs. Both Transforming growth factor- (TGF-) is a pleio-
OPN and IL-27 are expressed in DCs and macro- tropic cytokine with broad tissue distribution that
phages; thus, the outcome of Th17 accumulation plays critical roles during embryonic develop-
in tumor microenvironment may depend on the ment, normal tissue homeostasis, and cancer [105].
102 M. Gopal

Fig. 7.2 TGF--mediated Innate immunity

immunosuppression. TGF-
affects components of both
innate and adaptive immune

DC maturation
systems. TGF- inhibits NK
cell activation and its effector
function. In addition, TGF- Th1
Ma ti on
inhibits DC maturation and cro iva
pha ct
antigen-presenting function ge e ll a
while promoting polarization pol
ariz Tc Th2
atio 4+
of M2 macrophages. TGF- n CD
inhibits CD8+ T-cell-
mediated antitumor immune Treg
response. TGF- also has a
signicant impact on CD4+
T-cell differentiation and CD Th17
on 8+
function. TGF- induces vati Tc
cti ell
ll a

B cell activation
Treg and Th17 differentiation ac
ce tiv
while inhibiting Th1 and Th2 NK on
differentiation. Furthermore,
TGF- inhibits B-cell
proliferation and antibody

Adaptive immunity

Elevated TGF- serum concentrations were TGF- growth inhibitory signals which is an
observed in patients with different malignancies important reason for the shift from being a tumor
and were associated with poor prognosis. TGF- suppressor to a tumor promoter. Subsequently,
is released not only by a variety of cells in human cancer cells start secreting nonphysiological lev-
and murine tumors including T cells, macro- els of TGF- in an autocrine and paracrine man-
phages, and DCs but also by tumor cells them- ner which may affect the differentiation of the
selves [106]. Almost all human cell types are tumor cells and the surrounding cellular environ-
responsive to TGF-, which signals through type ment, respectively, leading to development of the
I and type II TGF- receptors (TRI and TRII, tumor and metastasis [108]. Notably, TGF-
respectively). Upon binding of TGF- to TRII, induces epithelial-mesenchymal transition
TRI is recruited and activated to phosphorylate (EMT), whereby epithelial tumor cells acquire an
the downstream mediators, SMAD2 and SMAD3. invasive, mesenchymal-like phenotype accompa-
Phosphorylated SMAD2 and SMAD3 combine nied by changes in the expression of cell-cell
with SMAD4 to enter the nucleus to modulate adhesion molecules and secretion of metallopro-
gene transcription [107]. teinases, leading to metastasis [109, 110]. The
The function of TGF- in cancer is complex. potent regulatory activity of TGF- on immune
TGF- can act as a tumor suppressor or a tumor cell functions represents an important mechanism
promoter depending on the stages of tumor devel- of immune tolerance to tumors. The presence of
opment. Initially, it acts as a tumor suppressor TGF- in the microenvironment of the develop-
since it induces apoptosis and inhibits the growth ing tumor is predicted to disable effective immu-
of normal and premalignant tumor cells [108]. At nosurveillance by multiple mechanisms, most of
later stages of tumor progression, TGF- acts as which converge on the impairment of tumor cell
a tumor promoter. At this stage, cancer cells pro- killing by innate and adaptive immune cells
tect themselves and tend to acquire resistance to (Fig. 7.2).
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 103 Effect of TGF- on Innate Macrophages, the predominant leukocyte,

Immunity to Tumors play a key role in tumor growth. The role of
TGF- is an important regulator of NK cell tumor-associated macrophages (TAMs) in
function, being a potent antagonist of IL-12- tumors is controversial [123]. TAMs origi-
induced production of IFN- in NK cells [111]. nate from recruited myeloid cells, such as
In addition, TGF- inhibits the activity of NK blood monocytes or MDSCs by a number of
cells by limiting expression of the activating chemoattractants produced by tumor cells
receptor NKG2D, NKp30, and DNAM-1 [112]. and stromal cells. Tumor-derived chemokine
In fact, reduced expression of NKG2D is associ- CCL2 is critical for the recruitment of macro-
ated with elevated levels of TGF- in patients phages to the tumor site [124]. Macrophages
with cancer. It has been shown that surface- can exert different properties when polarized
bound TGF- on MDSCs can inhibit NK cell with distinct inducers. Differential cytokine
cytolytic activity against mammary adenocarci- production is a key feature of polarized mac-
noma [113, 114]. Moreover, TGF- has been rophages. When stimulated with IFN-, M1
shown to suppress MHC class I and MHC class macrophages secrete high levels of IL-12, but
II expression in a number of cell populations low levels of IL-10. In contrast, M2 macro-
[115117]. Importantly, the TGF--dependent phages express high levels of IL-10 but low
decrease of MHC class I expression in tumor levels of IL-12 [125]. Due to their different
cells has been shown to result in reduced tumor cytokine proles, these polarized macrophages
cell lysis by NK cells [117]. Although NK cells have distinct functions. For example, the IL-12
are the major innate effectors, they require acti- produced by M1 macrophages can promote the
vation by DCs. TGF- has been demonstrated to differentiation of Th1 cells, which can improve
impair DC function both in vitro and in vivo. antigen phagocytosis and contribute to antitu-
TGF- inhibits upregulation of critical co-stim- mor immunity. Whereas, the IL-10 expressed
ulatory molecules on the surface of DCs and by M2 macrophages can promote the produc-
reduces cytokine production and their antigen- tion of IL-4 and IL-13 by Th2 cells. Both IL-4
presenting capacity [118, 119]. TGF- can and IL-13 have been shown to impair antitu-
immobilize DCs, thereby interfering with their mor T-cell responses. TGF- promotes tumor-
migration and the transport of antigen to drain- associated macrophage polarization to an M2
ing lymph nodes for presentation to T cells. vs. M1 phenotype, which further promotes
Moreover, TGF- can also induce apoptosis of TGF- production and deepens immunosup-
DCs [120]. In recent years, more correlative pression [126]. In most tumors, the inltrated
clinical data has supported the inhibitory role of macrophages are considered to be of the M2
TGF- in the observed defects in cancer. phenotype. TAMs orchestrate various aspects
Increased serum TGF- in human colorectal of cancer, such as tumor progression, angio-
cancer correlates with reduced circulating DCs genesis, metastasis, and immunosuppression
[121]. Moreover, tumor-inltrating DCs both [127]. It has been shown that NKT cells can
secrete and respond to TGF-, in either an auto- suppress CTL responses through mechanisms
crine or paracrine manner. These TGF-- involving TGF-. Therefore, blockade of
secreting DCs promote the formation of Tregs TGF- signaling not only enhances the fre-
[122] that potently inhibit the function of other quency of antitumor CTLs but also restores the
T cells, and that Treg production of TGF- can activities of the cytolytic machinery and pre-
inhibit NKG2D-mediated NK cell cytotoxicity, vents NKT cell-mediated immunosuppression
thereby enhancing tumor growth and metastasis. [128]. Furthermore, TGF- also inhibits effec-
In addition to DCs, TGF- can suppress or alter tor functions of other innate immune cells such
the activation and function of other innate as -T cells [106]. Thus a dampened innate
immune cells such as NKT cells, macrophages, immune response leads to poor adaptive immu-
and neutrophils [106]. nity, resulting in persistence of the tumor.
104 M. Gopal Effect of TGF- on Adaptive limit antitumor resistance. In contrast, depletion

Immunity to Tumors of Tregs with anti-CD25 Ab in animal models
The presence of TGF- in the tumor microenvi- enhances antitumor immunity and tumor regres-
ronment can have a profound impact upon antitu- sion, further suggesting the involvement of Tregs
mor activity of T cells. It has been shown that in tumor growth. Furthermore, when tumor-spe-
TGF- can suppress CTL differentiation and cic CD8+ T cells were adoptively transferred
CTL-mediated lysis of tumor cells [129, 130]. with either Tregs or CD4+CD25 T cells into
TGF- acts on CTLs to specically repress the hosts with tumor, CD8+ T-cell-mediated immu-
expression of different cytolytic effector mole- nity was abolished in those receiving Tregs but
cules such as perforin, granzyme A, granzyme B, not CD4+CD25 T cells [137, 138]. Collectively,
Fas ligand (FasL), and IFN-, which are collec- these studies provide strong evidence that Tregs
tively responsible for CTL-mediated tumor kill- can attenuate the antitumor immunity by down-
ing [131]. Blockade of TGF- in tumor models regulating the antitumor immune responses and
has been shown to reduce tumor burden by ultimately facilitating the development of cancer.
improving CD8+ T-cell-mediated tumor immu-
nity [131]. Furthermore, TGF- could suppress TGF-, Treg, and Tumor
IL-2 production and IL-2-induced T-cell differ- Angiogenesis
entiation [132]. Tumor cells transfected with Angiogenesis and tumor-associated immuno-
TGF- were shown to attenuate the efcacy of suppression are hallmarks of tumorigenesis. The
DC-based tumor vaccines [118]. In addition, association between angiogenesis and immunosup-
TGF- functionally regulates the differentiation pression is related to hypoxia which induces both
of T helper cell subpopulations both in vitro and angiogenesis and immunosuppression via activa-
in vivo. TGF- inhibits Th1 and Th2 cells, tion of hypoxia-induced factor 1 (HIF-1). The
whereas it promotes Treg and Th17 cell differen- induction of VEGF during hypoxia is primarily
tiation [133]. Most recently, TGF- has also been mediated by HIF-1. HIF-1-induced VEGF pro-
shown to play an important role in the develop- motes angiogenesis by inducing the recruitment
ment of IL-9-secreting Th9 cells [134]. of various proangiogenic bone marrow-derived
Although there are many sources of TGF- in cells including endothelial progenitors and
the tumor microenvironment, it has been shown myeloid cells [139]. Although hypoxia-VEGF
that Tregs can provide a signicant source of axis has been thought to be solely involved in
TGF- responsible for attenuation of tumor anti- vascular growth and permeability, recent stud-
gen expanded CTLs. Tregs hamper the functions ies suggest its role in immunosuppression in
of Th1, CD8+ T cells, NK cells, DCs, and other the tumor microenvironment. Within the tumor
key effector cells of antitumor immunity [106]. microenvironment multiple cell types with
Accordingly, Treg-mediated immunosuppression established roles in immunosuppression have
has been proposed to be one of the important been shown to promote angiogenesis. Among
mechanisms involved in tumor immune evasion. the immunosuppressive cell types found in the
An accumulation of Tregs in tumors can dampen tumor microenvironment, Tregs are consid-
T-cell immunity to tumors and is thus the main ered pivotal regulators of immunosuppression.
obstacle to successful immunotherapy [59]. The Tregs can trafc to tumors from the periphery
frequency of Tregs present in the peripheral blood under the inuence of chemokines in the tumor
of patients with various cancers is higher than microenvironment. It has been shown that tumor
that of normal population [135]. Notably, Tregs hypoxia leads to the recruitment of Tregs via
isolated from peripheral blood and solid tumors chemokine CCL28 [140]. Forced expression of
remain suppressive to T-cell activation in vitro CCL28 in mouse tumor cells resulted in accel-
[136]. Likewise, Tregs from tumor-bearing mice erated tumor growth and Treg accumulation
inhibited tumor rejection, indicating that Treg associated with increased VEGF levels and
cells suppress tumor-specic immunity and angiogenesis. In addition, Tregs were shown
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 105

to express CCR4, the receptor for CCL22, and improve tumor immunity. There are many TGF-
can therefore migrate to CCL22 present in the signaling antagonist agents under development at
tumor microenvironment [141, 142]. Beyond both the preclinical and clinical stages. Mice with
recruitment of Tregs through chemokines, the fully or partially disrupted TGF- function have
tumor microenvironment promotes the continued phenotypes with severe self-reactive immune
expansion of Tregs and the generation of Tregs responses [146, 147]. However, clinical trials of
due to a tumor microenvironment rich in TGF-. TGF- antagonists, such as a monoclonal Ab or
The recruited Tregs in turn dampen the antitumor small molecules that interfere with TGF- recep-
immune response and promote angiogenesis. The tor signaling, in cancer patients have been tested
accumulation of Tregs at tumors has been cor- and are ongoing. In phase I/II clinical trials, intra-
related with VEGF overexpression and increased tumoral administration of AP-12009, an anti-
angiogenesis in cancers, providing evidence for sense oligonucleotide to TGF-, resulted in a
an association between Tregs and angiogenesis signicant increase of survival time [148]. Some
[143, 144]. Tregs can contribute to tumor angio- clinical benet without apparent induction of
genesis through different mechanisms. They pro- autoimmune disease was found in a clinical trial
mote angiogenesis indirectly by suppressing Th1 of a human monoclonal anti-TGF- in melanoma
cells that release angiostatic cytokine IFN-, as patients [149, 150]. In addition, a vaccine that
well as interferon-induced chemokines such as contains allogeneic tumor cells that are modied
CXCL9 and CXCL10. Indeed, Tregs have been to express antisense TGF- has been tested in a
shown to promote tumor angiogenesis by spe- phase I/II clinical trial. Using this approach, a
cically inhibiting tumor-reactive T cells. Tregs response rate of 30 % has been reported in non-
can signicantly contribute to the direct promo- small cell lung carcinoma (NSCLC), with no
tion of tumor angiogenesis through the induc- serious toxicities observed [151]. LY2157299 is a
tion of VEGF and endothelial cell proliferation small molecule inhibitor which is selective for
[144]. Additional therapeutic opportunities may the kinase domain of the type 1 TGF- receptor.
be provided by Tregs capability in suppress- LY2157299 is currently being evaluated in
ing tumor-specic immunity while promoting patients with metastatic malignancies.
tumor angiogenesis by well-planned manipu-
lations of Tregs, including depletion, block-
ing trafcking into tumors, and reducing their 7.3.2 IL-17
differentiation and suppressive mechanisms. It
will be benecial to tumor eradication by com- Overview
bining this strategy with various current thera- IL-17 is a proinammatory cytokine produced by
peutic approaches. In an early phase I clinical Th17 cells [152]. In addition to Th17 cells, IL-17
trial in patients with metastatic breast cancer, can also be produced by cells other than T helper
the anti-CD25 Ab daclizumab signicantly cells, such as iNKT, CD8+ T, and -T cells [153
depleted Tregs and enhanced the immunogenic- 155]. Since Th17 cells produce large quantities of
ity of a cancer vaccine [145]. In addition, block- IL-17A, most Th17-mediated effects are attrib-
ing Treg function using Abs targeted against uted to this cytokine. Many factors are required
glucocorticoid-induced tumor necrosis factor for the induction and stabilization of Th17 cells.
receptor-related protein (GITR) and cytotoxic Of these, TGF- and IL-6 are the most crucial
T-lymphocyte antigen 4 (CTLA-4) is under cytokines for its differentiation. IL-6 induces
clinical evaluation in patients with cancer. production of IL-21, which subsequently favors
Th17 differentiation in an autocrine manner [156, TGF- in Clinical Trials 152]. These cells require CD40-induced IL-23 to
As a result of the wide variety of effects of TGF- maintain their Th17 phenotype in vivo. The dif-
on tumorigenesis, blockade of TGF- and its sig- ferentiation of Th17 cells into IL-17-secreting
naling pathway can be a potent approach to cells requires the expression of the transcription
106 M. Gopal

factor ROR-t [157]. It has been shown that Th17 T cells inhibits angiogenesis and induces major
cells are gradually increased in the tumor micro- histocompatibility complex I in tumor cells, thus
environment during tumor development. In addi- favoring immune recognition and subsequent
tion, Th17 cells have been found in patients with arrest of tumor growth [166]. In contrast, IL-17
different tumors. The frequent association of favors angiogenesis and tumor growth; therefore,
raised IL-17 concentrations with negative prog- replacing IFN- with IL-17 in the tumor micro-
nosis links the increased systemic IL-17 con- environment may have severe consequences for
centrations with the later stages of cancer. Many immune recognition and surveillance.
factors released by the tumor cells and molecules
secreted by tumor-inltrating immune cells such Tumor-Promoting Functions
as TGF-, IL-6, prostaglandin E2 (PGE2), IL-21, of IL-17
IL-23, osteopontin, IL-1, and TNF- can play Many functions of IL-17 in the tumor micro-
major roles in the induction of Th17 differentia- environment contribute to tumor progression,
tion [158161]. besides their minor direct effect on the prolifera-
tion and survival of tumor cells [167]. The major Th17 Differentiation in the Tumor pro-tumor role of IL-17 in cancer relies on its
Microenvironment proangiogenic property of the surrounding endo-
There are many sources for Th17 cells in the thelial cells and broblasts. For example, IL-17-
tumor microenvironment. Th17 cells found in the overexpressing human cervical cancer cells and
tumor microenvironment can either be migrated NSCLC cells show greater ability in developing
from the periphery or differentiated from nave tumors in immunocompromised mice compared
T cells under the inuence of tumor microen- with control cells with no IL-17 expression [168,
vironmental factors. Th17 cells can trafc to 169]. In addition, IL-17 overexpression in bro-
tumors from the periphery under the inuence of sarcoma cells enhances their tumorigenic growth
tumor microenvironmental chemokines such as in syngenic mice, primarily owing to the proan-
RANTES and monocyte chemotactic protein-1 giogenic activity of IL-17. Moreover, the levels
(MCP-1) [162]. In addition, high levels of che- of Th17 cells were positively correlated with
mokines CXCL12 and CCL20 have been found microvessel density in tumors [170]. By acting
in tumor microenvironments, which could facili- on stromal cells and broblasts, IL-17 induces a
tate Th17 cell trafcking and migration into the wide range of angiogenic mediators [171, 172]
tumor sites. Moreover, Th17 cells in the tumor including VEGF. In addition, they markedly
microenvironment might clonally expand follow- promote inammatory and tumor angiogenesis
ing stimulation by tumor-associated macrophages [173]. IL-17 is able to upregulate VEGF pro-
[163]. In addition, Th17 cells can be induced duction by broblasts and therefore promotes
and differentiate in the tumor microenvironment broblast-induced new vessel formation in the
[164]. It has become clear that IL-17 producing inammatory microenvironment and tumors. The
Th17 cells and Tregs share a common pathway. IL-17-VEGF loop that modulates angiogenesis
Although TGF- favors differentiation of nave T includes another angiogenic factor, TGF-. IL-17
cells into Tregs, the simultaneous presence of both induces VEGF, which in turn induces TGF- fol-
TGF- and IL-6 promotes the differentiation of lowed by VEGF-mediated angiogenesis [174].
Th17 cells. Given the tight association of TGF- TGF- enhances the VEGF receptivity of endo-
and IL-6 with tumor incidence and progression, thelial cells by increasing VEGF receptor expres-
nave T cells entering an established tumor are sion [175]. IL-17 also induces IL-6 and PGE2
more likely to be exposed to conditions favor- and enhances intercellular adhesion molecule
ing Th17 differentiation. Upon stimulation with (ICAM)-1 expression in broblasts. All these
TGF- and IL-6, CD8+ T cells not only lose their molecules were known to have a major role in
cytotoxic ability but are also induced to secrete angiogenesis and tumor invasion. IL-17 appears
IL-17 [165]. IFN- expressed by Th1 or CD8+ to stimulate production of IL-8 [176]. IL-8 sig-
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 107

naling promotes angiogenic responses in endo- of IL-17. Th17-polarized cells were found to be
thelial cells, increases proliferation and survival more effective than Th1 cells in eliminating large
of endothelial and cancer cells, and potentiates established tumors [182]. However, the Th17-
the migration of cancer cells and inltrating mediated tumor responses were highly dependent
neutrophils at the tumor site. Moreover, IL-17 on IFN--based mechanisms. Indeed, the effects
was found to induce IL-1 and TNF- in mac- of Th17-polarized cells were completely abro-
rophages and cytokines which can further syn- gated by the administration of IFN--depleting
ergize with IL-17 to activate neutrophil-specic Ab and not by IL-17- or IL-23-depleting Abs.
chemokine, thereby recruiting neutrophils to the Adoptively transferred IL-17-secreting CD8+ T
site of inammation [177]. cells also enhanced antitumor immunity resulting
One of the most important mechanisms under- in regression of B16 melanoma [183]. In addi-
lying IL-17 regulation of inammation which tion, IL-17 has been shown to inhibit the growth
orchestrates the tumor microenvironment is of hematopoietic tumors such as mastocytoma
through NF-B signaling, the master regulator of and plasmocytoma by enhancing CTL activity
the inammation [178]. IL-17R signaling results [184]. Different mechanisms have been proposed
in the activation of NF-B and regulates the activi- for the IL-17 enhancement of tumor-specic
ties of extracellular-regulated kinase 1 (ERK1), CTLs. IL-17 has been shown to induce IL-6 from
ERK2, c-Jun N-terminal kinase, and p38 mitogen- a variety of cells. Moreover, IL-17 stimulation
activated protein kinases [179, 180]. While the can induce IL-12 production from macrophages
IL-17-mediated cytokine expression is regulated [185]. Both IL-6 and IL-12 have been associated
primarily by NF-B, the same cytokines can fur- with induction of tumor-specic CTL. IL-17 pro-
ther stimulate NF-B-mediated transcription of motes maturation of DC progenitors as indicated
themselves in tumor cells and tumor-associated by increased expressions of co-stimulatory mol-
stromal cells, thereby creating a sustained chronic ecules, MHC-II antigens, and allostimulatory
inammatory state within the tumor microenvi- capacity [186]. This may lead to further improve-
ronment. In support of this notion, enhanced cervi- ment in T-cell priming by tumor cells producing
cal cancer growth elicited by IL-17 was associated IL-17. In addition, IL-17-transduced brosar-
with increased expression of IL-6 and macrophage coma cells induced tumor-specic antitumor
recruitment to the tumor sites [169]. Therefore, immunity by augmenting the expression of MHC
IL-17 might also function through IL-6 to promote class I and class II antigens [187]. These studies
tumor development. Chemokines can stimulate or were focused on the effects of exogenous IL-17 in
inhibit proliferation and chemotaxis of the blood established mouse tumor cell lines. A recent
vessel endothelial cells which serve the tumor. demonstration shows that tumor growth in subcu-
IL-17 has been shown to selectively enhance the taneous and lung tumor metastasis are enhanced
production of angiogenic chemokines such as in IL-17-decient mice [188]. The effect is
CXCL1, CXCL5, CXCL6, and CXCL8 from accompanied by reduced IFN- levels in tumor-
tumor cells and epithelial cells [168, 181]. In addi- inltrating NK cells and T cells.
tion, IL-17 is also known to inhibit angiostatic The evidence reviewed here demonstrates
chemokine secretion by broblasts [168]. Thus, that IL-17-secreting Th17 cells can either stim-
IL-17 may shift the local biologic balance between ulate or inhibit tumor growth and progression.
angiogenic and angiostatic chemokines toward a The benecial effects of IL-17 on upregulating
predominance of angiogenic chemokines in order host immune response may be present early in
to enhance the net angiogenic activity. inammation, but is eventually overcome by
increasingly large tumor burden. Clearly, as dis- Antitumor Functions of IL-17 cussed above, many of the inammatory func-
Although IL-17 seemed to be a potential tumor- tions of IL-17 can benet the tumor. The shift
promoting cytokine, a considerable number of from benecial inammatory functions of IL-17
reports have described tumor-inhibitory effects to a detrimental one may depend on the tumor
108 M. Gopal

type and inammatory mediators in the tumor contrast to the antitumor role of IL-12, IL-23
microenvironment. The pro-tumor vs. antitumor upregulates inammatory processes, including
effects of IL-17 are thus functions of a number of matrix metalloproteinase expression and angio-
combinations of all these IL-17-induced inam- genesis and reduces inltration and the function
matory mediators and, perhaps, mediators which of CTLs, thus contributing to tumor growth
counter-regulate IL-17 production as well, oper- [196]. Indeed, the mice lacking IL-23/p19 are
ating in tandem. completely resistant to carcinogen-induced
tumor. The absence of tumor formation in these
mice correlated with the absence of various
7.3.3 IL-23 markers including IL-17, GR-1+, and CD11b+
myeloid cells which are indicative of tumor- Overview associated inammation [196]. Recently, tumor-
IL-23 is a heterodimeric protein composed of two secreted lactic acid has been shown to activate
subunits IL-23p19 and IL-12p40. IL-23 is secreted the IL-23/Th17 pathway [159].
by activated DCs and macrophages. IL-23 binds In contrast, IL-23-overexpressing tumors
the IL-23R complex, composed of IL-23R and show reduced growth and metastasis [197201].
IL-12R1. Upon binding IL-23, IL-12R1, and The antitumor effects of IL-23 in these studies
IL-23R associate, marking the beginning of the were found to be mediated through enhancement
IL-23 signal-transduction cascade [189]. IL-23 of CD8+ T-cell response. In addition, intra-
plays an important role in bridging innate and tumoral injection of IL-23-overexpressing DCs
adaptive responses. Therefore, IL-23 has been resulted in a similar phenotype [201]. Articial
described as a key cytokine promoting inamma- overexpression of IL-23 could induce potent anti-
tion in peripheral tissues. The activity of IL-23 in tumor immunity through various mechanisms.
the regulation of tumor immunity is just begin- IL-23 can mediate myeloid inltration consisting
ning to be elucidated [190]. of DCs, macrophages, and granulocytes, which
instead may contribute to the inhibition of tumor Pro- vs. Antitumor Functions growth and boost an immune reaction to these
of IL-23 immune-sensitive tumors. In addition, overex-
Belonging to the IL-12 family, IL-23 performs pression of IL-23 is likely to increase the sys-
both pro-tumor and antitumor functions. IL-23 is temic IL-23 levels that could lead to the growth
spontaneously produced by TAM in several and survival of memory CD8+ T cells.
mouse tumor models. Tumor-secreted PGE2
enhances the production of IL-23 and IL-1 in
macrophages and DCs while downregulating 7.3.4 IL-35
IL-12 production [191193]. While IL-12 pro-
duction is decreased, IL-23 production is Overview
increased in tumors [194]. PGE2, together with IL-35 is a recently discovered IL-12 family cyto-
IL-23, favors the expansion of human Th17 cells kine composed of an IL-12 p35 subunit and an
from PBMCs; on the other hand, PGE2 enhances IL-12 p40-related protein subunit, EBI3 [202].
IL-17 production from memory CD4+ cells IL-35 is not constitutively expressed in tissues
induced by IL-23 [161]. The involvement of and is produced mainly by Tregs. IL-35 induces
IL-23 in the induction of Th17 was established the transformation of CD4+ effector T cells into
when investigators showed that IL-23 promotes Tregs, which in turn express IL-35 but lack the
the production of IL-17 by activated T cells expression of conventional Treg markers such as
[195]. Although IL-23 is not involved in the ini- Foxp3, TGF-, and IL-10 (Treg35 cells) [203].
tial differentiation of Th17 cells, it is crucial for The Treg35 cells generated in vitro can pre-
the function, survival, and propagation of this vent the development of autoimmunity in vari-
T-cell population in the inamed environment. In ous mouse models [204207]. Most recently, it
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 109

has been shown that human Tregs express and factor [214]. The function of IL-10 in cancer is
require IL-35 for maximal suppressive function. enigmatic. Depending on the experimental
Substantial upregulation of EBI3 and IL12A, model, IL-10 displays both immunosuppressive
but not IL10 and TGF-, was observed in acti- and immunostimulating activities. On the one
vated human Tregs compared with conventional hand, IL-10 promotes an antitumor CTL response
T cells [208]. leading to tumor regression. On the other hand,
IL-10 induces immunosuppression and assists in Pro-tumor Functions of IL-35 the escape from tumor immune surveillance,
Evidence on the role of IL-35 in tumor immunity hence promoting tumor growth.
is beginning to emerge. IL-35 subunit EBI3 is
expressed in Hodgkin lymphoma cells, acute IL-10-Mediated
myeloid leukemia cells, and lung cancer cells Immunosuppression in Cancer
[209211]. Small interfering RNA silencing of The cellular sources of IL-10 are Th2, Treg, Tr1,
EBI3 in lung cancer cells inhibits cancer cell pro- and Th17 cells; however, cytotoxic CD8+ T cells
liferation, whereas stable expression of EBI3 in can also produce IL-10, as can some subsets of
lung cancer cells confers growth-promoting DCs, macrophages, B cells, granulocytes, mast
activity in vitro [211]. High EBI3 expression in cells, keratinocytes, and epithelial cells. In addi-
human lung cancer cells was shown to be associ- tion, various cancer cells produce IL-10; among
ated with poor prognosis [211]. Recently, IL-35- those are multiple myeloma, melanoma, human
secreting Ag-specic Tregs have been observed colon carcinoma, lung cancer, oral squamous cell
in patients with prostate cancer [212]. Treg- carcinoma, renal cell carcinoma, non-Hodgkin
derived IL-35 was shown to inhibit antitumor lymphoma, and chronic lymphocytic leukemia
T-cell responses. In vitro generated Treg35 cells [15, 215]. Circulating concentrations of IL-10
accelerate the development of B16 melanoma were raised in patients with different cancer types
and prevent the generation of antitumor CD8+ and were associated with adverse disease stage or
T-cell responses [203]. In addition, T cells that with negative prognosis in those patients. It has
secrete IL-35 and have suppressive functions can been shown that serum levels of IL-10 increased
be induced in the tumor beds of melanoma and in parallel to clinical disease progression in
colorectal adenocarcinoma. Blockade of IL-35 patients with metastatic melanoma, as well as
has been shown to relieve suppression mediated colon cancer. Moreover, preoperative serum lev-
by Tregs [212]. Forced expression of IL-35 in els of IL-10 predicted the likelihood of colon
tumor cells leads to signicantly increased cancer recurrence [215, 216]. IL-10 can be
tumorigenesis in mice. IL-35 in the tumor micro- induced and sustained in the tumor microenvi-
environment signicantly increased the numbers ronment by a variety of cytokines. Macrophage-
of CD11b+Gr1+ myeloid cells in tumors and sub- derived IL-6 has been shown to induce production
sequently promoted tumor angiogenesis [213]. of IL-10 by cancer cells. Similarly, IL-6 in asso-
Furthermore, IL-35 renders tumor target cells ciation with TGF- can induce IL-10 production
more resistant to CTL destruction. in Th17 cells. However, TGF- alone can induce
IL-10, whereas IL-10 enhances the expression of
TGF- in a positive feedback circuit [217].
7.3.5 IL-10 TNF- promotes proinammatory reactions
while upregulating IL-10 in macrophages and Overview monocytes as a negative feedback, thereby termi-
IL-10 is an important immunoregulatory cyto- nating the inammatory response. In addition,
kine produced by many cell populations. Due to IL-12 and IL-27 can also induce IL-10 produc-
its ability in inhibiting the production of IL-2 and tion from T cells [99, 218].
IFN- by murine and human Th1 cells, IL-10 was IL-10 inhibits NKG2D ligand expression on
initially named a cytokine synthesis inhibitory tumor cells and suppresses cytotoxicity mediated
110 M. Gopal

Fig. 7.3 IL-10-mediated

tumor immunosuppression.
IL-10 can be induced in the
tumor microenvironment by
many cell types including
Th2 cells, Tr1 cells, Tregs,
DCs, TAM, and tumor cells. Th2
IL-10 has a multitude of
suppressive effects on the DC
antitumor immune response. Tr1 B
For example, IL-10 can
inhibit the maturation of DCs
and disrupt the differentia- CD8+
tion of CTLs and Th1 cells. Tr1
on IL-10 CTL activation
IL-10 can also inhibit the
cytolytic activity of NK lar
1 po CTL
cells. In addition, IL-10 can Tr T
promote tumor growth Nave
through the promotion of Th T
IL-10-producing Tr1 cells 1 Nave



NK activation



by NK cells. Furthermore, IL-10 induces HLA-G vaccine results in immune suppression and tumor
molecules that prevent the attack by NK cells progression, in line with a predominant inhibi-
[219]. These changes allow tumor cells to survive tory activity of IL-10 on DC-mediated antigen
from immunological attack by immune cells and to presentation. Moreover, IL-10-decient DCs are
grow exponentially. IL-10 can act as a negative reg- shown to be more effective in inducing protec-
ulator in the crosstalk between innate and adaptive tive antitumor immune response in mice [60].
antitumor immunity (Fig. 7.3): For instance, T cells Exposure of DCs to tumor cell lysates resulted
suppress NK and NKT cells by elaborating IL-10, in increased IL-10 production and expansion of
which ultimately leads to impaired activation of regulatory Tr1 cells. Tr1 cells have been shown
CTL and Th1 CD4+ T cells and tumor immune to down-modulate immune responses through the
privilege [220]. In vitro, IL-10 pretreatment can production of IL-10. In addition, IL-10 has been
convert different types of tumor cells to a CTL- shown to mediate the immunosuppressive activ-
resistant phenotype by decreasing the expression ity of Tregs [223]. Therefore, DCs that encounter
of HLA class I molecules on their surface [221]. tumor antigens in the presence of IL-10 in vivo
IL-10 acts on DCs and macrophages and inhib- acquire tolerogenic properties and subsequently
its the differentiation and the antigen-presenting induce T-cell tolerance to tumor antigens. In
properties of these cells. IL-10 inhibits essential addition, IL-10 signicantly suppresses other
steps in immune detection such as the expres- inammatory cytokines such as IL-1, IL- 6, and
sion of HLA-DR and co-stimulatory molecules, TNF expression in DCs. Moreover, inhibition
CD80 and CD86, on DCs. IL-10 also prevents the of IL-10 production by T cells or malignant cells
production of the Th1-polarizing cytokines IL-12 using anti-IL-10-/IL-10R-blocking Abs or anti-
and IFN- from DCs [222]. Administration of IL-10 antisense oligonucleotides improves anti-
IL-10 before and immediately after DC cancer tumor immune responses in animal models.
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 111 Antitumor Functions of IL-10 References

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Role of Chemokines
and Chemokine Receptors 8
in Cancer

Mathieu Paul Rodero, Christophe Combadire,

and Alexandre Boissonnas

Contents 8.6 Clinical Aspect ........................................... 133

8.6.1 Prognosis...................................................... 133
8.1 Introduction................................................ 121 8.6.2 CC Chemokines/Chemokine Receptors ...... 133
8.6.3 CXC Chemokines ........................................ 135
8.2 Chemokines and Chemokine Receptors .. 123
8.6.4 CX3C Chemokine Receptors....................... 135
8.3 Control of Tumor Cell Behavior ............... 125 8.6.5 Chemokine Circulating Expression ............. 135
8.3.1 Chemokines and Chemokine Receptor 8.6.6 Therapeutic Strategies ................................. 136
Alterations During Neoplastic
8.7 Concluding Remarks ................................. 137
Transformation............................................. 125
8.3.2 Metastasis/Homing ...................................... 126 References ............................................................... 138
8.3.3 Senescence, Proliferation, and Survival....... 127
8.4 Control of Immune Cell Behaviors .......... 128
8.4.1 Chemokines Involved in T-Cell
Antitumor Immune Response ...................... 128
8.4.2 Chemokines in Innate Immune
Components ................................................. 130 8.1 Introduction
8.4.3 Chemokine and Tumor-Induced
Tolerance...................................................... 131 Living tissues are highly organized and dynamic
8.5 Alternative Tumor-Associated structures at the cellular level. Tissue renewal,
Physiological Functions of Chemokines... 132 remodeling, and repair, immunosurveillance, and
8.5.1 Angiogenesis................................................ 132
8.5.2 Fibrosis and Extracellular Matrix
cell-to-cell interaction and communication are
Remodeling .................................................. 132 examples of physiological processes relying on
the ne recruitment and displacement of numer-
ous cell types. This equilibrium is strictly depen-
dent on the principle of recruiting the right cell at
M.P. Rodero, PhD C. Combadire, PhD the right place and the right moment. One major
A. Boissonnas, PhD (*)
Sorbonne Universits, UPMC Univ Paris 06, component of this principle is the chemokine and
CR7, Centre dImmunologie et des Maladies chemokine receptor system. Chemokines (CKs)
Infectieuses CIMI-Paris, Institut National de la, for chemoattractant cytokines are small, secreted
Sant et de la Recherche Mdicale (INSERM), molecules historically dened on the basis of their
U1135, Centre National de la Recherche
Scientique (CNRS), ERL 8255, functional chemotactic activity [13]. They con-
Institut Universitaire de Cancrologie (UPMC-IUC), stitute a family of over 50 members which inter-
CHU Piti-Salptrire, 91 Bd de lhpital, act with about 20 dened corresponding/cognate
Paris 75013, France receptors (CKRs). This discrepancy highlights
e-mail:;; the complexity of this system as several CKs can bind to a single receptor. Conversely, one receptor

N. Rezaei (ed.), Cancer Immunology: A Translational Medicine Context, 121

DOI 10.1007/978-3-662-44006-3_8, Springer-Verlag Berlin Heidelberg 2015
122 M.P. Rodero et al.

Fig. 8.1 Fine modulation

of cellular recruitment by
chemokines. The chemokine
network is organized around Cell types
several levels of complexity. 1 2 3
(a) Most of the cell types
(1, 2, 3) express several
chemokine receptors and a
same receptor is found on
several cell types. Moreover,
different chemokines can
bind to a same receptor and
most of the receptors can
bind several chemokines CKR CK
with distinct afnity
(color gradient represent
differential afnity). This Affinity
apparent complexity allows
for the ne control of cell
population recruitment. b Mix cells CK gradient Fine recruitment
(b) The schematic
representation illustrates
the selective recruitment
of cell populations according
to the respective colored CK
gradient. The number of cell
recruited is related to the
afnity of the respective CK
for its receptor

can bind several different CKs. This redundancy Cancer constitutes a very complex pathology
associated with differential avidity of the CK in many aspects. Neoplastic cells result from the
for their CKR and the specic expression by the environmental, viral-induced, or inherited dereg-
different cell population contributes to the ne ulation of genes known as oncogenes or tumor
tuning of cell migration (Fig. 8.1) and explains suppressor genes. This primary modication
that a modest deregulation of the system can often leads to uncontrolled expansion of undif-
lead to severe pathological conditions. In addi- ferentiated cells for which the transcriptome and
tion, there is overwhelming evidence describing the proteome are highly modied in comparison
alternative functions of the CK/CKR couple in with the original cell. Nevertheless, it is impor-
hematopoiesis, reproduction, angiogenesis, and tant to note that tumor development does not
immune-associated functions such as cell activa- result from the simple expansion of neoplastic
tion, proliferation, effector function, and survival cell. Indeed, solid tumors (primary tumor as well
[4, 5]. Numerous reports from the past two as metastasis) are also constituted by a wide vari-
decades have validated the importance of the CK/ ety of stromal cells. Stroma is composed of non-
CKR network with its diverse range of physi- hematopoietic cells, such as healthy cells of the
ological properties and its involvement in various affected tissue, broblasts, or endothelial cells, as
physiopathological disorders [68]. well as hematopoietic cells. Hematopoietic cell
8 Role of Chemokines and Chemokine Receptors in Cancer 123

populations are mainly composed of innate specialized tissue and nally generate metasta-
immune cells, such as tumor-associated macro- ses. Inammation generated by neoplastic trans-
phages (TAMs), dendritic cells (DCs), natural formation contributes to the recruitment of
killers (NK cells), neutrophils, and partners of protumoral population and the production of
the adaptive immune response such as T and B growth factors as well as the recruitment of
lymphocytes. immune component with antitumor activity.
The relative importance of the stroma compared Thus, tumorigenesis is a dynamic process involv-
to tumor cells depends on the type of cancer [9], ing important tissue remodeling and angiogene-
but it is now well described that several stromal sis, recruitment and local migratory mechanisms,
cells are important predictive markers of cancer and survival and cell death for both tumor and
evolution (macrophages, regulatory T cells, and stromal cells in which the CK/CKR network has
endothelial progenitor cells). Even though the major implication.
stroma cannot be characterized properly in circu- The CK/CKR network appears to be a promis-
lating hematological tumors, leukocytes will have ing target in cancer therapy and has already been
an important impact on the expansion, survival, used in standard therapeutic approaches, as well
and potential homing of tumor cells to the specic as in immunotherapy. Numerous basic and clini-
tissue. This phenomenon is distinguishable from cal interventions rely on the development of ago-
the metastatic process where the tumor cells need nist or antagonist CKR in order to manipulate
to cross the endothelial barrier from a primary their critical biological function toward antitu-
tumor site and home to a distant tissue. The stroma mor activity.
contributes to the global organization and progres- In this chapter, the role of the CK/CKR net-
sion of the tumor known as tumor microenviron- work in these aspects of cancer development, as
ment through the production of growth factors, well as its potential application in the improve-
cytokines, CKs, exchange of nutrients, and tissue ment of cancer therapy, is described in detail.
remodeling and repair. In contrast, immune cells
are responsible for the control of tumor growth.
The concept of immunosurveillance proposed by 8.2 Chemokines and Chemokine
Burnett et al. [10] in the early 1970s has been Receptors
widely debated. Recently, Schreiber and colleagues
provided experimental evidence for the clinical Chemokines are small cytokines initially
emergence of cancer as a result of strong selection described for their chemotactic properties on leu-
and modeling of tumors by the immune system in a kocytes. During cell recruitment from the blood
process termed as tumor editing [11]. In this pro- to inamed tissues, CKs initiate the activation of
cess, neoplastic transformation occurs, and tumor circulating cells, promoting cell rolling, adhesion
cell expansion is detected by the innate and adap- to activated endothelium, and extravasation
tive immune systems, which either succeed in com- (Fig. 8.2). In tissues, CKs determine cell direc-
plete tumor elimination or maintain a state of tional migration, by establishing a concentration
equilibrium between tumor cell expansion and gradient (Fig. 8.3). Evidence from previous stud-
elimination. This phase leads to the immune selec- ies has shown that the control of cell mobility by
tion of tumor cell variants that develop immune CKs is implicated in developmental mechanisms
resistance and immunesuppressive mechanisms and cell homeostasis, as well as in the induction
resulting in tumor escape and cancer progression to and tuning of acute and chronic inammation
a clinical outcome. and control of the immune response. Numerous
Cancer is a complex process whereby undif- reviews have extensively described the CK clas-
ferentiated tumor cells expand locally in special- sication, structural organization, and their asso-
ized tissues, migrate in an active manner by ciated biological properties [12, 13]. CKs are
leaving the primary tumor site through the endo- subdivided in four subfamilies based on the
thelial barrier, establish in a distant and different number and spacing between conserved cysteine
124 M.P. Rodero et al.

Fig. 8.2 Chemokine-

associated extravasation
process. (a) Circulating cell a b
within the bloodstream.
(b) Chemokine presented by
proteoglycan on activated c d
endothelial cells, induce the
expression of adhesion
molecules implicated in the
slow rolling and the capture
process. (c) Once stuck to
the endothelium, cell exerts
crawling behavior on the
luminal side of the blood
vessel and (d) extravasates Endothelial cells Chemokine receptor
and migrates through the
tissue toward a chemokine Adhesion molecules Chemokine

Proteoglycan loaded with chemokine

a b c

Stromal cells Chemokines Migrating

Immune cells
immune cells
Inflammed cells

Fig. 8.3 Interstitial migration. (a) Upon activation, cells will migrate through the tissue toward the higher
(b) stromal cells will produce chemokines forming a concentration of chemokine
gradient within the tissue. (c) Tissue-inltrated immune

in the primary amino acids sequence [14]. CKRs is in reality a tool to tightly regulated leukocytes,
are seven transmembrane G-protein-coupled stem cells, and other cell types migrations during
receptor classied according to the CK family physiological and pathological condition.
they bind. As previously mentioned, most CKs It is now well established that CK function is
bind to several receptors, and most of the recep- not limited to cell migration. It has been clearly
tors can bind several CKs with different afni- demonstrated that CKs directly control cell
ties. Additionally, one cell subset can express proliferation, survival and senescence, as well as
different CKR and the same CKR is expressed by cytokine secretion and phagocytic properties
different cell subsets. This apparent redundancy (Fig. 8.4). It is the balance between these
8 Role of Chemokines and Chemokine Receptors in Cancer 125

Fig. 8.4 Control of cell

biology by chemokines.
Besides cell migration,
chemokines are implicated Inhibition
in multiple cellular functions Activation
including apoptosis,
proliferation, and
senescence. Chemokines
Cytokine secretion
are also directly implicated
in cell activation, cytokine
secretion, or phagocytosis


Proliferation Senescence

migratory, secretion, phagocytic, survival, and resistant tumor variants. CK and CKR are not
proliferation signals which explains the central oncogenes per se; however, modulation in the
roles of CK in development, tissue homeostasis, production of CK or their receptors by tumor
repair, inammation, and immunity. cells is often the result of oncogenic modica-
tions and immune selection (Fig. 8.5). The rst
evidence came from a human papillary thyroid
8.3 Control of Tumor Cell cancer. The authors showed that RET (rearranged
Behavior during transfection)-tyrosine kinase rearrange-
ment promotes the secretion of numerous inam-
The biological property controlled by the CK/ matory cytokines, including CCL2, CCL20,
CKR recognition system is not restricted to che- and CXCL12, and increases the expression of
motactism. Several important processes involved CXCR4 [15]. Later studies have shown that Myc
in the behavior of tumor cells will be affected by overexpression in pancreatic cancer has been
these axes. In this section, the effect of CK/CKR associated with increased CK expression [16, 17].
expression on tumor cell behavior and cancer Nevertheless, the predictive outcome of onco-
progression is discussed. genic modications on the regulation of CK and
CKR expression is difcult to assess. While
RAS-RAF signaling pathway promotes CXCL8
8.3.1 Chemokines and Chemokine and CXCL1 transcription in pancreatic and ovar-
Receptor Alterations During ian cancer, it inhibits CCL27 transcription in skin
Neoplastic Transformation cancer [1820]. Similarly, Von Hippel-Lindau
tumor suppressor mutation in renal cancer [21]
Primary neoplastic transformation leads to strong and TP53 mutation in cancer stem cells promote
modication of the transcriptome and proteome CXCR4 expression [22] while downregulating its
which is mainly shaped by immune selection of expression in breast cancer cells [23].
126 M.P. Rodero et al.

Fig. 8.5 Oncogenes induce

altered chemokine and
chemokine receptor Inhibition
expression by tumor cells. Activation
Common oncogene mutations Sc
are associated with Ras CXCL1/8
modication of chemokine Pc Oc
or chemokine receptor
transcription, resulting in Pc
tumor promotion. RET/PTC Myc
rearranged RET tyrosine RET/PTC
kinase, VHL Von Hippel-
Lindau tumor suppressor
gene, Sc skin cancer, Pc Mutated
pancreatic cancer, Oc ovarian Tp53 cSC

cancer, HPTc human papillary Rc

thyroid cancer, cSC cancer VHL
stem cell, Rc renal cancer

Through modication in the prole of CKR gradients may induce tumor cell chemotaxis, but
expression, tumor cells will change their sensitiv- the direct implication of CKs in this specic pro-
ity to the microenvironment and acquire new cess is poorly documented. We can distinguish
migratory and homing capabilities. the indirect contribution of CK to the chemotaxis
activity of cancer cells through angiogenesis,
brogenesis, and matrix remodeling mediated by
8.3.2 Metastasis/Homing stromal cells.
CXCL12/CXCR4 is the major axis directly
The metastasis index is undoubtedly the major involved in tumor cell metastases. Overexpression
factor of prognosis and determines the therapeu- of CXCR4 in rat mammary adenocarcinoma
tic attitude. Metastasis denes the process enhances the motility of tumor cells in the pri-
through which tumor cells leave a primary site to mary tumor [26]. This receptor is widely involved
settle in a distant location and creates a new in the epithelial-to-mesenchymal transition
colony. This phenomenon is a characteristic of (EMT) process, which is a major step leading to
tumor malignancy including tumor invasion, metastasis [27, 28]. Few studies have reported the
intravasation, and homing to different sites. This implication of other CKs and CKRs such as
has to be distinguished from the potential sec- CCL18, CCL2, or CXCR7 [2931] through the
ondary localization of circulating tumor cells activation of EMT-implicated signaling path-
which only involves the homing mechanism. ways. IL8/IL8R axis might also favor mainte-
nance of the mesenchymal status of the tumor Tumor Invasion cell [32]. Interestingly, the integration of multiple
The rst step of metastasis spreading relies on CKR axes adds complexity to the tumor invasion
either tumor cell or stromal cell-mediated brosis process. Indeed, overexpression of CXCR4 pro-
activity and the ability of tumor cells to acquire motes invasion. However, coexpression of
migration and intravasation capabilities, in order CXCR7 which binds the same ligand CXCL12
to leave the primary tumor site and reach the impairs invasion but favors angiogenesis and pri-
bloodstream. Chemotaxis of tumor cells is well mary tumor growth [26].
characterized [24]. This process requires a para-
crine loop between tumor cells and stromal cells, Homing
such as macrophages shaping the microenviron- Once in the bloodstream, the tumor cell needs to
ment to favor metastasis [25]. Different chemical migrate to a site that will allow its engraftment,
8 Role of Chemokines and Chemokine Receptors in Cancer 127

Table 8.1 Metastases implantation of various cancer types based on their chemokine receptor expression

survival, and proliferation. In 2001, Muller et al. CXCL8 can result in senescence induction [37].
demonstrated for the rst time that the expression CXCR2 activation is thus able to act as a suppres-
of specic CKRs by tumor cells could predict the sor of malignancy in prostate and breast cancer
implantation of malignant cells in tissues express- [38, 39].
ing high levels of the receptor ligands [33]. Since Inhibition of tumor proliferation by CXCR2
then, several other studies have established asso- ligand is probably limited to tumor models and to
ciations between metastases, CKR expression, early stages of tumor development. Indeed, the
and implantation sites for various cancer types same CK axes display opposite effects in other
(Table 8.1). Consistently with their homeostatic tumor models. CXCR1 and CXCR2 activation by
functions, CCR7 expression by tumor cells is CXCL8 promotes the proliferation of gastric can-
associated with lymph node metastases; CCR10 cer, esophageal cancer, non-small lung cancer,
with skin metastasis; CX3CR1 with brain, liver, and melanoma cell lines [4043]. Other receptors
and bone metastases; CCR9 with intestine metas- of the CXC receptor family are involved in tumor
tases; and CXCR4 with bone and liver metastases cell proliferation. CXCR6 is involved in cell pro-
[3336]. liferation of pancreatic cancer cells [44], and
Overall, these observations show that CK CXCR4 is associated with tumor proliferation in
axes generate a complex relationship between numerous models, including ovarian, melanoma,
tumor cell and the environment and deserve glioma, renal, lung, and thyroid cancer cells [27,
further attention in preclinical studies as it 45]. Few studies have investigated the implication
represents an important target with clinical of CCRs in the control of tumor cell proliferation.
application. CCR6 favors colon tumor cell proliferation upon
CCL20 activation [46], and CCR9 favors
pancreatic cancer cell proliferations upon CCL25
8.3.3 Senescence, Proliferation, activation [47].
and Survival Another role of CK in tumor cell biology is
the ability to control tumor cell survival, essen-
Tumor expansion results in the capacity of tumor tially mediated through the CC receptor family.
cells to proliferate innitely without developing CCR10 activation promotes phosphatidylinositol-
senescent mechanisms. Several CKs have dem- 3-kinase-mediated protection from apoptosis of
onstrated the ability to activate signaling path- melanoma cells [48]. The same mechanisms are
ways in favor of this goal. observed in squamous cell carcinoma of the head
Cellular senescence is generally dened as an and neck after CCR7 activation [49]. CCR7
irreversible state of G1 cell cycle arrest in which engagement by CCL21 is also implicated in the
the cell is refractory to growth factor stimulation. prevention of apoptosis in NLCLC, through
Activation of CXCR2 by either CXCL1 or ERK-dependant activation pathways [50].
128 M.P. Rodero et al.

CK direct promotion of tumor cell survival is feature of this maturation is the downregulation
not limited to CC chemokines; CXCL12 through of peripheral tissue-associated CKR like CCR1,
CXCR4 activation promotes hepatoma, ovarian, CCR5, and CCR6 and the upregulation of CCR7.
and chronic leukemia tumor cells survival [51], Due to the constitutive expression of CCR7
and CXCR7 activation increases cell survival by ligand, CCL19, and CCL21 by peripheral lymph
reducing apoptosis [52]. nodes, this switch of CKR expression by APCs
Overall, these observations highlight extended promotes their migration toward the priming site.
functional contributions of the CK system to Once in the draining lymph node, APCs will
tumor development and reveal that they are not locate in the preferential area to present the tumor
merely restrained to chemotaxis. Ag to the CCR7 expressing naive lymphocyte. Ag Presentation to T
8.4 Control of Immune Cell Lymphocytes
Behaviors Despite the fact that APCs display low dynamic
activity, nave lymphocytes have a high basal
As described previously, the immune system is mobility favoring scanning of thousand APCs per
known to shape the tumor through the tumor hour [55, 56]. This behavior requires CCR7
editing phenomenon. In this context, CKs are expression by T lymphocytes [57]. An additional
directly or indirectly implicated in the control of CKR-dependant mechanism favors the probabil-
immune cell activation, migration to the priming ity of encounter between APCs and T lympho-
site, and immune response induction. It is now cytes. Encounter of Ag-specic CD4+ or CD8+ T
clear that in most cases, the CK network is cells with an APC bearing their cognate Ag
shunted by the tumor, favoring its escape from induces the secretion of CC-chemokines by the
immunosurveillance and tumor progression. conjugate, namely, CCL19, CCL5, CCL3, and
Nevertheless, the production of some CKs pro- CCL4. These CKs will promote nave T-cell
motes the antitumor immune response and has scanning behaviors and attraction toward the
been associated with improved patient outcome, conjugate [5860], which is known to favor the
including lower recurrence rate or increased establishment of memory immune response, in
patient survival [53]. addition to the induction of polyclonal responses
against different tumor Ags [61].
CKs are also implicated in the improvement
8.4.1 Chemokines Involved in T-Cell of APC/T-cell adhesion mechanism as well as in
Antitumor Immune Response immunological synapse stabilization, promoting
T-cell priming (Fig. 8.6). CCR7 ligands secreted
Induction of antigen (Ag)-specic antitumor in the lymph node promote immunological syn-
immune response requires the uptake of tumor Ag apse formation by T cells [62]. CXCR4 and
by professional antigen-presenting cells (APCs) CCR5 expressed by T cells are recruited toward
and migration from the tumor site to the corre- the immunological synapses made with the
sponding draining lymph node, in order to present APC. This polarization results in desensitization
the processed tumor-Ag to T lymphocytes. These of T cells from external sources of CKs and
major immune functions can be divided into dif- improves synapse stability. A similar mechanism
ferent steps for which the CKR network has is observed during the interaction between tumor-
important regulatory implications [54]. inltrated lymphocytes (TILs) and tumor cells.
Indeed, the recruitment of CCR5 at the immune Migration of APCs synapse formed between the TIL and the tumor
to the Priming Site cell results in defective responses to TIL toward a
Encounter with tumor Ag induces maturation of CCR5 gradient [63]. This mechanism allows for
APCs present in the tumor environment. One the modulation of the GO signals generated by
8 Role of Chemokines and Chemokine Receptors in Cancer 129

a b c
Immune synapse

Chemokine receptors TCR MHC Co-stimulatory molecules

Fig. 8.6 Control of cell polarization toward immune syn- sequestration of CKR leads to reduced sensitivity to dis-
apse. (a) T-cell scan for their cognate antigen-presenting tant CK gradient and may participate in the stabilization
cell. (b) Upon recognition, T cell will polarize chemokine of the immune synapse
receptors toward the immune synapse. (c) This

CKs, competing with the STOP signals medi- periphery; however, the underlying mechanisms
ated by the TCR-MHC interaction [64]. remain unclear. Several clues could help us specu-
late on the mechanism of trapping the TILs at the Migration of Effector T tumor periphery. The recent contribution of real-
Lymphocytes to Tumor time imaging showed that dense peripheral extra-
Naive T cells, after clonal expansion and differ- cellular matrix might restrain TILs access to the
entiation into effector T cells, migrate toward the tumor parenchyma [68]. Whether specic niches
tumor site, implying that T cells downregulate of CKs are expressed on collagen bers is unclear
the expression of the CKRs implicated in the and needs further investigation. In addition,
retention at the priming site like CCR7. In addi- dynamic analysis showed that Ag-specic CTLs
tion, they upregulate various CKRs including are trapped in the network of tumor-associated
CCR1, CCR3, CCR5, and CXCR3 allowing their APCs restraining their inltration and probably
movement toward the tumor site [65]. Cytotoxic favoring immunosuppression [69, 70]. The role of
T lymphocytes (CTLs) recruitment to the tumor CKs in this trapping is not dened, but Ag expres-
site is consistent with this pattern of CKRs sion by APC at least induces stable engagement
expression and is mainly mediated by CCL3, between the CTL and the APC. In addition,
CCL5, CCL20, CXCL9, and CXCL10 [54]. experimental evidences showed that non-tumor-
Membrane-anchored CKs expression such as Ag-specic TIL cannot inltrate the tumor deeply
CXCL16 and CX3CL1 have also been shown to without the prior tumor cells destruction by
correlate with greater numbers of tumor- Ag-specic CTL. These results suggest that deep
inltrated lymphocytes and improved prognosis inltration of the tumor by TIL might be favored
in colorectal cancer [66, 67]. The antitumor effect by chemotactic agents secreted upon tumor cell
of the membrane-bound CK form vs. the soluble destruction by CTL or on extensive ECM remod-
form is yet to be clearly established. eling to allow their interstitial migration [71].
The control of TIL localization within the Overall, considering the numerous CKs
tumor is ill-dened. It is obvious that in most expressed by the various cell subsets of the tumor
cases, TILs are mainly found at the tumor microenvironment, it is very difcult to address
130 M.P. Rodero et al.

specic contributions of CK/CKR couple in the derived from the yolk sac or fetal liver [72].
interstitial migration and positioning of T lym- Within neoplastic tissues, it is suggested that
phocytes within the tumor parenchyma. The TAMs are mostly recruited from the periphery.
various properties of these molecules have dem- Nonetheless, knowledge of the relative propor-
onstrated that this positioning is controlled by tion of native resident macrophages remains a
sensitivity to the chemotactic gradient and the poorly investigated eld in oncology. CCL2, also
subsequent desensitization upon polarization called MCP-1 for monocyte chemoattractant pro-
toward the synapse or the downregulation of the tein-1, is probably the most frequently found
expression of CKRs. CC-CK in tumors involving recruitment of circu-
lating monocytes [73]. Interestingly, in a mela-
noma system where tumorigenesis is dependent
8.4.2 Chemokines in Innate on an external growth factor CCL2, there is a
Immune Components biphasic effect depending on its secreted quan-
tity. High amounts are associated with a massive
Innate immune cells constitute a rst barrier recruitment of TAM into the tumor with domi-
against tumor development. However, due to nant antitumor activity, while lower amounts
their plasticity and capacity to produce a myriad induce lower inltration into the tumor resulting
of cytokines, chronically activated innate immune in tumor promotion through the secretion of
cells are key modulators of cell activation and growth factor by the macrophages [74]. These
survival, as well as regulators of the ECM metab- results point out the importance of the ratio
olism. Several physiological processes necessary between protumor and antitumor macrophages
for tumor development, such as increased cell recruited into the tumor.
survival, tissue remodeling, angiogenesis, and Other CKRs implicated in TAM recruitment
suppression of antitumor adaptive immune are CX3CR1 and CCR1. In human glioblastoma,
responses, are regulated by innate immune cell the level of tumor inltration by microglial cells
inltrate in the tumor. is dependent on CX3CR1. Patients with a func-
Macrophages are the main stromal cell popu- tional mutation in the CX3CR1 gene associated
lation present in the tumor parenchyma. They can with impaired monocyte migration have a
account for more than 50 % of the tumor mass. reduced TAM inltration into the tumor [75].
The role of TAM in tumor development is criti- Injection of a thymoma tumor cell line (EL4)
cal, as these cells, depending on their state of with a liver tropism to mice results in an increased
activation, can display antitumor properties asso- inltration of the liver by immune cells, includ-
ciated with production of Th1 cytokine, high ing macrophages. In CCR1 KO mice, this recruit-
quantity of reactive oxygen species, and efcient ment during the rst stage of the tumor
Ag presentation or they could display protumor development is massively reduced [76].
properties mediated by the secretion of Th2 cyto- CXC chemokine receptors could also be
kine, proangiogenic factors, growth factors that implicated in TAM recruitment. In humans, IL-4
support tumor survival, and proliferation and the and IL-13, two cytokines secreted in the tumor
secretion of MMP which promote tumor invasion environment, sensitize monocytes to CXCL1 and
and metastases. Consistently, the impact of TAM CXCL8 by upregulating their receptors (CXCR1
on tumor development and metastases will and CXCR2). Thus, these cytokines indirectly
depend on the balance between M1 antitumor promote the recruitment of TAM into the tumor
macrophages and M2 protumor macrophages. through CXC chemokine receptors [77].
Depending on the tissue, resident macro- As previously discussed, CKs not only con-
phages are in a small proportion derived from trol leukocyte recruitment into the tumor but
the recruitment of circulating monocytes assur- also organize their localization within the
ing immunosurveillance and mainly origin from tumor. Lack of proper vascularization at the
self-renewal of interstitial resident macrophages center of the tumor induces the secretion of
8 Role of Chemokines and Chemokine Receptors in Cancer 131

several hypoxic factors like hypoxia-inducible 8.4.3 Chemokine and Tumor-Induced

factors (HIFs). HIFs promote the expression of Tolerance
CXCR4 by macrophages, favoring their recruit-
ment toward tumor hypoxic areas [78]. On the Recruitment of tolerogenic cells such as regula-
other hand, tumor environment decreases CKR tory T cells or immunosuppressive myeloid sub-
expression on monocytes. Indeed, macrophages sets is a feature or immune escape. Tumor cells
from tumor sites express low levels of CKR secrete ligands of CKRs expressed by immature,
[79]. Time-lapse imaging of TAMs in experi- regulatory or Th2 polarized cells. CCL22 and
mental murine model revealed that TAMs dis- CCL17 produced by tumor cells recruit mono-
play reduced displacement but intense cytes, as well as Th2 lymphocytes and regulatory
protrusive activity [69, 70]. Downregulation of T cells through CCR4 signaling [91]. This strat-
CKR might explain this retention at the tumor egy of immune escape has been also selected in
site. viral-induced oncogenesis process. HHV8 virus,
CKs do not only act on leukocyte attraction the pathogen of Kaposis sarcoma, encodes three
but are also implicated in their activation. viral CKs which bind to CCR3, CCR4, and CCR8
Induction of copper/zinc-superoxide dismutase involved in the recruitment of Th2 and regulatory
by CCL5/CCR5 activation causes tumor necrosis T cells [92].
factor-alpha and reactive oxygen species produc- Stromal cells produce CKs which promote the
tion by macrophages [80], promoting tumor recruitment of protumoral cells. Amongst others,
destruction. Inversely, in human monocytes, CC TAM produces CCL18 which is induced by IL10
chemokines induce the transcription of metallo- [93]. CCL18 favors the recruitment of nave T
proteinase, implicated in tumor invasion and cells through activation of an unknown receptor.
spreading. The fact that both TAM recruitment It is proposed that these nave T cells acquired
and activation are regulated by CK increases the tolerogenic properties in contact with the tumor
potential interest of targeting TAM for antitumor environment. CCR6+ immature lymphoid DCs
therapies. recruitment into the tumor is favored by the
NK cells represent another component of the secretion of CCL20 from both tumor cells and
innate immune system highly involved in antitu- TAM [94]. CCL5 recruits immature DCs as well
mor immune responses. NK cell recruitment to by binding CCR1 and CCR5 [95]. Immature DCs
the tumor is mainly mediated through the acquire tolerogenic properties in the tumor envi-
CXCL10-CXCR3, CX3CL1/CX3CR1, and ronment and participate in the immune tolerance
CCL3-4-5/CCR5 axes. High CX3CL1 quantity is loops against tumor Ags [96].
associated with increased NK cell recruitment Subversion of tumor immune component is
into the tumor in both human and mice [81, 82]. a central point of tumor outcome. The above
Similar phenomenon is observed with increased described implication of CK in cellular mech-
CCL5 and CCL3 expression by tumor cells in anisms should provide the basis to better
mouse models [83, 84]. CXCR3 is implicated in understanding the clinical implication of CK
the recruitment of human NK cells to breast can- network in cancer pathology. The regulation
cer tumor, which is mediated by CXCL10 secre- of the balance between immunogenic and
tion from tumor cells in response to IFN- tolerogenic components has deserved major
produced by the NK cells themselves [85, 86]. attention for a long time and is the basis of
Thus, CKs not only control NK cell recruitment immunotherapy which represents an apparent
but also regulate their antitumor properties. inexhaustible field of innovative anticancer
CX3CR1 activation by CX3CL1 results in strategies. Targeting the CK system in this
improved antitumor cytotoxicity of NK cells [87, goal is in the course of important investigation
88]. CCL3, CCL4 and CCL5 have been shown to through the development of pharmaceutical
activate NK cytotoxicity through induction of compounds able to stimulate or antagonize
degranulation [89, 90]. CKR axes.
132 M.P. Rodero et al.

8.5 Alternative Tumor- In contrast, ELR chemokine secretion is

Associated Physiological often associated with attenuation of angiogene-
Functions of Chemokines sis. ELR CXC chemokines are described by
their angiostatic properties. ELR CXC chemo-
8.5.1 Angiogenesis kine secretion is induced by IFN- and IFN-.
Through CXCR3 binding, these CKs mediate
One of the features of CKs is their dual role in the their angiostatic properties by inhibition of ELR+
angiogenic process. In the tumor environment, chemokine, VEGF, and FGF proangiogenic
there is increased production of proangiogenic effects in vitro [103]. Interestingly, the expres-
CK, while angiostatic CKs are downregulated. sion of CXCR3 is dependant of the cell cycle
In addition to a direct angiogenic effect of CKs, phase, limiting the angiostatic properties of ELR
this activity is indirectly potentialized by the CXC chemokines to the S/G2-M phase [104].
CK-induced recruitment of leukocyte display- This important association of CKs and angio-
ing angiogenic properties such as neutrophils or genesis within the tumor environment sets the
macrophages [97]. inhibition of ELR+ chemokine as a robust antitu-
CK from the CXC family are probably the mor therapy.
most described for their direct implication in
tumor-associated angiogenesis. CXCLs 1, 2, 3, 5,
6, 7, and 8 display angiogenic properties. All 8.5.2 Fibrosis and Extracellular
these CKs contain a specic amino acid sequence Matrix Remodeling
of glutamic acid-leucine-arginine (or ELR for
short) immediately before the rst cysteine of the The association of CKs in EMT leading to bro-
CXC motif (ELR-positive). This ELR sequence sis activity has been previously suggested by
absence from the other CXC chemokines is studies; however, there is no clear evidence that
responsible of the proangiogenic properties of CKs play a direct role in this process.
most of the CXC chemokine [98]. Fibrosis and extracellular matrix remodeling
ELR+ chemokines mediate angiogenesis are continuous processes present in the tumor
through binding to the CXCR2 receptor. ELR+ parenchyma reecting the intense dynamic and
chemokines are able to recruit endothelial pre- migratory activity of the neoplastic tissue. Two
cursor cells, induce cell proliferation, and pro- different types of migratory activity are dened,
mote maturation. These mechanisms could be namely, the amoeboid and mesenchymal migra-
negatively regulated by a decoy CKR expressed tion. The amoeboid migration does not require
by endothelial cells called duffy antigen receptor extracellular matrix (ECM) remodeling through
for CK (DARC). Unlike most of the other CKR, matrix metalloproteinases (MMPs) activity due
DARC is not linked to G protein, and its activa- to the ability of the cell to squeeze through the
tion does not induce calcium ux. DARC reduces ECM. The mesenchymal migration relies on pre-
angiogenesis by sequestering all the ELR+ CKs. vious proteolysis and degradation of the ECM to
One specicity within ELR chemokines is generate sufcient space for cell displacement.
attributed to CXCL12 which is the only ELR CK-mediated induction of MMP is mostly medi-
chemokine with proangiogenic activity. CXCL12 ated by CC chemokines; CCL5 and CCL9 pro-
mediates its proangiogenic effect by directly pro- duced by mesenchymal stem cell promote tumor
moting the recruitment of endothelial progenitor cell invasion in a MMP-dependant manner [105,
cells [99, 100] or indirectly by promoting tumor 106]. CCL25 promotes MMP secretion in ovar-
angiogenesis through the recruitment of CXCR4+ ian cancer cells through CCR9 binding and favors
proangiogenic monocyte [78, 101] and through tumor cell invasion [107]. CCL21/CCR7 interac-
the secretion of vascular endothelial growth fac- tion favors MMP-9 secretion, tumor invasion,
tor (VEGF) by CXCR7 activation [102]. and metastases in colon cancer cells and in B-cell
8 Role of Chemokines and Chemokine Receptors in Cancer 133

chronic lymphocytic leukemia cells [108, 109]. correlation between functional variants and
At least, one CXC chemokine has been related to tumor risk or progression (Table 8.2).
MMP activity; thus, CXCL12 is implicated in The paragraphs below focus on the most com-
increased MMP2 activation and increased cell monly described polymorphisms, their functional
invasion in a pancreatic cancer cell line [110]. relevancies, and their subsequent prognostic
Studies have suggested that the extracellular value in tumor risk and/or progression.
matrix promotes tumor escape from the immune
system by trapping antitumor leukocytes at dis-
tance from tumor cell niches [111]. However, 8.6.2 CC Chemokines/Chemokine
tumor progression and metastases require degra- Receptors
dation of this extracellular matrix surrounding
the tumor. The main protagonists of these physi- CCL2
ological activities are represented by mesenchy- A single-nucleotide polymorphism (SNP) in the
mal stem cell (MSC)-derived cell populations. CCL2 promoter, based on the substitution of an
CXCL12 is implicated in the recruitment of mes- adenine by a guanine in position 2518
enchymal stem cells (MSCs) from the bone mar- (A < 2518 < G), is associated with increased
row. Bone marrow-derived MSCs can account CCL2 secretion [113]. This polymorphism with
for up to 25 % of the cancer-associated bro- an allelic frequency close to 30 % is associated
blasts, the main source of brosis within the with an increased susceptibility to the develop-
tumor [112]. ment of breast, gastric, and oral squamous can-
There is ongoing evidence that targeting pro- cer. However, it is not associated with an
teolysis activity in combination with chemotaxis increased risk of developing hepatocellular and
would provide promising results in the strategy to prostate cancer, glioblastoma, and melanoma.
inhibit tumor cell invasion and metastasis. Despite this lack of association with the devel-
opment of melanoma, CCL2 polymorphism is
associated with increased Breslow index, sug-
8.6 Clinical Aspect gesting its link with melanoma progression
[114]. CCL2-2518G variant is also associated
CKs are implicated in several aspects of tumor with increased metastases development in naso-
development. Due to these pivotal roles in tumor pharyngeal and breast cancer. In the former
biology, CKs have been frequently associated case, the deleterious effect of the polymorphism
with tumor evolution and clinical outcomes and is observed only after radiotherapy [115].
have been highlighted for their potential use as Overall, the deleterious effect of the CCL2-
prognostic or diagnostic markers. Therefore, they 2518G allele-associated increase of CCL2
represent a promising target with a potential for a expression is consistent with the protumoral
diverse range of therapeutic strategies. effect of TAM in most tumors, as previously
described above.

8.6.1 Prognosis CCL5

Conicting data arises from the study of the
Due to its importance across a wide range of CCL5 G < 403 < A polymorphism on cancer
physiological mechanisms, CK/CKR network risk. This mutation is thought to be responsible
alteration could impact tumor development. for the decreased secretion of CCL5 and is asso-
Correlative studies using genetic polymorphisms ciated with decreased risk for leukemia and gas-
provide essential information for prognosis. tric cancer in women [116], as well as an
Several functional polymorphisms in CKs or increased risk for prostate and pancreatic cancer
CKRs have been studied in order to establish [117]. This discrepancy could reect the balance
134 M.P. Rodero et al.

Table 8.2 Association between

chemokines and chemokine
receptor polymorphisms and
tumor risk and/or progression

Prog prognosis, + good indicator, poor indicator, = no association, * meta-analysis

between the antitumor effects of CCL5 through CCR5

recruitment of cytotoxic CTL and the protumoral CCL5 main receptor (CCR5) is also subject to
effect of CCL5 through recruitment of immature another relevant polymorphism. A deletion of
DC. Nonetheless, there is no evidence supporting 32 base pairs named CCR5 delta 32 results in a
an association between CCL5 polymorphism and reading frame shift, associated with complete
tumor progression. defect in receptor expression. The impact of the
8 Role of Chemokines and Chemokine Receptors in Cancer 135

polymorphism in tumor risk and progression is ture make any interpretation challenging. Several
not well documented. Most studies conclude a meta-analyses have been performed in order to
lack of association; however, one report suggests gain some clarity, and despite some variation in
that CCR532 could be associated with higher the conclusion, it appears likely that the rare vari-
risks of the development of gallbladder cancer ant of CXCL8 promoter region is associated with
[118]. In melanoma, CCR532 is associated with increased risk of gastric and oral cancer [121123].
reduced survival of patients with grade 4 tumor
treated by immunotherapy strategies [119]. These CXCL12
observations might reect the role of CCR5 in the CXCL12 is subject to a polymorphism in a 3
induction of T-cell priming and memory. untranslated region named CXCL12 3 G801A. The
rare variant is associated with increased secretion CCR2 of CXCL12. Consistent with the protumoral effect
CCR2 V64I polymorphism has also been studied of CXCL12 mentioned above, studies essentially
for its implication in tumor risk and progression. report that CXCL12 801A variant is associated
There is no known effect of the genetic variation with an increased risk for several cancers (lung,
on the CCR2/CCL2-signaling pathway, but it is breast, oral, prostate, hepatocellular, and colorectal
associated with CCR5 instability, which could be cancer). It is also thought to favor tumor progres-
explained by stability alteration of the CCR2/ sion or metastases in lung cancer, hepatocellular
CCR5 dimer. Most of the studies conclude that carcinoma, colorectal cancer, and myeloid leuke-
there is an increased risk for people carrying the mia. The only three meta-analyses performed to
rare variant. This is the case for cervical, oral, date conclude that there is an increased risk for
bladder, prostate, and endometrial cancer. A recent breast and lung cancer, without any signicant
meta-analysis with 2,661 cancer patients and effect on other cancer types [124126].
5,801 healthy controls found an overall signicant
association between the CCR2-V64I polymor-
phism and cancer risk [120]. In the subgroup anal- 8.6.4 CX3C Chemokine Receptors
ysis stratied by cancer types, there was a
signicant association between this polymorphism The only receptor for the CX3C chemokine fam-
and the risk of bladder, cervical, and oral cancer. ily is CX3CR1, which is also subject to poly-
morphisms associated with cancer outcome.
Substitution of a valin by an isoleucine in position
8.6.3 CXC Chemokines 249 results in increased adhesion of the couple
CX3CR1/CX3CL1 and defective migration of
Two CXC chemokines, CXCL8 (also referred as CX3CR1+ cells. The rare variant is associated with
interleukin-8) and CXCL12 (SDF-1), have been increased risk of colorectal cancer, but not hepa-
intensively investigated for their association between tocellular cancer, melanoma, and glioblastoma.
polymorphisms and tumor risk and development. In this last case, the rare variant is associated with
improved patient survival after tumor biopsies and CXCL8 decreased inltration of the tumor by microglial
CXCL8 T < 251 < A polymorphism is prob- cells [75]. This is consistent with the promotion
ably one of the most studied CK polymorphism of glioblastoma invasion by microglial cells [127].
in cancer. Its physiological effect and its impact
on CXCL8 expression remain to be elucidated.
There is an apparent discrepancy between stud- 8.6.5 Chemokine Circulating Expression
ies on these effects; however, this may reect
specicity depending on the cell type or the cell CK circulating levels have also been related to can-
activation status. The implication of CXCL8 poly- cer progression. A high concentration of CCL17 is
morphism in cancer risk and outcome remains associated with the progression of Hodgkin lym-
unclear. Unfortunately, controversies in the litera- phoma (HL) after treatment [128]. Interestingly,
136 M.P. Rodero et al.

Table 8.3 Current clinical trials evaluating the benets of targeting chemokines or chemokine receptors cancer
Inclusion criteria Phase Treatment
Colorectal cancer Phase I/II Chemokine-modulatory regimen
Stage IV adenocarcinoma of the lung Phase I/II GM.CD40L and CCL21
Metastatic castrate-resistant prostate Phase II Anti-CCL2 carlumab
Solid tumors Phase I Human monoclonal antibody against CCL2 (CNTO 888)
Colorectal cancer patients with hepatic Phase I CCR5 antagonist (Maravirok)
liver metastases
Previously treated peripheral T-cell Phase II Anti-CCR4 monoclonal antibody KW-0761
lymphoma (Mogamulizumab)
CCR4-positive adult T-cell Phase II Anti-CCR4 (KW-0761)
High-grade glioma Phase I CXCR4 antagonist (Plerixafor/AMD3100) and
Multiple myeloma previously treated Phase III Filgrastim with or without CXCR4 antagonist
with lenalidomide (plerixafor/AMD3100)
Non-Hodgkin lymphoma Phase III CXCR4 antagonist (Plerixafor/AMD3100) and G-CSF
Multiple myeloma Phase Ib Anti-CXCR4 (BMS-936564) alone or plus lenalidomide/
dexamethasone or bortezomib/dexamethasone
Multiple myeloma Phase I/IIA CXCR4 antagonist (BKT-140)

opposite effects are observed in melanoma, where been developed to target CKs or CKR as innova-
high CCL17 expression is associated with progres- tive strategies in cancer treatment. To date, there
sion-free survival in patients with immunotherapeu- is no molecule targeting macrophage release;
tic treatment [129]. Elevated concentrations of however, multiple clinical trials from phase I to
CXCL10 in the serum before treatment (monoclonal phase III are recorded at clinical website
antibody therapy together with combination chemo- (Table 8.3). Some strategies aim to promote the
therapy) are associated with an increased likelihood production of CKs implicated in the recruitment
of clinical relapse and an inferior survival in patients of immune-competent cells to the tumor by injec-
with diffuse large B-cell lymphoma [130].Despite tion of IFN, celecoxib, and rintatolimod
numerous promising results, CK and CKR genes (NCT01545141). In another trial, patients with
and molecules are not currently used in clinical lung adenocarcinoma were directly injected with
settings to evaluate a patients risk of developing CKs implicated in the recruitment of antitumor
cancer or to predict tumor progression. This effector T cells, in combination with vaccination
could be explained in part by the nonhomoge- approach (NCT01433172). Inversely, another
neous distribution of the polymorphism variants trial aimed to inhibit the recruitment of protu-
amongst ethnic communities. Additionally, in moral leukocyte using an Ab against CCL2 in
most cases, CK and CKR gene polymorphisms order to control metastatic castrate-resistant pros-
are not singularly powerful predictive tools. Their tate cancer (MCRPC) (NCT00992186). However,
clinical utility is most likely to be dependent on this strategy failed as all the patients were
their association with other markers. removed from the study, due to progression of the
tumor despite anti-CCL2 treatment.
Another approach aimed to directly target
8.6.6 Therapeutic Strategies CKR expressed by neoplastic cells in order to
control tumor or metastases development. The
As discussed throughout this chapter, CKs are CCR5 antagonist, named maravirok, originally
implicated in all steps of the tumor development, commercialized for AIDS treatment, is under
invasion, and dissemination. Several tools have evaluation for its antitumor property in colorectal
8 Role of Chemokines and Chemokine Receptors in Cancer 137

cancer (NCT01736813). Promising results have immune system maturation and surveillance, and
been obtained with an anti-CCR4 Ab named tissue remodeling functions like angiogenesis or
KW-0761. Injection of KW-0761 in subjects brosis are shunted in most cases toward tumor
with CCR4-positive adult T-cell leukemia- promotion. The central role of the CK network
lymphoma resulted in the stabilization of tumor in these processes positions the CK system as an
progression in half of them. This molecule is now attractive target against tumor development, pro-
under evaluation in cutaneous T-cell lymphoma gression, and dissemination. Clinically, CK and
(NCT01728805) and in second-phase treatment CKR polymorphisms or serum levels are already
for peripheral T-cell lymphoma (NCT01611142). associated with susceptibility or prognostic mark-
CXCR4 antagonists are probably the most ers. Current investigations aiming at controlling
widely used molecules in trials targeting the CK tumor development by targeting the CK network
network. plerixafor is a FDA-approved CXCR4 are not limited to the direct effect on tumor cells.
antagonist for use in patients with non-Hodgkin For instance, it is proposed that CKs could modu-
lymphoma (NHL) and multiple myeloma. It is late the involvement of TAMs in tumor eradica-
used as a preconditioning regimen for its ability to tion or protection after chemotherapy suggesting
mobilize bone marrow resident hematopoietic that chemoattractant molecules could be used
stem cells and tumor stem cells toward circulation in combination with standard chemical chemo-
before chemotherapy. Plerixafor and other mole- therapies to favor tumor eradication through
cules targeting CXCR4 are now evaluated in sev- modulation of the TAM activity. Despite numer-
eral clinical trials from grades I to III phase in ous promising results, few molecules targeting
combination with other treatment, in various forms CKRs have received FDA approval. The CXCL12
of leukemia and myeloma. Evaluation of CXCR4 antagonism is already being used in patients
targeting in cancer therapies is not limited to blood with leukemia or myeloma to promote tumor
tumors. Plerixafor is currently being evaluated in a cell mobilization toward the bloodstream before
phase I trial in conjunction with bevacizumab for treatment, and the CCR5 antagonist maravirok
patients with high-grade glioma (NCT01339039). is currently being evaluated in colorectal cancer.
These low numbers of molecules targeting CKs
in the market could be explained by the relatively
8.7 Concluding Remarks recent discovery and characterization of the CKs.