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Current Cancer Drug Targets, 2014, 14, 000-000

The Role of CD40 Expression in Dendritic Cells in Cancer Biology; A

Systematic Review
Gui Han Lee1,2, Alan Askari1, George Malietzis1,2, David Bernardo2, Susan K. Clark1,
Stella C. Knight2,* and Hafid Omar Al-Hassi2
1

Department of Surgery, St Marks Hospital, Harrow, UK; 2Antigen Presentation Research Group, Department of
Medicine, Imperial College London, UK
Abstract: CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor superfamily and is essential in
activation of dendritic cells. Dendritic cells (DCs) are antigen-presenting cells capable of initiating cytotoxic Tlymphocyte immune response against cancer cells. However, there are few studies on the characterization of DCs in
cancer, specifically their expression of CD40, despite its implication in cancer immunotherapy. We reviewed available
data on the expression of CD40 on DCs in various cancers, and its implications for cancer immunotherapy.
A systematic review on CD40 expression on DCs in cancer was performed with reference to preferred reporting items for
systematic reviews and meta-analyses (PRISMA). Studies that satisfied the inclusion and exclusion criteria were 21 out of 927.
Variations in type and status of the cancers, source of DCs and methodology for detecting CD40 expression amongst the
studies resulted in contrasting results. DCs generally expressed low CD40 in tumor infiltrating DCs (tiDCs), in DCs
derived by in vitro culture from blood monocytes using cytokine stimulation (MoDCs) and in DCs exposed in vitro to
tumor cells lines; the studies suggested that CD40 expression in DCs is impaired in cancer particularly in metastatic
disease. However, DCs identified in fresh peripheral blood mononuclear cells (PBMC) expressed higher numbers of
CD40 positive cells in some cancer patients, which could be due to tumor-derived factors leading to partially-stimulated
DCs. The results provide evidence that some cancer patients may show partial systemic DC activation and expression of
increased CD40 in response to the presence of tumor but that such activity may become abortive in the presence of factors
produced by the tumor.
This review has thus identified key papers on CD40 expression on DCs in various cancers and discusses the limitations
and contrasting results of these studies in relation to variations in methodology. The results highlight the need for further
studies on the role of CD40-CD40 ligand pathway to inform cancer treatment.

Keywords: Antigen presenting cells, cancer, CD40, CD40 ligand, dendritic cells, immunotherapy
INTRODUCTION
CD40 is a type 1 membrane glycoprotein co-stimulatory
molecule that belongs to the tumor necrosis factor
superfamily. CD40 is expressed on a variety of immune and
non-immune cells, including B lymphocytes, macrophages,
dendritic cells, epithelial cells and endothelial cells [1, 2].
In cancer, CD40 expression is found on hematological
cancer cells, such as those in lymphoma, leukemia and
multiple myeloma cells [3-5]. There is growing evidence that
CD40 is also expressed on solid tumors including bladder,
breast and gynecological cancers [6-8].
CD40 and CD40-ligand (CD40L or CD154) pathway is
of particular interest due to its potential implication in
pathogenesis and immunotherapy for cancer. For example,
CD40 receptors on B cells, upon activation by CD40-L

*Address correspondence to this author at the Antigen Presentation

Research Group, Department of Medicine, Imperial College London,
Northwick Park and St Marks Hospital Campus, Level 7W, Watford Road,
Harrow, HA1 3UJ, United Kingdom; Tel: +44 20 8869 3534; Fax: +44 20
8869 3532; E-mail: s.knight@imperial.ac.uk
1568-0096/14 $58.00+.00

expressed mainly on activated T cells, induce clonal

expansion of B cells. Hence CD40 is implicated in the
survival and proliferation of B cells engaged in secondary
immune responses. In macrophages, activation leads to
amplification of adaptive immune responses to intracellular
and extracellular pathogens [9].
The Relationship Between DCs and CD40
Dendritic cells (DCs) are the most potent antigen
presenting cells, capable of sampling antigens and initiating
cytotoxic T-lymphocyte response against cancer cells [10].
DCs express receptors for CD40. Upon up-regulation by
CD40-L, DCs become mature and activated followed by
both a strong innate immune response and T-cell priming [2,
11, 12]. CD40 ligation enhances the survival of DCs and
induces the production of anti-tumor cytokines such as IL-8,
TNF- and IL-12, which is critical for activation of Th1
response and cytotoxic T-lymphocyte activities [2, 13].
Therefore, CD40/CD40L interaction is critical in DC
maturation, and for effective cell-mediated tumor immunity.
This process is regulated by expression of the co-stimulatory
molecules, CD80 and CD86 [14].
2014 Bentham Science Publishers

Current Cancer Drug Targets, 2014, Vol. 14, No. 7

Characteristic consequence of CD40 activation on DCs

has led to research on utilizing CD40-CD40L pathway to
develop protective immunity against cancer. In 1999, the
first animal studies showed that CD40 agonists promote
effective tumor immunity by overcoming T cell tolerance
and enhancing cytotoxic T cell responses in tumor-bearing
mice. Subsequent animal studies further proved that CD40
ligation leads to effective anti-tumor immune responses as a
direct result of activation and maturation of DCs [15].
Currently, the findings from such studies have been
applied in developing a drug formulation targeting
CD40 pathway in treating patients with advanced-stage
cancer. Various route of administration and methodologies
have been used in current randomized control studies, either
as a direct CD40 agonist or via adenovirus gene therapy
[16].
Aim of the Review
Despite promising findings from animal studies and
preliminary clinical trial of CD40 agonists, there have been
few in vivo and in vitro studies describing the phenotype of
DCs in cancer, specifically the expression of CD40. Such
information is vital in fully understanding the systemic and
local immunological effect of cancer on CD40 expression in
DCs, especially because of the importance of CD40/CD40L
pathway in future immunotherapy for cancer. Therefore, the
aim of this systematic review is to summarize the available

Lee et al.

data on the characterization of DCs, specifically the

expression of CD40, in various solid cancers and to
determine the implication of findings in pathogenesis and
potential future immunotherapy. For the purposes of this
review, both systemic (blood) and tumoral CD40 expression
on DCs of solid tumors was investigated; hematological
malignancies were excluded due to difference in mechanisms
of cancer pathogenesis and treatment.
LITERATURE SEARCH AND REVIEW
A systematic review on CD40 expression on DCs in
cancer was performed with reference to preferred reporting
items for systematic reviews and meta-analysis (PRISMA)
[17] (see diagram 1).
Search Strategy
Pubmed/MEDLINE, Cochrane and Google scholar
databases were searched using combination of terms:
CD40/Bp50/p50/CDW40/TNFRSF AND dendritic cell/
antigen-presenting cells AND cancer/neoplasm/tumor/
malignancy published between September 1947 and March
2014. Duplicates and selected abstracts were reviewed using
EPPI Reviewer 4.0 by three researchers (GHL, AA and
GM). Selected abstracts and full papers were further
reviewed by GHL, AA, GM and HOA with any discordance
resolved by discussion with HOA as moderator.

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The Role of CD40 Expression in Dendritic Cells in Cancer Biology

Eligibility Criteria
Experimental and original (in vivo, ex vivo and in vitro)
studies describing the expression of CD40 on DCs in cancer
were included. Studies on the effect of cancer cell lines on
expression of CD40 on DCs were also included.
Studies were limited to those published in the English
language and conducted using human cells. Reviews, letters,
comments, conference proceedings, abstracts, non-English
language and animal studies were excluded. Studies on
CD40L without description of CD40 expression on DCs in
cancer were excluded. For reasons mentioned above, studies
on hematological cancers were excluded.
Data Extraction and Synthesis
Main areas evaluated include: 1) type of cancer, 2)
number of patients studied, 3) type of DCs studied: systemic
versus tumor derived, 4) method of identifying DCs:
technique, type of analysis used, 4) CD40 expression on
DCs, 5) conclusion of study.
MAJOR FINDINGS REPORTED IN LITERATURE
Included Studies
In total, 927 articles were identified using the search
strategy with duplicates removed. 21 original studies were
included in the qualitative synthesis after application of
inclusion and exclusion criteria.
Type of Cancer Studied and Patient Demographics
Of 21 studies included, 3 studies were on pancreatic
cancer, 4 on skin and soft tissue cancer, 1 on head and neck
cancer, 2 on gynecological cancer, 4 on breast cancer, 2 on
renal cell cancer, 1 on colorectal cancer, 1 on thyroid cancer
and 3 studies on various cancers (breast cancer, lung cancer,
gastric cancer, sigmoid cancer, prostate cancer and glioma)
and cell lines (A549 lung cancer, SW480 colorectal
cancer, HeLa cervical cancer, MCF-7 breast cancer,
Jurkat T-cell lymphoma and Raji B-cell lymphoma) (see
Table 1). Number of patients participated ranged from none
(for studies using cancer cell lines) to 173. In some studies,
there was no mention of total number of patients, but
commented on the number of specimens used [18, 19].
Depending on the type of cancer studied, there were
variations in types of DCs studied and methods in
identifying DCs. Broadly, DCs studied were either systemic
(blood) or tumor derived/infiltrating (tissue).
Eight out of 21 studies looked into blood DCs of patients
with cancer and 8 studies described DCs in cancer tissues.
Five studies looking into the effect of cancer cell lines on
DCs consisted of 3 studies on effect of cancer cell lines on
blood DCs, one study on the effect of ganglioside cell lines
derived from melanoma on Langerhans cells, and one study
on the effect of proteolytic enzymes on expression of CD40
on DCs derived from blood and ascites of ovarian cancer.
From 12 studies describing the phenotype or utilizing
blood DCs, 8 studies used monocyte derived DCs (MoDCs)
using granulocyte-macrophage colony-stimulating factor

Current Cancer Drug Targets, 2014, Vol. 14, No. 7

(GM-CSF) and IL-4. Despite minor differences in protocol

used to obtain MoDCs, there was overall consistency in
reagents, methods and quality check in production and
labeling of MoDCs. Four out of 12 studies used Ficoll
gradient to obtain peripheral blood mononuclear cells
(PBMC) and classified DCs as lineage negative and HLADR+ on flow cytometry. Despite some variations in lineage
cocktail, all except one study used CD3, CD14, CD19, CD20
and CD56 antibodies to distinguish DCs from other
immunological cells expressing HLA-DR. In one study, DCs
were isolated using cell sorting for experiments with cancer
cell lines [19].
There were also variations in method used to identify
DCs in tumor tissues. The commonest method used was
immunohistochemistry (6 out of 8 studies), while other
studies used a combination of immunohistochemistry and
reverse transcription polymerase chain reaction (RT-PCR)
or flow cytometry in identifying expression of CD40 on DCs.
In identifying DCs using immunohistochemistry, the
commonest marker used was CD83 (n=3) [18, 20, 21].
However there were some variations in antibodies used to
identify DCs because there are no DC-specific markers using
immunohistochemistry. In one study, qualitative description
of DCs was stated using tissue electron microscopy, while
expression of CD40 was determined using flow cytometry of
tumor tissue [22].
Systemic CD40 Expression on DCs in Cancer
Of the 8 studies looking into systemic CD40 expression
in blood DCs in various cancers, 3 studies using MoDCs
showed decreased CD40 expression [23-25]. Bellone et al.
assessed the effect of tumor-associated cytokines on DC
maturation and function in pancreatic cancer [23]. Their
study concluded that pancreatic tumor produces cytokines,
which suppress DC survival and proliferation. Studying
differences in systemic DC phenotype in pancreatic cancer,
Bellone et al. reported significantly decreased expression of
CD40 on MoDCs from advanced pancreatic cancer patients
compared with healthy control. Similarly, Hou et al.
assessed the expression of CD40 on MoDCs in renal cell
carcinoma (RCC) [24]; there was little or no expression of
CD40 on MoDCs from RCC patients, and when DC were
co-cultured with TNF-, the expression of CD40 was
significantly lower than that on cultured MoDCs from
healthy controls. This study concluded that DCs from RCC
exhibited a less mature phenotype and decreased T-cell
stimulatory function in response to TNF- stimulation.
Hasebe et al. investigated the MoDC phenotype of nine
patients with advanced metastatic cancer with previous
surgery and chemotherapy, which included four patients with
breast cancer, three with lung cancer, one with gastric cancer
and one with sigmoid cancer [25]. Mean fluorescence
intensity of CD40 was significantly lower in MoDCs from
the metastatic cancer group compared to healthy control,
leading to the conclusion that low levels of cell surface
expression of CD40 may be responsible for defective
allogeneic T cell stimulating capacity.
In contrast with studies on CD40 expression on MoDCs,
2 studies using DCs from PBMC (fresh DCs) showed

Current Cancer Drug Targets, 2014, Vol. 14, No. 7

Table 1.

Lee et al.

Summary of CD40 expression on DC in various cancers.

Number of Patients

Source of DC

Method of Identifying/Generating DC

CD40 Expression

Kalady 2004 [28]

13

Blood

Monocyte derived DC using GM-CSF and IL-4

No difference

Bellone 2006 [23]

24

Blood

Monocyte derived DC using GM-CSF and IL-4

Reduced

Tjomsland 2010 [26]

20

Blood

PBMC obtained using Ficoll gradient.

Lineage negative, HLA-DR positive population
identified as DC

Increased

Viac 1997 [31]

8 BCC
6 SCC
11 benign

Tissue

Immunohistochemistry
(CD1+ stained Langerhan cells)

Reduced

Amo 2000 [32]

10 SCC
10 Bowens disease

Tissue

Immunohistochemistry and RT-PCR

Reduced

20 melanoma

Tissue

RT-PCR

Reduced

n/a

Tissue

Langerhan cell purified and

isolated from epidermal tissue

Reduced

n/a

Blood DC + cell
lines

PBMC obtained using Ficoll gradient.

Lin- CD11c- CD123+ HLA-DR+ population identified
as pDC

Reduced

Zavadova 2001 [37]

15

Peritoneal fluid +
blood DC

Mononuclear leukocytes from peritoneal fluid

obtained after Ficoll gradient. Blood DCs were
monocyte derived using GM-CSF and IL-4

Increased upon
stimulation

Jia 2012 [22]

45

Tissue

Qualitative description of DC using tissue electron

microscope and flow cytometry of tumor cells

Reduced

Pockaj 2004 [29]

25

Blood

Monocyte derived DC using GM-CSF and IL-4

No difference

Thomachot 2004 [52]

n/a

Blood DC + cell
lines

Monocyte derived DC using GM-CSF and IL-4 used

to test with cancer cell lines

Reduced

Matsuura 2006 [21]

70

Tissue

Immunohistochemistry and RT-PCR

Reduced

Pinzon-Charry 2007
[30]

173

Blood

PBMC obtained using Ficoll gradient.

Lineage negative, HLA-DR positive population
identified as DC

No difference

Schwaab 1999 [18]

17

Tissue

Immunohistochemistry (CD83+ cells identified as

tumor infiltrating DC)

Increased

Hou 2010 [24]

30

Blood

Monocyte derived DC using GM-CSF and IL-4

Reduced

57

Tissue

Immunohistochemistry (CD83+ cells identified as DC)

Reduced

8 papillary
3 medullary

Tissue

Immunohistochemistry (S-100+ and CD68+ identified

as DC and macrophages)

Reduced

Pancreatic cancer

Skin cancer

Essner 2001 [33]

Bennaceur 2006 [19]
Head and neck cancer
Bekerdjlan-Ding 2009
[36]
Gynaecological cancer

Breast cancer

Renal cell cancer

Colorectal cancer
Schwaab 2001 [20]
Thyroid cancer
Scarpino 2000 [34]

The Role of CD40 Expression in Dendritic Cells in Cancer Biology

Current Cancer Drug Targets, 2014, Vol. 14, No. 7

Table 1. contd.

Number of Patients

Source of DC

Method of Identifying/Generating DC

CD40 Expression

Hasebe 2000 [25]

4 breast, 3 lung,
1 gastric, 1 sigmoid

Blood

Monocyte derived DC using GM-CSF and IL-4

Reduced

Pinzon-Charry
2005 [27]

120 breast,
10 prostate, 6 glioma

Blood

PBMC obtained using Ficoll gradient.

Lineage negative, HLA-DR positive population identified as DC

Increased

n/a (cell lines for various

cancer used)

Blood DC +
cell lines

Monocyte derived DC using GM-CSF and IL-4 used to test with

cancer cell lines

No difference

Various cancer

Cancer cell lines

Seo 2011 [35]

increased CD40 expression in cancer [26, 27]. Tjomsland

et al. investigated 20 patients with pancreatic ductal
adenocarcinoma before and after 8 to 12 weeks of curative
surgery, comparing DC phenotype with that in age-matched
healthy controls; CD40 expression on systemic DCs in
pancreatic cancer patients was significantly higher than that
in healthy controls, both in the myeloid and putative
plasmacytoid DCs. CD40 expression was unchanged after
curative surgery in pancreatic cancer patients. The study
concluded that inflammatory factors (namely CXCL8 and
PGE2) were significantly raised in plasma from patients with
pancreatic cancer, leading to DC activation. However, the
authors emphasized that activated DCs in pancreatic cancer
patients are semi mature, and express low HLA-DR, which
could explain impaired ability of DCs from pancreatic cancer
patients to stimulate T cells. Similarly, Pinzon-Charry et al.
studied DC phenotypes of 120 patients with breast
adenocarcinoma at various stages (stage I n=37, stage II
n=59, stage IV n=24), 10 patients with prostate cancer and 6
patients with high-grade malignant glioma [27]. In the cohort
breast cancer patients, the percentage of DCs expressing
CD40 and the intensity of labeling were significantly higher
only in the stage IV metastatic group compared to healthy
controls. However, further analyses demonstrated that DCs
expressing high CD40 were HLA-DR+ immature cells
(HLA-DR+CD11c-CD123-). DR+ immature cells had lower
antigen capture, presentation and phenotypic maturation in
response to inflammatory mediators, suggesting a role in
tumor escape in cancer.
In other studies, there were no differences in CD40
expression between cancer and healthy control [28, 29].
Kalady et al. studied MoDCs from 13 patients with
pancreatic adenocarcinoma who had undergone neoadjuvant
chemotherapy and compared them with 10 healthy
volunteers [28]. Analyses showed similar cell surface marker
profiles from MoDCs of study groups, with no difference in
CD40 expression on MoDCs between cancer and control
groups. The study concluded that DCs from patients with
pancreatic cancer retained normal immunological function.
However, the patient group underwent chemoradiotherapy
prior to the study, which may have an effect on the DC
phenotype and function, and, therefore, may not be a true
representation of circulating blood DC phenotype in
pancreatic cancer. Pockaj et al. demonstrated that there was

reduced expression of MoDC co-stimulatory markers

including CD80 and CD86 [29]. However, there was no
significant decrease in CD40 expression on MoDCs from
patients with breast cancer. It is notable that the study group
consisted of patients with varying stages and grade of breast
cancer; subgroup analysis was not done due to small
numbers of patients (n=25).
In another study by Pinzon-Charry, there was no
significant change in expression of CD40 on DCs before (2
weeks prior to surgery) and after curative breast cancer
operation (at 6, 24 and 48 weeks after surgery), suggesting
that removal of cancer tissue may not affect the expression
of CD40 on DCs [30].
CD40 Expression on DCs of Cancer Tissue
Despite variation in methods and definition of DCs in
cancer tissue, there was general consensus that in cancer
tissues DC expressed less CD40 compared with those in
surrounding healthy tissue. In skin cancer, CD40 expression
on DCs decreased with increasing severity of cancer, i.e.
there was less CD40 expression in squamous cell carcinoma
compared to Bowens disease, which is squamous cell
carcinoma in-situ [31, 32]. In melanoma, there was reduced
expression of CD40 in sentinel nodes compared to nonsentinel nodes [33].
Interestingly in renal cell carcinoma, Schwaab et al.
demonstrated that there was increased expression of CD40 in
tumor infiltrating DCs (tiDCs) expressing CD83. CD40
expression in tiDCs further correlated with increased
expression of CD1a. Schwaab et al. concluded that increased
expression of CD40 and CD1a in tiDCs of renal cell
carcinoma could suggest antigen presentation occurring
within tumor tissue rather than in lymph nodes [18].
In colorectal cancer, the majority of primary tumor
derived DCs expressed CD40. However, the density of
mature DCs co-expressing CD83 and CD40, was reduced in
metastatic tumor, consistent with findings from skin cancer
[20].
Scarpino et al. noted that tiDCs (S-100+ and CD68+) in
thyroid cancer were immature, being moderately positive for
CD40 and negative for CD86 [34].

Current Cancer Drug Targets, 2014, Vol. 14, No. 7

Effect of Cancer Cell Lines on Expression of CD40 on

DCs
Various cell lines were used to test their effects on
expression of CD40 on DC. Bennaceur et al. extracted
Langerhans cells using density gradient enrichment from
normal skin and tested the effects of ganglioside cell lines
[19]. Their experiments showed that ganglioside cell lines
decreased the expression of CD40 in Langerhans cells,
giving an indication that melanoma potentially decreases
expression of CD40 on DCs. Similar results were shown
using breast cancer cell lines T47-D, CLB-SAV and
MCF-7 - where expression of CD40 in MoDCs decreased
upon conditioning with cancer cell lines. However, Seo et al.
demonstrated no change in expression of CD40 in MoDCs
upon conditioning with MCF-7 and Jurkat cell lines [35].
In head and neck cancer, tumor derived factor,
prostaglandin E2 (PGE2) decreased expression of CD40 in
plasmacytoid DCs isolated from healthy controls, indicating
a potential mechanism by which CD40 expression on DCs is
decreased in cancer [36].
Zavadova et al. tested the effect of proteolytic enzymes
on DCs derived from blood and ascites from patients with
ovarian cancer [37]. Previous studies had shown that
proteolytic enzymes consisting of chymotrypsin, papain and
trypsin have immunostimulatory properties, leading to
maturation of DCs. In this study, CD40 expression on DCs
derived from blood and ascites of ovarian cancer patients
were used as an indication of maturation. Proteolytic
enzymes increased expression of CD40 on DCs both from
blood and ascites, indicating a potential therapeutic target for
ovarian cancer.
CD40 Expression on DCs in Metastatic Disease
Four out of 21 studies commented on CD40 expression
on DC in metastatic cancers. Hasebe et al.s study
concentrated on metastatic cancer patients of various
primaries including breast, lung, gastric and sigmoid colon
[25]. Their study reported significantly reduced expression
of CD40, CD11c, CD86 and HLA-DR on MoDCs in
metastatic cancer patients, compared to healthy controls. In
contrast, Pinzon-Charry et al. demonstrated increased
expression of CD40 on circulating DCs in metastatic stage
IV breast cancer patients (n=9) compared to stage I (n=20)
and stage II (n=12) breast cancer patients, using flow
cytometry to identify the DC population in PBMC (Lin /HLA-DR+ cells) [27]. Bellone et al. further subdivided his
pancreatic cancer group into early (n=14) and advanced
(n=10) stage pancreatic cancer [23]. Expression of CD40,
CD80, HLA-DR on MoDCs was significantly decreased in
patients with advanced stage pancreatic cancer, but not in
early stage pancreatic cancer, when compared with healthy
controls. The study concluded that advanced but not early
stage pancreatic cancer produced significant amounts of
tumor-derived cytokines to profoundly affect the phenotype
and function of DC
Schwaab et al. compared expression of CD40 in tiDCs
between non-metastatic (n=44) and metastatic (n=13)
colorectal cancer patients [20]. Mucosal DC density was
significantly lower in metastatic cancer, compared with non-

Lee et al.

metastatic cancer or normal mucosa. Significantly fewer

tissue cells expressed CD83 and CD40 ere in the metastatic
group compared to the non-metastatic group. However,
when patients were regrouped according to tumor stage,
there was no difference in CD40 expression on tiDCs
between stage I-III and stage IV cancer groups. The authors
commented that it is likely that lower DC density and CD40
expression in tiDCs reflects a difference in immune response
to metastasis rather than an immune deficit associated with
metastasis.
CRITICAL COMMENTS ON REPORTED RESULTS
A consensus from these studies is that tumor or tumorderived factors have direct or indirect effects on the function
of DCs in cancers, but that the phenotypic expression of
CD40 varies depending on type and severity of cancer,
source of DCs and techniques used in identifying DC.
Reduction in CD40 in tiDCs and MoDCs in cancer
contrasted with increased numbers of CD40 positive
circulating DCs in some patients; partially activated DCs in
the circulation provides encouragement that cancerrelated
pro-inflammatory activity can be induced and identified
systemically in some patients. This concept of a relationship
between tumor immunity and CD40 expression on DCs is
supported by evidence that CD40 was reduced with
increasing disease severity in skin cancer [31, 32] and was
lower in colorectal cancer tiDCs with metastatic disease [20].
There are multiple mechanisms by which the increasing
presence of tumor may incapacitate the functioning of the
DCs, for example on the role of myeloid cell immaturity
and suppressor cell on DCs in cancer, but few of these
effects have been studied for their impact on CD40
expression [38].
Variations in Results and Explanation for the Expression
of CD40 on DC in Cancer
Increased expression in CD40 on DCs implies activation
of DCs. Theoretically, this would lead to increase in Th-1
immune response and consequently increased cytotoxic Tcell activity. Blood MoDCs showed reduced expression of
CD40 in cancer compared to control. Hou et al. found that
there was little or no expression of CD40, CD80 and CD83
expression in MoDCs from renal cancer patients [24].
Furthermore, upon culturing with TNF-, there was further
reduction in expression of CD40 on DCs from cancer
patients; tumor antigens rendered DCs less mature and
functionally less capable of initiating cytotoxic T-cell
activity. Similar conclusions were drawn from Hasebe et al.,
where reduced expression of CD40, CD86 and HLA-DR on
MoDCs from cancer patients was responsible for defective
allogenic T-cell stimulating capacity. It was suggested
that the presence of mature antigen presenting cells
expressing CD40 in renal carcinomas meant that local
antigen presentation was occurring [18]. However,
accumulation of more mature DCs in the tumor tissue might
result from lack of migration of these cells to the lymph
nodes; without presentation of antigen to nave T cells
located within the lymph nodes effective adaptive immune
activity may not occur but leave a less effective innate
immune activity within the tumor.

The Role of CD40 Expression in Dendritic Cells in Cancer Biology

However, studies from Tjomsland et al. and PinzonCharry et al. demonstrated increased CD40 expression on
blood DCs in cancer patients compared to healthy controls
[26, 27]. Tjomsland et al. demonstrated that despite increased
CD40 expression, DCs from pancreatic cancer patients were
mostly semi-mature with impaired immunostimulatory
function. Furthermore, they studied the effect of inflammatory
factors such as CXCL8 and PGE2, which are increased in
pancreatic cancer, as the prime cause of activation of DCs in
plasma.
Pinzon-Charrys group also investigated the various
subtypes of DCs including HLA-DR+, CD11c- and CD123 DC subtype, which was classified as immature DR+ cells.
CD40 expression was increased in genuine blood DCs
(Lin- and HLA-DR+ cells), but the immature subtype
demonstrated even higher expression of CD40. These
findings suggested that CD40+ DCs in cancer maybe
predominantly immature as a direct result of cytokines and
factors produced by the tumor. This study added that such an
abundance of immature DCs was a consequence of resistance
to apoptotic effects of tumor, and this accumulation leads to
decline in numbers of mature blood DCs, which will
eventually favor tumor evasion.
Functional studies on tiDCs were rarely done to
investigate the mechanisms leading to decreased CD40
expression. In skin cancer studies, there were no reports of
functional experiments to explain such findings, but
hypotheses were based on previous in vitro studies. Viac et
al. mentioned that production of cytokines from the tumor
microenvironment, such as TNF-, affected CD40
expression in immunological cells [31]. Jia et al.s group
gave a descriptive analysis of tumor-derived DCs in
endometrial cancer without functional studies [22]. Their
discussion was based on previous in vitro studies showing
increased production of IL-10 by cancer cell lines resulting
in suppression of DCs maturation, which may not be fully
representative of live tumor-derived DCs [39-41].
Only one study by Schwaab et al. [20] investigated
possible mechanisms to explain reduced CD40 expression in
tiDCs in metastatic colorectal cancer. Schwaabs team
looked into the correlation of cytokines in tumor microenvironment such as TNF-, IL-10, TGF- and VEGF on
DC density and maturation status; only increased TNF-
expression on tumor infiltrating lymphocytes correlated with
increased maturation of DCs in primary colorectal cancer
compared to metastatic colorectal cancer.
Cultured DCs Versus Circulating DCs in Human
Experimental human DC research is frequently based on
the in vitro study of MoDC using GM-CSF and IL-4 or other
cytokines [42]. However, this method is based on previous
research on mouse model, where subtypes of DCs could be
derived from common granulocyte-macrophage progenitors
[43]. However, it is unclear whether there are committed DC
progenitors in humans [44] and there are no published
human studies demonstrating in vivo differentiation of
monocytes to DCs in situ [43].
There are limitations in using MoDCs because they are
different from blood and tissue DCs in vivo. They can be

Current Cancer Drug Targets, 2014, Vol. 14, No. 7

used as a model for studying effects of drugs and reagents.

However, MoDCs are generated using large boluses of
cytokines in a culture for 5 to 7 days, which may have an
effect on the characteristics of these cells. For example, we
found that, not only do monocytes express different tissue
homing markers from those of DCs, but MoDC also lose
their homing markers during culture and may not therefore
be able to direct the T cells they stimulate to a suitable tissue
location [45]. In addition, MoDCs are immature, express low
levels of CD80 and CD86 and are weak stimulators of T cell
in vitro [46, 47].
Therefore, it is not surprising to observe differences in
CD40 expression in DCs depending on how DCs were
identified or cultured. Despite the use that has been made of
MoDCs in studying mechanisms and signaling pathways,
they may not be a true representation of circulating
DCs. Despite heterogeneity in various preparations of
DCs in PBMC, MacDonald et al. provides a summary of
characterization of human blood DC subsets demonstrating
distinct differences between lineage negative/HLA-DR
positive DCs, MoDCs and monocytes [48].
Taken together, MoDCs may not be representative of DC
present in vivo. Hence, this could be the reason for the
variations in results between studies using fresh DCs versus
MoDCs in cancer.
Variation in Methodology in Identifying tiDC
There was even more variation in studies looking into
tissue DCs. There was no consensus in the antibodies used to
identify DCs in tumor tissue. This variation could have been
inevitable because there is no specific marker for DCs in
tissue. Therefore most studies compromised by choosing the
best suitable antibodies for tiDCs or used additional
techniques such as RT-PCR or flow cytometry.
Tissue immunohistochemistry has its inherent limitation.
It is normally a qualitative or semi-quantitative study,
describing the pattern and presence of antigen within tissue
architecture. This technique limits the number of antibodies
used to stain immune cells, thus making it challenging to
identify tiDCs confidently. In 6 studies using immunohistochemistry to identify tiDCs, various antibodies were
used to identify DCs, including CD1a, CD83 and S100.
However, none of these markers are specific for DCs. The
commonest marker used, CD83, is not exclusive for DCs,
and is mostly used as a marker for DC maturation [49, 50].
In one study, CD68 was used to distinguish DCs and
macrophages [34]. However, CD68 is not exclusively
expressed in macrophages and some tissue myeloid DCs
express CD68 [51].
Some studies on tiDCs have utilized RT-PCR or flow
cytometry, in addition to immunohistochemistry work,
giving quantitative and more robust data on the expression of
CD40 on DCs in cancer tissue [21, 32]. However, studies
such as Jia et al., only mentions qualitative description of
suspected tiDCs and its expression of CD40 [22].
In studies using cell lines, there were again variations in
types of DCs used to test the effects of cancer cell lines.
Bennaceur et al. used a unique gradient-separation protocol

Current Cancer Drug Targets, 2014, Vol. 14, No. 7

to isolate Langerhans cells and tested the effects of

ganglioside cell lines to gain an indication of possible effects
of melanoma on DCs. Some studies used MoDCs to test the
effect of cancer cell lines, while others used freshly isolated
DCs [35-37, 52]. In most studies the conclusion was that the
tumor cell lines reduced DC maturation as indicated from
CD40 expression.
Selection Criteria of Patients and Healthy Controls
In addition to the variation in data included in this
review, there was overall low patient numbers in most
studies looking at CD40 expression on DCs. With the
exception of Pinzon-Charrys group, which looked into over
100 patients, most studies had patient numbers less than 50
[27, 30]. Furthermore, most studies did not provide enough
demographic data on the patients they studied, such as age,
stage of cancer, medical co-morbidities and medication taken
by patient group. All these factors could be potential
confounding factors influencing the results.
Selection criteria for healthy controls were not robust
especially in studies comparing blood DC phenotype in
cancer. Apart from mentioning that controls were age and
sex-matched in some studies, no study disclosed the full
demographics of healthy controls, including medical comorbidities and current medications. However, in one study,
it mentioned that controls taking COX-2 inhibitors were
excluded [29]. Finally, there were inconsistencies in number
of controls used compared to patient group, where in some
cases, the number of cancer patients participated outnumbered
the healthy controls [24, 29].
Implication on DC Immunotherapy using CD40 Ligands
This review has highlighted the variations in results of
CD40 expression on DC depending on type and stage of
cancer. In assessing circulating DCs in patients with
metastatic breast cancer and non-metastatic pancreatic
cancer, there was increased expression of CD40 [26, 27].
However MoDCs derived by in vitro culture of circulating
monocytes from patients with metastatic pancreatic, breast,
lung, gastric, sigmoid and non-metastatic renal cancer
expressed low CD40 [23-25]. Despite contrasting results, all
studies identified and provided mechanistic pathways to
explain DC dysfunction in patients with various solid
tumors, but these pathways did not necessarily correlate with
increased or decreased expression of CD40 on DCs.
In assessment of CD40 expression in tumor-derived DCs,
there was some consistency in results, showing reduced
CD40 expression with cancer progression, especially in skin
cancers [20, 21, 31-34]. However, overall inconsistencies in
expression of CD40 on DCs in cancer highlight the
complexity of cancer biology, where different cancers
may have varying influences on systemic and tumoral
immunology. In cancer patients with circulating DCs
expressing high CD40, the response to immunotherapy using
CD40-CD40 ligand pathway may be different from that in
patients with DCs expressing low CD40. Also, studies on
tiDCs showing reduced expression of CD40 could suggest
possible improved outcome of using direct intratumoral
injection of agonistic anti-CD40 antibodies compared to
systemic administration [53].

Lee et al.

Results from our review show that human in vitro and ex

vivo studies may provide additional evidence in support of
agonistic anti-CD40 monoclonal antibodies (anti-CD40
mAb) in cancer immunotherapy, especially in cancers with
tiDCs expressing low CD40 demonstrated in studies on
skin cancers [31-33]. Vonderheide et al.s latest review on
agonistic CD40 antibodies in cancer therapy summarizes
the scientific background and clinical outcome on the
ongoing Phase I trials on patients with melanoma, pancreatic
carcinoma, mesothelioma and hematologic malignancies
[54]. Initial reports on the effectiveness of anti-CD40 mAb
in melanoma (n=29) described partial response rate of 14%
[55], whereas in pancreatic cancer (n=21), in adjunct to
chemotherapy, resulted in 19% partial response rate and 52%
stable disease [56]. Another Phase I trial on various tumor
types (n=27), reported that 26% of patients achieved stable
disease, but there was no comment on partial response rate.
However, the reviews on agonistic anti-CD40 in cancer
therapy extensively describe previous research on animal
models to assess efficacy and safety of anti-CD40 mAbs
[54, 57, 58]. They rarely comment on previous human
studies on CD40 expression on DCs in cancer, despite innate
differences in blood and tissue DC types between mice and
humans [44]. Previous research on expression of CD40 on
DCs in various cancers may provide insight into how
different cancers influence DC phenotype and function and
may provide valuable information on selecting cancers that
may be more suitable for therapeutic use of anti-CD40
mAbs.
The CD40/CD40L pathway is likely to play an essential
role in development of future cancer immunotherapy and is
especially promising in hematological malignancies [16, 58].
However, there are fewer studies showing enhanced
therapeutic benefit of CD40L in solid tumors compared to
hematological malignancies and it remains in early stages of
vaccine development [55, 59].
However, it must be emphasized that therapeutic
implication of CD40/CD40L pathway is not entirely based
on improving DC function. Additionally, although studies
on tiDCs show some consensus in reduced expression of
CD40, further research that is cancer-specific using more
standardized protocols is required to fully understand the
role of CD40 expression in DCs in cancer.
CONCLUSION
This review was conducted to highlight the importance of
CD40 expression on DCs in cancer. The CD40/CD40L
pathway has been identified as a potential key pathway for
cancer immunotherapy. Already there have been phase 1
clinical trials looking into the safety and effectiveness of
CD40L in treating various cancers [16]. Results show that in
blood DCs, there was generally increased expression of
CD40 in cancer patients when freshly isolated DCs were
studied, but reduced expression of CD40 when MoDCs were
used. In studies analyzing tumor tissue, there was a trend
towards reduced expression of CD40 in tiDCs. Finally,
cancer cell lines or tumor supernatants decreased expression
of CD40 in blood DC. There is variation in methodology and
general lack of consensus in identifying DCs. Also the
differences in results could indicate the potential effect of

The Role of CD40 Expression in Dendritic Cells in Cancer Biology

other confounding factors discussed in this review. There is

still need for further in vivo, in vitro and ex vivo studies on
various cancers where there is a potential diagnostic,
prognostic and therapeutic role of DCs. However, there is
also a need for consensus in research methodology and core
outcome set to enable future systematic review to confirm
the effects of cancer on DCs and to provide an evidence-base
for determining potential benefits of intervention in the
CD40/CD40L pathway in different types and stages of
cancer.

Current Cancer Drug Targets, 2014, Vol. 14, No. 7

[2]
[3]

[4]

[5]

CONFLICT OF INTEREST
The author(s) confirm that this article content has no
conflict of interest.

[6]
[7]

ACKNOWLEDGEMENTS
GHL literature search and wrote the manuscript; AA,
GM literature search; DB, SKC critically reviewed the
manuscript; SCK corresponding author, critically reviewed
the manuscript; HOA moderator and critically reviewed
the manuscript.

[8]

[9]

LIST OF ABBREVIATIONS
anti-CD40 mAb

agonistic
antibody

anti-CD40

monoclonal

BCC

basal cell carcinoma

CD40L

CD40 ligand

COX-2

cyclooxygenase 2

DCs

dendritic cell(s)

GM-CSF

granulocyte-macrophage
stimulating factor

[11]

[12]

colony[13]

Lin

lineage

MoDCs

monocyte derived dendritic cell(s)

PBMC

peripheral
cell(s)

pDC

plasmacytoid dendritic cell(s)

PGE2

prostaglandin E2

RT-PCR

reverse transcription
chain reaction

SCC

squamous cell carcinoma

TGF-

transforming growth factor beta

tiDCs

tumor infiltrating dendritic cell(s)

TNF-

tumor necrosis factor alpha

VEGF

vascular endothelial growth factor

blood

[10]

mononuclear

polymerase

[14]
[15]

[16]

[17]

[18]

[19]

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Accepted: July 11, 2012