Jurnal Reading

Mechanisms of allergen immunotherapy for inhaled allergens and predictive

biomarkers

Mohamed H. Shamji, PhD, FAAAI, and Stephen R. Durham, MD, FRCP London, United Kingdom

Dibacakan oleh:

Ni Nyoman Githa Setiani

17014101082

Massa KKM : 15 Januari 2018 – 11 Februari 2018

Pembimbing:

dr. Augustien Y. Tamus, SpTHT-KL

BAGIAN TELINGA HIDUNG TENGGOROK KEPALA LEHER

FAKULTAS KEDOKTERANUNIVERSITAS SAM RATULANGI

MANADO

2018
 LEMBAR PENGESAHAN

Jurnal Reading

Mechanisms of allergen immunotherapy for inhaled allergens

and predictive biomarkers

Telah dikoreksi dan disetujui pada Januari 2018.

Pembimbing

dr. Augustien Y. Tamus, SpTHT-KL

Mechanisms of allergic diseases
Mechanisms of allergen immunotherapy for inhaled allergens
and predictive biomarkers
Mohamed H. Shamji, PhD, FAAAI, and Stephen R. Durham, MD, FRCP
Allergen immunotherapy is effective in patients with IgE-
dependent allergic rhinitis and asthma. When immunotherapy is
given continuously for 3 years, there is persistent clinical benefit
for several years after its discontinuation. This disease-modifying
effect is both antigen-specific and antigen-driven. Clinical
improvement is accompanied by decreases in numbers of effector
cells in target organs, including mast cells, basophils, eosinophils,
and type 2 innate lymphoid cells. Immunotherapy results in the
production of blocking IgG/IgG4 antibodies that can inhibit IgE-
dependent activation mediated through both high-affinity IgE
receptors (FcεRI) on mast cells and basophils and low-affinity IgE
receptors (FcεRII) on B cells. Suppression of TH2 immunity can
occur as a consequence of either deletion or anergy of antigen-
specific T cells; induction of antigen-specific regulatory T cells; or
immune deviation in favor of TH1 responses. It is not clear whether
the altered long-term memory resides within the T-cell or the B-cell
compartment. Recent data highlight the role of IL-10–producing
regulatory B cells and ‘‘protective’’ antibodies that likely
contribute to long-term tolerance. Understanding mechanisms
underlying induction and persistence of tolerance should identify
predictive biomarkers of clinical response and discover novel and
more effective strategies for immunotherapy. (J Allergy Clin
Immunol 2017;140:1485-98.)
Key words: Immunotherapy, mechanisms, allergic rhinitis, allergic
asthma, long-term tolerance, T cells, B cells, type 2 innate
lymphoid cells, IgE-FAB, biomarkers
From the Immunomodulation and Tolerance Group; Allergy and Clinical Immunology;
Section of Inflammation, Repair and Development; National Heart and Lung
Institute; Imperial College London, and the MRC & Asthma UK Centre in Allergic
Mechanisms of Asthma.
Disclosure of potential conflict of interest: M. H. Shamji has received grants from
ALK, Regeneron, Merck, ASIT Biotech, and the Immune Tolerance Network, and
has received personal fees from ASIT Biotech, ALK, and Allergopharma. S. R.
Durham has received grants from Regeneron, Biotech Tools, ALK, Food Standards
Agency, the National Institute of Health Research, and the Immune Tolerance
Network/National Institute of Allergy and Infectious Diseases, and has received
personal fees from Anergis, Circassia, Biomay, Merck, Allergy Therapeutics, ALK,
and Med Update GmbH.
Received for publication September 12, 2017; revised October 25, 2017; accepted for
publication October 25, 2017.
Corresponding author: Stephen R. Durham, MD, FRCP, Allergy and Clinical Immunology,
National Heart & Lung Institute, Imperial College London, Dovehouse St, London SW3
6LY, United Kingdom. E-mail: s.durham@imperial.ac.uk.
The CrossMark symbol notifies online readers when updates have been made to
the article such as errata or minor corrections
0091-6749/$36.00
2017 The Authors. Published by Elsevier Inc. on behalf of the American Academy of
Allergy, Asthma & Immunology. This is an open access article under the CC BY-
NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
https://doi.org/10.1016/j.jaci.2017.10.010
Terms in boldface and italics are defined in the glossary on page 1486.
London, United Kingdom
Abbreviations used
Breg: Regulatory B
C1Q: Component 1Q
CRTH2: Chemoattractant receptor–homologous molecule expressed
on TH2 lymphocytes
DAO: Diamine oxidase
DC: Dendritic cell
DCreg: Regulatory dendritic cell
ELIFAB: Enzyme-linked immunosorbent–facilitated antigen-binding
assay
FOXP3: Forkhead box P3
ILC: Innate lymphoid cell
ILC2: Group 2 innate lymphoid cell
iTreg: Inducible regulatory T
LT: Leukotriene
nTreg: Natural regulatory T
RIPK4: Receptor-interacting serine/threonine-protein kinase 4
sIgE: Allergen-specific IgE
sIgG: Allergen-specific IgG
TFH: Follicular helper T
TFR: Follicular regulatory T
Treg: Regulatory T
VLA4: Very late antigen 4, integrin a4b1
Discuss this article on the JACI Journal Club blog: www.jaci-
online.blogspot.com.
Allergen immunotherapy is effective in selected patients with
allergic rhinitis, including those with mild/moderate asthma.1,2
There is heterogeneity in the populations studied, the different
allergen products and protocols used, and the clinical outcomes
used to document efficacy and safety. 3 Nonetheless, recent
guidelines4 confirm that immunotherapy is particularly
effective in patients with seasonal rhinitis, and recent data
strongly support its use in perennial allergy caused by house
dust mites.5
Subcutaneous immunotherapy involves weekly updosing
injections, followed by monthly maintenance injections for at
least 3 years.1,6,7 In view of occasional systemic allergic side
effects, subcutaneous immunotherapy requires administration in
a specialist allergy clinic with access to resuscitative measures.
Sublingual immunotherapy involves daily drops or tablets
placed under the tongue. Sublingual immunotherapy is effective
and safer than subcutaneous immunotherapy, such that it is self-
administered by the patient at home.1,8 Sublingual and
subcutaneous immunotherapy are effective generally within 2 to
4 months of initiating treatment and can be given
presesonally/coseasonally for short-term benefit. Indirect
1485
1486 SHAMJI AND DURHAM
comparisons have suggested that immunotherapy might be more
effective than antiallergic drugs. In contrast to antiallergic drugs
and currently available mAb therapies, when allergen immuno-
therapy is given continuously for 3 years, both routes have been
shown to be disease-modifying, manifest as long-term remission of
9,10
symptoms for at least 2 to 3 years after discontinuation.
In this review we explore historical and recent data on the
mechanisms of immunotherapy for inhalant allergens. Our
expectation is that a greater understanding of the underlying
mechanisms of tolerance will identify potential biomarkers that
could predict and/or monitor the response to treatment. Such
knowledge could inform new potential treatment strategies.
OVERVIEW OF MECHANISMS OF ALLERGIC
RHINITIS AND ASTHMA IgE and mast cells
The cardinal features of allergic rhinitis include increased
allergen-specific IgE concentrations to clinically relevant allergens,
IgE-dependent activation of mast cells, and local eosinophilia in
target organs. In addition to systemic and regional lymphatic
sources of IgE, specific IgE can be synthesized and produced
11
locally by B cells within the respiratory mucosa, thereby
accounting for the occasional phenomenon of ‘‘local
GLOSSARY
Bet v 1: A potent allergen from trees within the order Fagales, which is the
main cause of type I allergies observed in early spring and characterized
by hay fever, dermatitis, and asthma.
METHYLATED CpG SITES: CpG sites are regions of DNA in which a
cytosine nucleotide occurs next to a guanine nucleotide separated by only
1 phosphate. Methylation of the cytosine within a gene can turn the gene
off.
c-kit (CD117): A cytokine receptor most notably expressed on the surfaces
of hematopoietic stem cells and other cell types, including mast cells.
CD117 is a receptor tyrosine kinase type III protein that binds to stem cell
factor and forms a dimer that activates its intrinsic tyrosine kinase, resulting
in phosphorylation and activation of signal transduction molecules that
produce cell signaling.
CYTOTOXIC T LYMPHOCYTE–ASSOCIATED PROTEIN 4 (CTLA-4): A
receptor that functions as an inhibitory signal and downregulates immune
responses when bound to CD80 and CD86. CTLA-4 is constitutively
expressed in regulatory T cells but only upregulated in conventional T cells
after activation.
IFN-g: A type II interferon, IFN-g is a cytokine required for innate and
adaptive immunity against viral, bacterial, and protozoal infections. IFN-g
has been shown to be an important activator of macrophages and inducer
of class II MHC molecule expression. IFN-g is produced predominantly by
natural killer (NK) and NKT cells as part of the innate immune response
and by CD4 TH1 and CD8 cytotoxic T lymphocyte effector T cells once
antigen-specific immunity develops.
ImmunoCAP (A REGISTERED TRADEMARK OF PHARMACIA
DIAGNOSTICS AB): An in vitro quantitative assay that measures allergen-
specific IgE levels in human serum. ImmunoCAP assays can be performed
on hundreds of allergens by using cellulose polymer, which provides high
binding capacity of clinically relevant allergen proteins, including those
present at very low levels.
IL-3: A growth-promoting cytokine capable of supporting the proliferation
and activation of a broad range of hematopoietic cell types, including
basophils.
The Editors wish to acknowledge Kristina Bielewicz, MS, for preparing this glossary.
J ALLERGY CLIN IMMUNOL
DECEMBER 2017
allergic rhinitis’’ with symptoms on allergen exposure in the
absence of detectable serum specific IgE or positive immediate
skin test results to relevant allergens.12
IgE-dependent activation is detectable during the immediate
(0- to 60-minute) response after nasal allergen provocation.
Allergen cross-linking of adjacent surface IgE molecules on
mast cells and basophils triggers within seconds or minutes the
release of preformed mediators, such as histamine13 and
tryptase,14 contained within intracytoplasmic granules. Newly
formed mediators derived from arachidonic acid within the
membrane lipid include sulphidopeptide leukotrienes (LTs;
LTC4, LTD4, and the terminal metabolite LTE4),12 platelet-
activating factor, and prostaglandin D2. The biological
properties of these mediators are consistent with the local
vasodilatation, edema formation, local neurogenic stimulation,
and mucus secretion that characterize typical nasal allergen–
induced immediate type I hypersensitivity. In the lower airways
bronchial smooth muscle contraction, as well as edema and
mucus hypersecretion, contribute to acute bronchoconstriction.
A proportion of subjects have a late response at 2 to 10 hours
after challenge. The late response is characterized by tissue
eosinophilia, nasal congestion, and mucosal hyperreactivity to
both allergic and nonallergic triggers that can last for days or
even weeks after a single nasal allergen challenge. In contrast
IL-6: A cytokine also known as IFN-b2 and implicated in a wide variety of
inflammation-associated disease states, IL-6 has been associated with B-
cell maturation and has been shown to act as an endogenous pyrogen
capable of inducing fever in patients with autoimmune diseases or
infections.
IL-12: A cytokine produced by dendritic cells, macrophages, neutrophils,
and human B-lymphoblastoid cells (NC-37) in response to antigenic
stimulation and has been shown to be required for differentiation of naive T
cells into TH1 cells.
IL-21: A cytokine expressed in human CD41 T cells and found to be
upregulated in TH2 and TH17 subsets and follicular T cells, which induces
cell division/proliferation of various cells of the immune system, including
natural killer cells and cytotoxic T cells.
IL-25: A proinflammatory cytokine that shares sequence similarity with IL-
17 and has been shown to favor the TH2-type immune response. IL-25 can
induce nuclear factor kB activation and stimulate IL-8 production.
IL-33: A member of the IL-1 family of cytokines expressed on TH2 cells,
mast cells, and group 2 innate lymphocytes that potently drives production
of TH2-associated cytokines.
IL-35: An IL-12 family cytokine produced by regulatory, but not effector, T
and B cells that plays a role in immune suppression.
PROGRAMMED CELL DEATH 1 (PD-1): A cell-surface receptor that plays
an important role in downregulating the immune system and suppress-ing
inflammatory T-cell activation. PD-1 is an immune checkpoint that serves a
dual role of promoting apoptosis in antigen-specific T cells while
simultaneously reducing apoptosis in regulatory T cells.
THYMIC STROMAL LYMPHOPOIETIN (TSLP): A cytokine that stimulates
T-cell maturation through activation of antigen-presenting cells, such as
dendritic cells and macrophages.
TGF-b: A cytokine secreted by many cell types, including macrophages,
that controls proliferation, cellular differentiation, and inflammatory
processes in a variety of cells. It also plays a role in T-cell regulation and
differentiation.
J ALLERGY CLIN IMMUNOL
VOLUME 140, NUMBER 6
FIG 1. A and B, Mechanisms of allergic inflammation: summary of immunologic response to initial triggers of
allergic sensitization and allergic inflammation after re-exposure to inhalant allergens (see text). EC, Epithelial
cells.
to findings in patients with allergic asthma and nasal polyposis,
morphologic and immunohistochemical features of airway
remodeling are not a consistent feature of even moderate/severe
allergic rhinitis.15
TH2 lymphocytes and group 2 innate lymphoid cells
The above pathophysiologic events are under the regulation of
a distinct subset of TH2 cells. TH2 cells produce IL-4, the key
cytokine responsible for TH2 cell differentiation.16-18 IL-4 and
IL-13 induce B lymphocytes to produce ε-germline gene
transcripts,19 the first step in heavy chain gene rearrangement in
favor of IgE production. IL-4 and IL-13 upregulate vascular cell
adhesion protein 1 expression on the vascular endothelium,
promoting adhesion of very late antigen 4, integrin a4b1–
expressing eosinophils. Both stimulate mucus production from
glands in the upper and lower airways. IL-5 is responsible for
terminal differentiation and release of eosinophils from the
bone marrow and prolongs eosinophil survival by inhibiting
eosinophil apoptosis in tissues.20 Along with stem cell factor,
IL-9 is a key cytokine for the differentiation and maturation of
mast cells.21 Release of TH2 cytokines and tissue eosinophilia
are apparent during the late-phase response that occurs at 4 to
12 hours after allergen challenge.22
Group 2 innate lymphoid cells (ILC2s) represent an alternative
source of TH2 cytokines in the nasal mucosa. Innate lymphoid cells
(ILCs) are morphologically similar to lymphocytes, although they
are distinct in not expressing surface antigen
receptors or other cell lineage markers and act in an antigen-
independent manner.23,24 ILCs consist of 3 different groups
referred to as group 1 ILCs, ILC2s, and group 3 ILCs.
Group 1 ILCs constitutively express T-box transcription factor
and produce the TH1 cytokines IFN-g and TNF and provide
protection against intracellular bacteria and parasites. ILC2s
SHAMJI AND DURHAM 1487
constitutively express RAR-related orphan receptor a and
GATA-3; produce TH2 cytokines, particularly IL-5 and IL-13;
and provide immunity to helminths, as well as stimulating
allergic responses. Group 3 ILCs are characterized by the
transcription factor RAR-related orphan receptor gt, express IL-
17a and/or IL-22, afford protection against extracellular
bacteria, and are involved in tissue repair processes.
The role of ILC2s in allergic rhinitis was first identified in
patients with cat allergy, who showed increases in peripheral
blood ILC2 numbers at 4 hours after a cat allergen nasal
challenge.25 Subsequently, increases in circulating ILC2
numbers have been identified in both patients with grass allergy
and rhinitis26 and asthmatic patients27 during the grass pollen
season. ILC2s represent an abundant alternative source of TH2
cytokines and likely serve to amplify and maintain local TH2-
driven allergic inflammation. In view of recently identified
plasticity within ILC2s in tissues of patients with chronic
obstructive pulmonary disease and chronic rhinosinusitis,28 this
concept must be revisited in the context of allergic rhinitis. 18
The respiratory epithelium and dendritic cells
Although IgE-dependent mast cell activation and tissue
eosinophilia are driven by TH2 lymphocytes, TH2 cell
differentiation is dependent on the local cytokine milieu
provided by interactions between the respiratory epithelium,
local dendritic cells (DCs), and regional lymph nodes.
In an atopic subject aeroallergens pass through the inflamed
nasal epithelium, and activated epithelial cells release CCL2 and
CCL20, which recruit immature DCs (Fig 1, A). Activated DCs
migrate to regional draining lymph nodes and polarize naive T cells
into TH2 cells. DC migration is primed by IL-13 produced by
ILC2s and also by IL-4 produced principally by basophils. Within
the germinal center of the lymph node, a subset of TH cells
1488 SHAMJI AND DURHAM J ALLERGY CLIN IMMUNOL
DECEMBER 2017

FIG 2. Mechanisms of AIT. During the initial sensitization phase in patients with allergic rhinitis, low allergen
exposure at the nasal mucosal surface results in activation of epithelial cells, which then activate DCs. DCs
uptake and present antigens to naive T cells to induce allergic T H2 (Th2A) responses and IgE-facilitated antigen
presentation. Subsequent allergen re-exposure leads to mast cell and basophil degranulation, causing classic
early-phase reactions. Subsequent infiltration of other leukocytes leads to late-phase allergic inflammation. High-
dose allergen exposure by immunotherapy restores DC function, which produces IL-12, IL-27, and IL-10 and
promotes immune deviation from a TH2 to TH1 response and induction of Treg and Breg cells (including other B-
cell subsets) that produce IgA, IgG, and IgG4 blocking antibodies. Suppressive activities of Treg cells, Breg cells,
and IgG-blocking activity is indicated by red arrows. EC, Epithelial cells; TLR, Toll-like receptor.

differentiates into follicular helper T (TFH) cells. TFH cells
produce both IL-4 and IL-21, which, along with TH2 cell–
derived IL-4, promote immunoglobulin heavy chain class-
switching to IgE in B cells.
The respiratory epithelium of atopic allergic subjects
expresses cytokines that include IL-25,29 IL-33,30 and thymic
stromal lymphopoietin protein.31 These epithelial cytokines
favor development of a proallergic DC phenotype32,33 that
provides help for TH2 cell differentiation. Additionally, these
epithelially derived cytokines are major growth factors for
ILC2s that amplify and maintain local TH2-driven allergic
inflammation.34-36 During subsequent allergen exposure, IgE-
facilitated allergen recognition through FcεRI on DCs and
FcεRII on B cells amplifies the development of TH2 responses
to inhaled allergens (Fig 1, B).
DCs, depending on their maturation phase, their location, and
the associated local cytokine milieu, can either initiate and
maintain allergic inflammation (proallergic DC2s)32,33,37,38 or
alternatively promote a state of immune tolerance (tolerogenic
regulatory dendritic cells [DCregs])32,33,39-42 to sensitizing
allergens. DC2s express the markers CD141, GATA-3, OX40
ligand, and receptor-interacting serine/threonine-protein kinase
4 (RIPK4).33 When DC2s were exposed to allergen and
cocultured subsequently with T cells, they promoted
preferential TH2 T-lymphocyte responses.35-39
MECHANISMS OF ALLERGEN IMMUNOTHERAPY
Further details on mechanisms of allergen immunotherapy
can be found in Fig 2.
IgE, IgG, and IgA responses
Both sublingual and subcutaneous immunotherapy have been
associated with transient early increases in serum allergen-
specific IgE antibody levels that are followed by blunting of the
usual seasonal increases in IgE levels during natural allergen
exposure.43 These early increases are not accompanied by
untoward side effects, and it has been suggested that early TH2
priming by high allergen exposure might be important for
successful immunotherapy. Prolonged subcutaneous
immunotherapy over several years can result in a decrease in
allergen-specific IgE (sIgE) concentrations,44,45 an event that
might contribute to long-term tolerance.
In 1935, Robert Cooke and colleagues
46
demonstrated the
passive transfer of suppressive activity for immediate ragweed IgE
sensitivity in the skin by use of serum obtained from patients who
had undergone ragweed subcutaneous immunotherapy. Serum and
nasal inhibitory activity for IgE after subcutaneous immunotherapy
was subsequently shown to reside within serum IgG, IgG4, and IgA
47-51
fractions. Studies have shown 10- to 100-fold increases in
serum concentrations of IgG,
J ALLERGY CLIN IMMUNOL
VOLUME 140, NUMBER 6
7,52-55
particularly IgG4. Sublingual immunotherapy has also been
56
shown to induce allergen-specific IgG1, IgG4, and IgA
10,49,57-59
antibodies. These increases in levels of immunoreactive
antibodies have been observed after immunotherapy to both
seasonal pollens and perennial allergens, such as house dust
60,61
mite. Serum specific IgG4 levels have been shown to increase
in a time- and dose-dependent manner during grass pollen
immunotherapy.59
Several studies have highlighted the inhibitory capacity of IgG4
for IgE-dependent events. IgG4 antibodies are bispecific and have
the capacity to exchange F(ab) arms by swapping heavy-light chain
62
pairs between IgG4 molecules with diverse specificities. IgG can
63
compete with IgE for allergen, thereby blocking allergen-IgE
complex formation. This prevents cross-linking of high-affinity IgE
receptors (FcεRI) on basophils and mast cells, inhibiting histamine
release. Competition of IgG/IgG4 for IgE can also block binding of
allergen-IgE complexes to low-affinity receptors (FcgRIIb) on B
cells, thereby inhibiting IgE-facilitated antigen presentation to T
16,64-66
cells, a major driver of allergen-specific TH2 responses.
Paradoxically, although immunoreactive IgG/IgG4 levels
decreased by 80% to 90% within 1 year of stopping allergen
immunotherapy, IgG-associated serum IgE-inhibitory activity
persisted for several years and accompanied long-term clinical
49
efficacy. This suggests that despite lower levels, IgG antibodies
that persist after discontinuation of immunotherapy can have either
higher avidity or higher affinity. These data raise the possibility
that long-lived memory B cells induced by immunotherapy can
persist as a result of low-level environmental allergen stimulation,
thereby contributing to long-term tolerance.
IgG antibodies have been detected locally in both nasal fluid
and serum after immunotherapy.53 Both specific IgG4 levels
and associated inhibitory activity for IgE-facilitated antigen
binding (IgE-FAB) were increased in the nasal fluid of patients
undergoing sublingual immunotherapy compared with untreated
participants.43 The IgG4 dependency of IgE-inhibitory activity
has been shown in depletion experiments using IgG 4 affinity
chromatography. The magnitude of IgE suppression was greater
with nasal fluid than with serum, thereby highlighting the
potency of local IgG inhibitory antibodies. 52
Allergen immunotherapy and effector cells
The influence of allergen immunotherapy on effector cells has
been studied after nasal allergen provocation and during natural
seasonal pollen exposure. Both subcutaneous and sublingual
Immunotherapy inhibit early- and late-phase responses after
57,67
allergen challenge. Suppression is accompanied by a reduction
in early increases in local nasal histamine, tosyl L-arginine methyl
ester–esterase, and tryptase concentrations in nasal fluid. Inhibition of
late responses is associated with a
decrease in eosinophil numbers68 and levels of TH2 cytokines,
including IL-4, IL-5, IL-9, and IL-13.69,70 A reduction after
immunotherapy is also noted in nasal fluid levels of the CC
chemokine eotaxin, which contributes to eosinophil recruitment. A
double-blind trial of subcutaneous grass pollen immunotherapy
resulted in decreases in numbers of effector cells, including
71 72 73
CD1171 (c-kit1) mast cells, basophils, and eosinophils, in
the nasal mucosa compared with pretreatment numbers that were
72,74
significant compared with placebo-treated participants. These
local changes detected in nasal biopsy specimens were
SHAMJI AND DURHAM 1489
accompanied by improvements in seasonal symptoms and a
decrease in requirements for rescue medication. A direct
correlation was noted between reductions in IL-5 levels and
nasal mucosal eosinophil numbers and also between eosinophil
numbers and the severity of seasonal symptoms. In house dust
mite–sensitive patients sublingual immunotherapy with mite
extract inhibited local mucosal vascular intercellular adhesion
molecule 1 expression and also decreased local eosinophilia.72
These data illustrate that both sublingual and subcutaneous
immunotherapy result in decreases in recruitment, activation, or
both of effector cells at allergic tissue sites.
Allergen immunotherapy and T-lymphocyte
responses
Decreases in TH2 cell numbers. Suppression of allergen-
induced late nasal responses during subcutaneous grass pollen
immunotherapy has been associated with decreases in numbers of
CD41 T cells and local IL4 mRNA–positive T cells in the nasal
74
mucosa. These findings are supported by the observation of
decreases in TH2 cytokine levels in nasal lining fluid after nasal
69
challenge. Recent techniques that include ex vivo tetramer
75-78
analysis have enabled the phenotyping of peripheral
79-82
circulating allergen-specific T cells. This has permitted
identification of key T-cell surface markers, such as CD27,
chemoattractant receptor–homologous molecule expressed on TH2
lymphocytes (CRTH2), CD161, and chemokine receptor 4 (CCR4),
associated with type 2 proallergic responses. In patients with grass
pollen allergy, tetramer-specific T cells that did not express CD27
mostly expressed the surface markers CRTH2 and CCR4.
75-78
This
was in contrast to nonallergic subjects, whose T cells expressed low
levels of CRTH2 and CCR4 and high levels of CD27. Patients with
alder pollen allergy expressed a high frequency of CD27 2 TH2
cells that decreased after subcutaneous immunotherapy. Similarly,
in the Gauging Responses in Allergic Rhinitis to SCIT versus SLIT
Trial (GRASS),
45,70
both subcutaneous and sublingual
immunotherapy resulted in clinical improvement during 2 years
that was paralleled by a decrease in peripheral tetramer-positive
CRTH21CCR41CD272CD41 TH2 cell numbers. These changes
were paralleled by a decrease in levels of local nasal TH2
cytokines, including IL-4, IL-5, and IL-13, in nasal fluid after nasal
allergen provocation. Both numbers of circulating tetramer-positive
TH2 cells and levels of local nasal TH2 cytokines rebounded during
year 3, along with a deterioration in seasonal symptoms 1 year after
discontinuation of immunotherapy. The failure of 2 years of
9,10
immunotherapy (in contrast to 3 years of continuous treatment)
to induce durable tolerance might have been related to this re-
emergence of antigen-specific TH2 immunity.
Increases in numbers of regulatory T cells. Immune
tolerance during immunotherapy has been shown to be associated
with induction of allergen-specific regulatory T (Treg) cells.
83-88
Treg cells can be grouped into 2 subsets: natural regulatory T
(nTreg) cells, which express the transcription factor forkhead box
P3 (FOXP3), and inducible regulatory T (iTreg) cells, which
produce regulatory cytokines, such as IL-10, IL-35, and
TGF-b.59,79,83
nTreg cells. nTreg cells were first described by Sakaguchi et
al.80 In addition to the transcription factor FOXP3, nTreg cells
have increased expression of the IL-2 receptor (CD25) and low
1490 SHAMJI AND DURHAM
FIG 3. Time course of the effect of immunotherapy on surrogate clinical markers (early and late cutaneous
responses) and associated immunologic events during the induction and maintenance phases of immunotherapy
(desensitization), as well as their persistence after withdrawal of treatment (tolerance phase). Abs, Antibodies;
BAT, Basophil Activation Test; BHR, basophil histamine release response; EPR, early-phase response; LPR,
late-phase response.
expression of the IL-7 receptor (CD127). nTreg cells exert their
suppressive capacity in a direct cell-cell contact–dependent
manner.81,82 Functional roles have been proposed for
membrane cytotoxic T lymphocyte–associated protein 4,
surface-bound TGF-b, the glucocorticoid-induced TNF receptor,
and programmed cell death 1. nTreg cells have also been shown
to modulate allergen-specific T-cell responses in healthy nonatopic
89
subjects. Subcutaneous immunotherapy was associated with local
84
increases in FOXP31CD251 T-cell numbers in the nasal mucosa
compared with those in untreated control subjects. After sublingual
grass pollen immunotherapy, immunofluorescence studies on
sublingual biopsy specimens identified increases in FOXP3 1CD31
87
cell numbers in the sublingual mucosa. Human in vitro studies of
biopsy specimens of human buccal mucosa and associated lingual
tonsils and
adenoids identified the oropharyngeal mucosa as an environment
90,91
rich in protolerogenic DCs and Treg cells. Altered nTreg cell
function has been associated with epigenetic modification at the
FOXP3 promoter region. In a randomized controlled study of
dual sublingual immunotherapy in participants allergic to both
house dust mite and grass pollen, methylated CpG sites within
the FOXP3 locus of enriched peripheral memory Treg cells
were decreased after 12 months of treatment.92
TFH/TFR cells. TFH cells are characterized by surface CXCR5,
the transcription factor B-cell lymphoma 6 protein, and increased
expression of IL-4, IL-21, and IL-6. TFH cells reside in the
marginal zones of germinal follicles within regional lymph nodes,
where they provide essential help for B-cell maturation and class-
switching. In 2004, a distinct population
J ALLERGY CLIN IMMUNOL
DECEMBER 2017
of CXCR5-expressing FoxP31 Treg cells was identified, which
possessed the ability to migrate into germinal centers and
suppress T- and B-cell responses.93,94 However, it was not until
2011 that this population of cells was recognized as a distinct
subset of CD41 T cells with regulatory capacity, namely
follicular regulatory T (TFR) cells. One study has shown that
memory TFH cells were significantly reduced after
immunotherapy.95 Moreover, TFR cells from immunotherapy-
treated patients were shown to have higher capacity to produce
IL-10 compared with TFH cells. When CXCR51 TFH cells were
enriched from immunotherapy-treated donors and cultured in
the presence of T-cell receptor stimulation and IL-2 for 5 days,
flow cytometric analysis revealed an increase in TFR cell
numbers. These findings highlight the plasticity of T FR cells
and their likely role in suppressing TH2 responses and IgE
antibody production during allergen immunotherapy. 95
iTreg cells. iTreg cells produce either IL-10 (TR1) or TGF-b
(TH3) and have been shown to modulate allergen-driven T-cell
proliferative responses and TH2 cytokine release.83 Studies of
nasal biopsy specimens obtained before and at 2 years after
grass pollen immunotherapy identified a shift in favor of local
iTreg cell responses in the nasal mucosa. There was an increase
in numbers of IL-10–expressing T cells during the pollen
season that was associated with an increase in serum IgG 4
levels.96 Seasonal increases in numbers of TGF-b1 T cells in
the nasal mucosa correlated with increases in peripheral
circulating IgA concentrations.51
Induction of peripheral IL-101 Treg cells was reported after
grass and birch pollen sublingual immunotherapy. 56,85
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VOLUME 140, NUMBER 6
A time-course study during subcutaneous grass pollen
immunotherapy demonstrated that PBMCs obtained as early as
2 to 4 weeks during early updosing, when cocultured with grass
pollen allergen for 6 days, produced high levels of IL-10 in
supernatants.57 This early IL-10 signal was paralleled closely
by suppression of the allergen-induced late-phase response (Fig
3). Increases in IL-10 production and suppression of the late
response were followed sequentially by increases in serum IgG 4
levels at 6 to 8 weeks that peaked at 16 weeks, along with
parallel suppression of immediate skin responses.
Postimmunotherapy serum was shown to have IgG-associated
IgE-blocking activity for both basophil activation (increased
allergen stimulated basophil CD63) and IgE-FAB inhibition
that paralleled increases in IgG4 levels. The in vivo time course
of PBMC IL-10 production and associated changes in serum
blocking antibodies, allergen-induced skin responses and
hypothetical changes in Th2 lymphocytes during updosing, 57
and maintenance of grass pollen immunotherapy for 3 years and
during immunotherapy withdrawal49 are shown in Fig 3.
Regulatory B cells. Regulatory B (Breg) cells are a subset
of B cells that produce IL-10 and have the capacity to inhibit T
cell– and DC-mediated inflammatory responses and maintain
natural immunologic tolerance.97 Purified populations of IL-
10–producing Breg cells in bee venom–tolerant subjects
exhibited high surface expression of CD25 and CD71 and low
expression of CD73. These cells had the capacity to suppress
bee venom–specific T-cell proliferation.98 Moreover, the
provenance of allergen-specific IgG4 antibodies after bee
venom immunotherapy was shown to be from phospholipase
A2–specific IL-101 Breg cells. In addition to IL-10, Breg cells
have been shown to exert their suppressive capacity through
production of TGF-b and IL-35.97 It is likely that similar Breg
cell responses can be elicited during immunotherapy with grass
pollen or house dust mite allergens. Whether the same
phenotype is expressed by B cells after immunotherapy with
inhalant allergens remains to be determined.
TH1 immune deviation. Suppression of TH2 immunity
during both subcutaneous and sublingual immunotherapy has
also been associated with immune deviation and induction of
TH1 cells.9,99 In situ hybridization studies of the nasal mucosa
after successful subcutaneous immunotherapy demonstrated
increases in IFNG mRNA1 T cells after allergen challenge that
correlated with decreases in nasal symptoms during the pollen
season.9 Pollen immunotherapy was associated with decreases
in the ratio of IL5/IFNG mRNA1 cells in the mucosa and
increases in nasal IFN-g protein levels in nasal fluid during
natural seasonal allergen exposure.52 Similarly, subcutaneous
grass pollen immunotherapy resulted in increases in IL12
mRNA1 macrophages in the skin that accompanied suppression
of late cutaneous responses and correlated positively with local
IFN-g1 T cells and inversely with IL-4–expressing T cells.100
Evidence for/against TH1 deviation in peripheral blood studies has
101,102
been more controversial. One study suggested that the
shift from TH2 to TH1 responses might have been related to
103
activation-induced cell death of allergen-responder TH2 cells.
During birch pollen subcutaneous immunotherapy, a transient
increase in Bet v 1–specific IL-10–secreting cells at 3 months
was followed at 12 months by a reduction in the ratio of Bet v
1–specific IL-5/IFN-g–secreting T cells.104,105 Moreover,
survival of TH1 cells has been reported after deletion of TH2
cells.106
SHAMJI AND DURHAM 1491
Allergen immunotherapy and ILCs
The influence of immunotherapy on ILC2 cells has been studied
in peripheral blood but not in target organs, partly because of
difficulties in identifying these cells that do not express cell lineage
markers accessible to immunohistochemical localization in tissues.
After grass pollen subcutaneous immunotherapy, there was a
marked inhibition of seasonal increases in lineage-negative
CRTH21CD1271 ILC2s that correlated with the severity of self-
26
reported symptoms during the pollen season. These results were
highly significant when compared with seasonal increases in ILC2
numbers observed in matched untreated control subjects with
seasonal allergic rhinitis. These data were supported by inhibition
of seasonal increases in numbers of CD1171 (c-kit1) ILC2s and in
the proportion of IL-131 ILC2s, as determined by means of
intracellular cytokine staining. In a study of immunotherapy in
27
participants with seasonal asthma, there was no change in the
number of ILC2s, although this is likely explained by the
measurements having been performed out of season when the
participants were asymptomatic. To our knowledge, there have
been no reports of the influence of immunotherapy on innate
epithelially derived cytokines, which are known to be closely
involved in the regulation of both local TH2-mediated events and
ILCs.
Allergen immunotherapy and DCs
The buccal mucosa is exposed constantly to foreign proteins in
foods and represents a distinct protolerogenic environment. Ex vivo
studies of buccal mucosal biopsy specimens from patients with
grass pollen allergy have shown that oral mucosal Langerhans cells
bind the major grass pollen allergen Phl p 5 in a dose- and time-
dependent manner that plateaus at 5 minutes and leads to a
decelerated maturation of oral Langerhans cells in parallel with an
enhanced migratory capacity and increased pro-duction of
107
tolerogenic cytokines that include IL-10 and TGF-b.
In a randomized controlled trial of sublingual immunotherapy,
despite local increases in numbers of FOXP31 Treg cells in
sublingual mucosal biopsy specimens, there was no change in local
monocyte-derived DCs, although CD1a1 Langerhans cells were not
87
examined specifically. However, the influence of allergen
immunotherapy on subtypes of DCs in the blood circulation has
been studied. PCR studies of peripheral blood samples taken before
and after 4 months of sublingual grass pollen immunotherapy was
used to characterize changes in DC phenotype. A significant
increase in the numbers of DCs with a DCreg phenotype was
32
observed. The DCreg signature was reflected by an increase in
mRNA expression for stabilin-1 and complement component 1Q
32
(C1Q), as predicted from in vitro studies. Interestingly, this
DCreg signature was observed only in those ‘‘responders’’ to
immunotherapy, as reflected by a significant decrease in
32
rhinoconjunctivitis symptoms. In support of these findings, a 1-
year treatment with sublingual immunotherapy in children with
mite allergy resulted in peripheral DCs that showed an immature
phenotype and an increased capacity to produce IL-10 and
102
decreased IL-12 levels.
BIOMARKERS OF RESPONSE TO
ALLERGEN IMMUNOTHERAPY
International guidelines highlight the need for quantitative and
108
validated measurements for potential biomarkers. A European
1492 SHAMJI AND DURHAM J ALLERGY CLIN IMMUNOL
DECEMBER 2017

TABLE I. Six domains of biomarkers: (1) antibodies, (2) serum inhibitory activity for IgE, (3) basophil activation, (4) cytokines and chemokines,
(5) cellular biomarkers, and (6) in vivo biomarkers
Domains Biomarkers References
Antibodies IgE (sIgE, total IgE, sIgE/total IgE) 44, 51, 59, 96, 109, 110
sIgG4 83, 95, 106
IgA 51, 87
Serum Inhibitory activity for IgE IgE-FAB 59
ELIFAB 43
Basophil activation CD63 111, 115-117
CD203c 112, 118
DAO 16, 112, 113
Basophil histamine release 16, 112, 113
Cytokines and chemokines TH2: IL-4, IL-13, IL-9, IL-17, eotaxin, TNF-a 99, 119-122
TH1: IFN-g, IL-12 99
Regulatory: IL-10, TGF-b 83
Cellular biomarkers Treg cells 83-88
Breg cells 123, 124
DCs 32, 33, 102, 114
In vivo biomarkers Allergen provocation tests (SPT, ID, NPT, and EEC) 9, 72, 125-127
Chamber studies 128, 129

EEC, Environmental exposure chamber; ID, intradermal test; IgE-BF, IgE blocking factor; NPT, nasal provocation test; SPT, skin prick test.

Academy of Allergy and Clinical Immunology Task Force reported
a consensus statement on potential biomarkers of allergen
101
immunotherapy. These were classified into 7 domains (Table
I)*: (1) IgE (total IgE, sIgE, and sIgE/total IgE ratio), (2) IgG
subclasses (allergen-specific IgG [IgG]1 and sIgG4, including the
sIgE/IgG4 ratio), (3) serum inhibitory activity for IgE (IgE-FAB),
(4) basophil activation, (5) cytokines and chemokines, (6) cellular
markers (Treg cells, Breg cells, and
DCs), and (7) in vivo biomarkers, which include provocation
tests.108
IgE (total IgE, sIgE, and sIgE/total IgE ratio)
Inclusion criteria for initiation of immunotherapy rely on a
history of symptoms on exposure to allergen 1,130,131 and
increased serum sIgE levels to the clinically relevant allergen,
as measured by using the ImmunoCAP system. Patient
selection has been refined by the availability of recombinant
allergen technology to identify specific IgE to the major
allergen determinants and to recognize irrelevant cross-reacting
allergens.132 For example, a patient with high IgE levels to Phl
p 1 and Phl p 5 might be a good candidate for grass pollen
immunotherapy. In contrast, IgE sensitivity to the grass pollen
profilin Phl p 12 might result in high increased IgE levels and
false-positive skin test responses to whole birch pollen extract
because of cross-reactivity with the birch profilin Bet v 2.
An initial early increase in sIgE levels during both subcutane-
59 109
ous and sublingual pollen immunotherapy has been shown to
be followed by blunting of seasonal increases in sIgE levels. In
long-term studies of subcutaneous immunotherapy, a gradual
44
decrease in sIgE levels over several years was observed, although
there was no clear association between changes in sIgE levels and
51,96
the magnitude of the clinical response. The ratio of specific
IgE/total serum IgE at baseline was reported to correlate with
*References 9, 16, 32, 33, 43, 44, 51, 58, 72, 83-88, 95, 96, 99, 102, 106, and 109-129.
clinical response to immunotherapy,110,133 although
others53,60,134 have not replicated these findings.
IgG subclasses (sIgG1, sIgG4, and sIgE/IgG4 ratio)
Immune-reactive IgG1 and IgG4 antibodies can be measured by
using ImmunoCAP (Fig 4, A) and by using an allergen microarray
(eg, ImmunoSolid Allergen Chip Assay [ISAC]). Allergen-specific
IgG subtypes, including IgG1 and particularly IgG4 have been
shown to be increased in the range of 10- to 100-fold compared
with baseline values during immunotherapy, although with no
83,95,106
consistent correlation with clinical response to treatment.
ISAC can be performed by using very small volumes of serum or
nasal fluid. A large component of the observed increase in serum
allergen-specific IgG or IgG4 levels after allergen immunotherapy
is likely to reflect high allergen exposure and could be used
potentially to monitor patients’ adherence to immunotherapy
108
regimens. A decrease in the sIgE/IgG4 ratio has been reported
after subcutaneous immunotherapy and was associated with a
101
reduction in late cutaneous skin reactions. However, this finding
101
has not been reproduced in other studies.
Serum IgE inhibitory activity (IgE-FAB and
enzyme-linked immunosorbent–facilitated
antigen-binding assay)
IgG-associated IgE-inhibitory activity can be assessed by using a
flow cytometry–based assay (IgE-FAB) that has been validated
according to the International Conference on Harmonisation
135
guidelines. This assay measures the ability of IgG-containing
serum obtained after allergen immunotherapy to inhibit the FcεRII-
dependent binding of allergen-IgE complexes to B cells, a surrogate
for IgE-facilitated antigen presentation to T cells (Fig 4, B). An
alternative approach is the enzyme-linked immunosorbent–
43
facilitated antigen-binding assay (ELIFAB). The IgE-FAB assay
is reproducible but technically more complex and is currently
59
confined to specialized laboratories. Limited data available
suggest a modest correlation between IgE-FAB and
J ALLERGY CLIN IMMUNOL SHAMJI AND DURHAM 1493
VOLUME 140, NUMBER 6

FIG 4. Induction of IgG4 antibodies and associated IgE-inhibitory activity during immunotherapy. A, Specific IgG4
levels. B, IgE-facilitated allergen binding to B cells. C, Histamine release at the single-cell level using labeled
DAO (increased intracellular DAO demonstrates inhibition of histamine release). D, Histamine ELISA.
Measurements were from untreated patients with grass allergy (SAR), subcutaneous immunotherapy (SCIT)–
and sublingual immunotherapy (SLIT)–treated patients, and those who had completed 3 years of SLIT, followed
by discontinuation for up to 3 years (SLIT-TOL). Data are expressed as individual data (quintile box plots with
contour). *P < .05 and ***P < .001, Mann-Whitney U test.16

ELIFAB results and the clinical response to immunotherapy over and
above that observed when simply measuring immunoreactive IgG
levels.43,59 This is likely related to IgE-FAB and ELIFAB, providing
a further functional measure of affinity and/or avidity of
antibody binding.
Basophil activation
In flow cytometry–based assays using whole blood, basophil
activation can be studied by monitoring expression of surface
markers, such as CD63 and CD203c. Although CD63
expression measures basophil degranulation,111 CD203c is a
specific basophil marker that also measures IL-3–dependent
activation of basophils. A novel functional assay that detects
intracellular staining of phycoerythrin-conjugated diamine
oxidase (DAO) has also been validated. DAO binds tightly to
its substrate histamine, such that allergen stimulation results in a
reduction in basophil intracellular DAO levels proportional to
the amount of intracellular histamine released. This reduction
has been detected
during both subcutaneous and sublingual immunotherapy (Fig
4, C and D).112,113
Cytokines and chemokines
Recent advances in miniaturized multiplex cytokine analysis
with the Meso Scale Discovery and Luminex platforms have
enabled the measurement of cytokines and chemokines in nasal
fluid that increase in response to allergen provocation and are
modified by immunotherapy.69,70
Cellular and molecular markers
Cellular markers of potential use for assessing or predicting
response to immunotherapy include phenotypic markers for T cells
(TH2, Treg, TFH/TFR, and TH1 cells) and subpopulations of Breg
cells, all of which have been shown to be modified during
immunotherapy, principally by using flow cytometry. Although in
clinical trials these markers have been able to distinguish between
treatment groups and correlate overall with clinical outcomes of
1494 SHAMJI AND DURHAM
efficacy,69,83,88,136,137 they have been unable to distinguish
responders from nonresponders or predict response in
individual subjects. Furthermore, there is a need for optimal cell
processing and transfer and storage of samples such that
complex flow cytometry for multiple T and B cell–associated
markers is beyond the scope of routine clinical laboratories.
DCs express distinct molecular markers according to their T
cell–differentiating capacity. DCregs preferentially express C1Q
32
and FcgRIII, which favor preferential Treg cell development,
whereas DC2s express CD141, GATA-3, OX40 ligand, and RIPK4,
33
which favor polarizing naive T cells into TH2 cells. Expression
of these markers in PBMCs was evaluated before and at 2 and 4
months after sublingual grass pollen immunotherapy. This
quantitative RT-PCR–based method correlated with clinical
33,114
outcomes. Remarkably, an optimal combination of 5
molecular markers that included 3 DC2 markers (CD141, GATA-3,
and RIPK4) and 2 DCreg markers (C1QA and FcgRIIIA) was able
to distinguish clinical responders from nonresponders with a
sensitivity of 90.48% and a specificity of 61.9%. These interesting
results demand further evaluation in clinical trials and ultimately in
clinical practice.
In vivo biomarkers
In vivo biomarkers refer to the use of allergen provocation
tests to evaluate patients’ allergen-specific reactivity before and
after treatment. Provocation tests include skin prick tests,
intradermal tests, and nasal, conjunctival, and bronchial
provocation tests.101 For example, the magnitudes of early and
late responses after grass pollen nasal challenge and after
intradermal testing were inhibited by both subcutaneous and
sublingual immunotherapy. A modest correlation was observed
between recorded total nasal symptom scores after challenge,
the late skin response, and participants’ subjective assessment
of hay fever severity during the pollen season.69,70 The
European Medicines Agency has elaborated on the use of
provocation testing for proof of concept for novel approaches,
allergen dose finding, and use as supportive secondary efficacy
end points during clinical trials of allergen immunotherapy. 138
Summary
In the context of a clinical history of symptoms on exposure
to relevant inhaled allergens, the serum sIgE level is the single
most relevant biomarker for selection of patients for allergen
immunotherapy, and this has been refined by the availability of
recombinant allergen technology.132 However, at present, the
level of sIgE is unable to reliably predict or monitor the clinical
response to immunotherapy. At present, the ratio of IgE/total
IgE at baseline remains under evaluation as a possible predictor
of response. Functional assays of IgG-associated inhibitory
activity for IgE (IgE-FAB and ELIFAB) have better correlation
with clinical response in clinical trials than immunoreactive
IgG/IgG4 levels but do not predict efficacy in individual
subjects. Serum-based assays have the advantage of ease of
sample handling and storage, and it seems likely that IgG/IgG 4
levels might be more effective as a surrogate for compliance
with treatment, which could be of particular value for
monitoring patients receiving sublingual immunotherapy.
The various cellular assays reported are restricted to specialist
centers. Basophil responsiveness assays and T/B-cell phenotypic
J ALLERGY CLIN IMMUNOL
DECEMBER 2017
assays require flow cytometry and involve, respectively, either
processing of fresh blood or complex cell separation and
storage protocols. They are informative for proof of concept in
clinical trials but are not feasible for routine clinical practice.
Studies of DC phenotype by use of RT-PCR on whole blood
or PBMCs have been shown to separate clinical responders and
nonresponders to grass pollen sublingual immunotherapy, and
further studies are needed to replicate these findings and assess
their value in individual patients.
NOVEL APPROACHES FOR IMMUNOTHERAPY
A better understanding of mechanisms should translate ideally
1,139
into novel immunotherapy approaches. The aim has been to
improve efficacy over standard allergen extract–based extracts
while permitting shorter, safer, and more convenient strategies
for patients. Alternative routes, such as epicutaneous140 and
intralymphatic141,142 approaches, have proved safer than
conventional subcutaneous immunotherapy, although there are no
head-to-head trials to assess comparative efficacy. The intradermal
route was ineffective for grass pollen allergen and might have
143
exacerbated seasonal symptoms. Targeting immune deviation
using the Toll-like receptor 4 agonist monophosphoryl lipid A in
combination with subcutaneous grass pollen allergoid
immunotherapy was effective with 4 preseasonal injections without
an increase in side effects.
144
A trial of bacterial DNA
oligonucleotides rich in CpG sequences covalently linked to the
major ragweed allergen Amb a 1, was effective in a phase II trial,
possibly by inducing Treg cells and/or immune deviation,
145
94
although this approach failed at phase III. Targeting IgE or type 2
cytokines (IL-4 and IL-5) has been successful in reducing
exacerbations in asthmatic patients, although without durable
146
effects after discontinuation. Anti-IgE in combination with
allergen immunotherapy was highly effective in reducing the risk
of systemic allergic reactions.
147
The combination of anti–IL-4
with subcutaneous allergen immunotherapy was effective in
suppressing circulating TH2 cells and allergen-induced late
148
responses but showed no advantage over allergen extract alone.
Current novel approaches to reduce systemic adverse events
include the use of engineered recombinant hypoallergenic
132
molecules and
allergen peptide–based approaches that specifically target either
T-cell epitopes149,150 or B-cell epitope–based strategies that
selectively promote allergen-specific IgG responses.151,152 For
the future, targeting the innate immune response using
antibodies directed against the epithelial cytokines IL-33, IL-25,
or thymic stromal lymphopoietin in combination with allergen
immunotherapy would be an attractive combination strategy to
likely reduce inflammation, suppress ILC2s, and promote a
more tolerogenic DC phenotype.
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Mekanisme penyakit alergi
Mekanisme imunoterapi alergen
untuk alergen yang terhirup dan
biomarker prediktif
Mohamed H. Shamji, PhD, FAAAI, dan
Stephen R. Durham, MD, FRCP London,
Inggris
Imunoterapi alergen efektif pada pasien dengan
rinitis alergi dan asma yang bergantung pada
IgE. Ketika imunoterapi diberikan secara terus
menerus selama 3 tahun, ada manfaat klinis yang
ditimbulkan selama beberapa tahun setelah
imunoterapi dihentikan. Efek modifikasi pada
penyakit ini yaitu antigen-specific dan antigen-
driven. Kemajuan secara klinis disertai dengan
penurunan jumlah sel efektor pada organ – organ
yang ditarget, termasuk sel mast, basofil, eosinofil,
dan sel limfoid tipe 2. Imunoterapi menghasilkan
produksi antibodi IgG / IgG 4 yang menghalangi
aktivasi IgE-dependent yang dimediasi melalui
reseptor IgE dengan afinitas tinggi (Fc ε RI) pada
sel mast dan basofil dan reseptor IgE dengan
afinitas rendah (Fc ε RII) pada sel
B. Penekanan imunitas pada T H 2 dapat terjadi
sebagai konsekuensi dari penghapusan atau alergi
sel T yang spesifik-antigen; induksi pada sel T
regulasi antigen- spesifik; atau penyimpangan
kekebalan yang mendukung respon dari
T H 1. Tidak jelas apakah memori jangka panjang
yang berubah yang berada di dalam kompartmen
sel T atau sel B. Data terkini menyoroti peran IL-
10 – yang memproduksi sel B dan ' ' pelindung ' '
antibodi yang mungkin berkontribusi pada
toleransi jangka panjang. Memahami mekanisme
yang mendasari induksi dan keteguhan dari
toleransi harus mengidentifikasi prediktor
biomarker padarespons klinis dan menemukan
strategi baru dan efektif untuk imunoterapi. (J
Allergy Clin Immunol 2017; 140: 1485-98.)
Kata kunci: Imunoterapi, mekanisme, rhinitis
alergi, alergi asma, toleransi jangka panjang, sel T,
sel B, sel limfoid bawaan 2, IgE-FAB, biomarker
Alergi imunoterapi efektif pada pasien
tertentu yang menderita rhinitis alergi, termasuk
penderita asma ringan / sedang. 1,2 Ada
keberagaman yang terjadi pada populasi yang
dipelajari, beragam produk alergen dan protokol
yang digunakan, dan hasil klinis yang digunakan
untuk mendokumentasikan kemanjuran dan
keamanan. 3 Meskipun demikian, pedoman baru-
baru ini 4 mengkonfirmasi bahwa imunoterapi
sangat efektif secara khusus pada pasien dengan
rhinitis musiman, dan data terakhir sangat
mendukung penggunaannya dalam alergi
permanen yang disebabkan oleh tungau debu
rumah. 5
Imunoterapi subkutan melibatkan suntikan
mingguan yang ditingkatkan dosisnya, diikuti
dengan suntikan perawatan bulanan minimal
selama 3 tahun. 1,6,7 Mengingat sisi alergi sistemik
sesekali efeknya, imunoterapi subkutan
memerlukan pelaksanaan di klinik alergi spesialis
dengan akses ke tindakan resusitasi. Sublingual
imunoterapi dapat berupa tetesan atau tablet setiap
hari yang ditempatkan di bawah lidah. Sublingual
imunoterapi efektif dan lebih aman daripada
imunoterapi subkutan, sehingga dapat dilakukan
sendiri oleh pasien di rumah. 1,8 Imunoterapi
sublingual dan subkutan umumnya akan efektif
dalam waktu 2 sampai 4 bulan sejak memulai
pengobatan dan dapat diberikan presesonally /
coseasonally untuk manfaat jangka pendek.
Perbandingan yang dilakukan secara tidak
langsung telah menyarankan bahwa imunoterapi
mungkin lebih efektif daripada obat anti-
alergi. Berbeda dengan obat anti-alergi dan terapi
mAb saat ini, bila terapi immuno-allergen
diberikan terus menerus selama 3 tahun, kedua
rute telah terbukti mengubah penyakit,
bermanifestasi sebagai pengurangan dalam gejala
jangka panjang paling sedikit 2 sampai 3 tahun
setelah penghentian. 9,10
Dalam tinjauan ini, kami mengeksplorasi
data historis dan terkini mengenai mekanisme
imunoterapi untuk alergen yang terhirup. Harapan
kami adalah bahwa pemahaman yang lebih besar
tentang mekanisme dasar dari toleransi yang akan
mengidentifikasi biomarker potensial yang dapat
memprediksi dan / atau memantau respons
terhadap pengobatan. Pengetahuan semacam itu
member informasi mengenai potensi strategi
pengobatan baru.
TINJAUAN UMUM MEKANISME
RHINITIS ALLERGIK DAN ASTHMA
IgE dan sel mast
Gambaran kardinal pada alergi rhinitis
mencakup peningkatan konsentrasi IgE allergen-
spesifik yang relevan secara klinis, aktivasi sel
mast yang bergantung pada IgE, dan eosinofilia
lokal pada organ target. Selain sumber limfatik
sistemik dan regional IgE, IgE yang lebih spesifik
dapat disintesis dan diproduksi secara lokal oleh
sel B di dalam mukosa pernafasan, 11 dengan
demikian memperhitungkan fenomena
berkala “rhinitis alergi lokal” dengan gejala pada
paparan alergen dengan tidak adanya serum IgE
yang terdeteksi secara spesifik atau hasil tes kulit
positif langsung terhadap alergen yang relevan. 12
Aktifnya IgE-dependent dapat terdeteksi
pada respon seketika(0 sampai 60 menit) setelah
provokasi alergen pada hidung. Cross-linking
Alergen dari molekul IgE di permukaan yang
berdekatan pada sel mast dan basofil terpicu
dalam hitungan detik atau beberapa menit setelah
melepaskan mediator yang baru terbentuk, seperti
histamin 13 dan tryptase, 14 yang terkandung dalam
butiran intracytoplasmic. Mediator yang baru
terbentuk berasal dari asam arakidonat di dalam
lipid membran termasuk leukotrien sulfida
leukotrien (LTs; LTC 4 , LTD 4 , dan terminal
metabolit LTE ), 12 faktor pengaktif platelet, dan
prostaglandin D 2. Sifat biologis mediator ini
konsisten dengan vasodilatasi lokal, pembentukan
edema, rangsangan neurogenik lokal, dan sekresi
lendir yang mengkarakterisasi hipersensitivitas
tipe I yang disebabkan langsung pada alergi
hidung. Pada kontraksi otot polos bronkus di
saluran pernapasan bawah, serta edema dan
hipersekresi lendir, memberikan kontribusi pada
bronkokonstriksi akut. Beberapa bagian dari
subyek memiliki respons yang lambat pada 2
sampai 10 jam setelah tantangan. Respons
terlambat ditandai oleh eosinofilia jaringan,
kemampatan hidung, dan hiperaktivitas
rangsangan pada pemicu alergi dan non-alergi
yang dapat berlangsung selama berhari-hari atau
bahkan berminggu-minggu setelah suatu
penolakan pada alergen hidung.Sebaliknya untuk
temuan pada pasien dengan asma alergi dan
poliposis hidung, fitur morfologi dan
imunohistokimia dari remodeling saluran napas
bukanlah ciri khas yang konsisten bahkan pada
alergi moderat / parah sekalipun. 15
GLOSARIUM
Bet v 1: Alergen kuat dari pohon dalam ordo
Fagales, yang merupakan penyebab utama alergi
tipe I yang diamati pada awal musim semi dan
ditandai dengan demam, dermatitis, dan asma.
SITUS CpG TERPADU: Lokasi CpG adalah
daerah DNA dimana nukleotida sitosin terjadi di
samping nukleotida guanin yang dipisahkan hanya
oleh 1 fosfat. Metilasi sitosin dalam gen bisa
mematikan gen.
c-kit (CD117): Sebuah reseptor sitokin secara
garis besar dapat ditemukan pada permukaan sel
induk hematopoietik dan jenis sel lainnya,
termasuk sel mast. CD117 adalah protein reseptor
tirosin kinase tipe III yang berikatan dengan faktor
sel induk dan membentuk dimer yang
mengaktifkan tirosin kinase intrinsiknya,
menghasilkan fosforilasi dan aktivasi molekul
transduksi sinyal yang menghasilkan sinyal sel.
SITOTOKSIK limfosit T -ASSOCIATED
PROTEIN 4 (CTLA-4): Sebuah reseptor yang
berfungsi sebagai sinyal penghambatan dan
mengatur perlambatan respon imun ketika terikat
dengan CD80 dan CD86. CTLA-4 secara
konstitutif diekspresikan dalam regulatorsel T
tetapi hanya diregulasi dalam sel T konvensional
setelah aktivasi.
IFN- g : Interferon tipe II, IFN- g adalah sitokin
yang dibutuhkan untuk kekebalan bawaan
dan adaptif terhadap infeksi virus, bakteri, dan
protozoa. IFN- g telah terbukti menjadi aktivator
penting dari makrofag dan induser ekspresi pada
molekul MHC kelas II. ProduksiIFN- g
didominasi oleh pembunuh alami (NK) dan sel
NKT sebagai bagian dari respon imun bawaan dan
oleh CD4 T H 1 dan CD8 sitotoksik T sel limfosit
efektor T pada saat kekebalan antigen-spesifik
berkembang.
ImmunoCAP (PERDAGANGAN TERDAFTAR
DIAGNOSTIK PADA FARMACIA AB): Sebuah
uji kuantitatifin vitro yang mengukur kadar IgE
alergen spesifik pada serum manusia. Tes
ImmunoCAP dapat dilakukan pada ratusan alergen
dengan menggunakan polimer selulosa, yang
memberikan kapasitas pengikat protein alergen
yang relevan secara klinis, termasuk yang hadir
pada tingkat yang sangat rendah.
IL-3: Sitokin yang mendukung pertumbuhan yang
mampu mendukung proliferasi dan pengaktifan
berbagai jenis sel hematopoietik, termasuk basofil.
IL-6: Sebuah sitokin juga dikenal sebagai IFN- b 2
dan terlibat dalam berbagai negara penyakit
peradangan terkait, IL-6 telah dikaitkan dengan
pematangan sel B dan telah terbukti bertindak
sebagai pirogen endogen mampu merangsang
demam pada pasien dengan penyakit autoimun
atau infeksi.
IL-12: Sitokin yang diproduksi oleh sel dendritik,
makrofag, neutrofil, dan sel B-limfoblastoid
manusia (NC-37) sebagai respons terhadap
stimulasi antigenik dan telah terbukti diperlukan
untuk diferensiasi sel T naif ke sel T H 1.
IL-21: Sitokin yang dinyatakan dalam
CD4 1 manusia Sel T dan ditemukan diregulasi
dalam T H 2 dan T H 17 subset dan sel T folikel,
yang menginduksi pembelahan sel / proliferasi
berbagai sel sistem kekebalan tubuh, termasuk sel
pembunuh alami dan sel T sitotoksik.
IL-25: Sitokin proinflamasi yang berbagi
kesamaan urutan dengan IL-17 dan telah terbukti
mendukung respons imun tipe T H 2. IL-25 dapat
menginduksi aktivasi faktor nuklir k B dan
merangsang produksi IL-8.
IL-33: Seorang anggota keluarga sitokin IL-1 yang
diekspresikan pada sel T H 2, sel mast, dan limfosit
bawaan 2 yang berpotensi mendorong
produksi sitokin T H 2.
IL-35: Sitokin keluarga IL-12 yang diproduksi
oleh regulator, tapi bukan efektor, sel T dan B
yang berperan dalam penekanan kekebalan.
PROGRAMMED CELL DEATH 1 (PD-
1): Reseptor permukaan sel yang berperan penting
dalam meregulasi sistem kekebalan tubuh dan
menekan aktivasi sel T inflamasi. PD-1 adalah pos
pemeriksaan kekebalan yang berfungsi ganda
dalam mempromosikan apoptosis pada sel T
spesifik antigen sekaligus mengurangi apoptosis
pada sel T yang resisten.
THYMIC STROMAL LYMPHOPOIETIN
(TSLP): Sitokin yang merangsang pematangan
sel-T melalui aktivasi sel penyajian antigen,
seperti sel dendritik dan makrofag.
TGF- b : Sitokin yang disekresikan oleh banyak
jenis sel, termasuk makrofag, yang mengendalikan
proliferasi, diferensiasi sel, dan proses inflamasi
dalam berbagai sel.
Gambar 1. A dan B, Mekanisme peradangan
alergi: ringkasan respons imunologis terhadap
pemicu awal sensitisasi alergi dan peradangan
alergi setelah terpapar kembali ke alergen
inhalan (lihat teks). EC, sel epitel.
T H 2 limfosit dan kelompok 2 sel limfoid
bawaan
Kejadian patofisiologis di atas berada di
bawah peraturan sebuah subset yang berbeda
dari sel T H 2. Sel T H 2 menghasilkan IL-4,
sitokin kunci yang bertanggung jawab
untuk diferensiasi sel T H 2. IL-4 dan IL-13
mendorong limfosit B untuk
menghasilkan ε transkrip gen -germline, langkah
pertama dalam rantai berat penataan ulang gen
dalam mendukung produksi IgE. IL-4 dan IL-13
upregulate vaskular protein adhesi sel 1 ekspresi
pada endotel vaskular, mempromosikan adhesi
sangat terlambat antigen 4, integrin 4 b 1 -
expressing eosinofil. Keduanya merangsang
produksi lendir dari kelenjar di saluran udara
bagian atas dan bawah. IL-5 bertanggung jawab
atas diferensiasi terminal dan pelepasan eosinofil
dari sumsum tulang dan memperpanjang
kelangsungan hidup eosinofil dengan menghambat
apoptosis eosinofil dalam jaringan. Seiring dengan
faktor sel induk, IL-9 adalah sitokin kunci untuk
diferensiasi dan pematangan sel mast. Pelepasan
T H 2 sitokin dan eosinofilia jaringan yang jelas
selama respon akhir-fase yang terjadi pada 4
sampai 12 jam setelah tantangan alergen.
Kelompok 2 sel limfoid bawaan (ILC2s)
mewakili sumber alternatif sitokin T H 2 pada
mukosa hidung. Sel limfoid bawaan (ILC) secara
morfologis mirip dengan limfosit, walaupun
berbeda dalam hal tidak menunjukkan antigen
permukaan. Reseptor atau spidol garis keturunan
sel lainnya dan bertindak secara antigen-
independen. ILC terdiri dari 3 kelompok yang
berbeda yang disebut kelompok ILC, ILC2, dan
kelompok 3 ILCs.Kelompok ILCs secara
konstitutif mengekspresikan faktor transkripsi T-
box dan menghasilkan sitokin T H 1 IFN- g dan
TNF dan memberikan perlindungan terhadap
bakteri intraselular dan parasit. ILC2s secara
konstitutif mengekspresikan reseptor yatim piatu
terkait RAR a dan GATA-
3; menghasilkan sitokin T H 2, terutama IL-5 dan
IL-13; dan memberikan kekebalan terhadap
cacing, serta merangsang respons
alergi. Kelompok 3 ILC dicirikan oleh faktor
transkripsi yang berhubungan dengan RAR yatim
reseptor g t, mengungkapkan IL-17a dan / atau IL-
22, mampu memberikan perlindungan terhadap
bakteri ekstraseluler, dan terlibat dalam proses
perbaikan jaringan.
Peran ILC2 pada rhinitis alergi pertama
kali diidentifikasi pada pasien dengan alergi
kucing, yang menunjukkan peningkatan angka
ILC2 darah perifer pada 4 jam setelah tantangan
alergi alergen kucing. 25 Selanjutnya, peningkatan
jumlah ILC2 yang beredar telah diidentifikasi
pada kedua pasien dengan alergi rumput dan
rinitis 26 dan pasien asma 27 selama musim serbuk
sari rumput. ILC2s merupakan sumber alternatif
yang melimpah dari T H 2 sitokin dan
kemungkinan berfungsi untuk memperkuat dan
mempertahankan T H 2 didorong peradangan
alergi lokal. Mengingat plastisitas yang baru
diidentifikasi dalam ILC2s pada jaringan pasien
dengan penyakit paru obstruktif kronik dan
rinosinusitis kronis, 28 konsep ini harus ditinjau
kembali dalam konteks rhinitis alergi. 18
Epitel respirasi dan sel dendritik
Meskipun IgE-dependent aktivasi sel mast
dan eosinofilia jaringan didorong oleh T H 2
limfosit, T H 2 diferensiasi sel tergantung pada
lingkungan sitokin lokal yang disediakan oleh
interaksi antara epitel pernapasan, sel dendritik
lokal (DC), dan kelenjar getah bening regional.
Pada subjek atopik aeroalergen melewati
epitel nasal yang meradang, dan sel epitel yang
diaktifkan melepaskan CCL2 dan CCL20, yang
merekrut DC yang belum matang ( Gambar
1 , A ). DC yang diaktivasi bermigrasi ke kelenjar
getah bening pengeringan regional dan sel T naif
polar ke sel T H 2. Migrasi DC dipersonalisasi oleh
IL-13 yang diproduksi oleh ILC2 dan juga oleh
IL-4 yang diproduksi terutama oleh basofil. Di
dalam pusat germinal kelenjar getah bening,
subset sel T H
berbeda dengan sel T (T FH )
sel folikel . Sel T FH menghasilkan IL-4 dan IL-
21 , yang bersama dengan sel T H 2
yang mengalami IL-4, mempromosikan
pengalihan kelas berat imunoglobulin ke IgE
dalam sel B. Epitel pernafasan subjek alergi atopik
mengekspresikan sitokin yang mencakup IL-
25 , 29 IL-33 , 30 dan stroma
timus protein limfopoietin 31 Sitokin epitel ini
disukai pengembangan fenotipe DC
32,33
proallergic yang memberikan bantuan untuk
diferensiasi sel T H 2. Selain itu, sitokin
epithelially berasal adalah faktor utama
pertumbuhan ILC2s yang memperkuat dan
mempertahankan T H 2 didorong peradangan
alergi lokal. 34-36 Selama paparan alergen
berikutnya, IgE-difasilitasi pengakuan alergen
melalui Fc ε RI pada DC dan Fc ε RII pada sel B
menguatkan pengembangan T H 2 tanggapan
terhadap alergen hirup ( Gambar 1 , B).
DC, tergantung pada fase pematangannya,
lokasi mereka, dan lingkungan sitokin lokal yang
terkait, dapat memulai dan mempertahankan
peradangan alergi (DC2s proalergik) atau sebagai
alternatif mempromosikan keadaan toleransi
kekebalan (sel dendritik peraturan tologenik
[DCregs]) untuk membedakan alergen. DC2s
mengekspresikan penanda CD141, GATA-3,
OX40 ligand, dan protein serin / protein kinase 4-
reseptor reseptor (RIPK4). 33 Saat DC2 terkena
alergen dan cocultured kemudian dengan sel T,
mereka dipromosikan preferensial T H 2 tanggapan
T-limfosit.
MEKANISME IMUN ALLERGEN
Rincian lebih lanjut tentang mekanisme
imunoterapi alergen dapat ditemukan
pada Gambar 2 .
Gambar 2. Mekanisme AIT. Selama tahap
sensitisasi awal pada pasien dengan rhinitis
alergi, paparan alergi yang rendah pada
permukaan mukosa hidung menghasilkan
aktivasi sel epitel, yang kemudian
mengaktifkan DC. Serapan DC dan antigen
yang ada pada sel T yang naif untuk
menginduksi tanggapan TH2 (Th2A) yang
alergi dan presentasi antigen yang difasilitasi
IgE. Keterpaparan alergen selanjutnya
menyebabkan degranulasi sel mast dan basofil,
menyebabkan reaksi fase awal klasik.
Selanjutnya infiltrasi leukosit lain
menyebabkan inflamasi alfa fase akhir.
Paparan alergen dosis tinggi dengan
imunoterapi mengembalikan fungsi DC, yang
menghasilkan IL-12, IL-27, dan IL-10 dan
meningkatkan penyimpangan kekebalan dari
respon TH2 ke TH1 dan induksi sel Treg dan
Breg (termasuk subset sel B lainnya) yang
menghasilkan antibodi IgA, IgG, dan IgG4.
Aktivitas penekan sel Treg, sel Breg, dan
aktivitas pemblokiran IgG ditunjukkan oleh
panah merah. EC, sel epitel; TLR, sejenis
reseptor seperti tikus.
Respon IgE, IgG, dan IgA
Baik imunoterapi sublingual dan subkutan
telah dikaitkan dengan peningkatan awal transien
pada tingkat antibodi IgE spesifik alergen serum
yang diikuti oleh penyumbatan kenaikan musiman
pada tingkat IgE selama paparan alergen
alami. 43 Peningkatan awal ini tidak disertai
dengan efek samping yang tak diinginkan, dan
telah menyarankan bahwa awal T H 2 priming oleh
paparan alergen yang tinggi mungkin penting
untuk kesuksesan imunoterapi. Imunoterapi
subkutan yang berkepanjangan selama beberapa
tahun dapat menyebabkan penurunan konsentrasi
alergen IgE (sIgE) , 44,45 merupakan peristiwa
yang mungkin berkontribusi terhadap toleransi
jangka panjang.
Pada tahun 1935, Robert Cooke dan
rekan 46 menunjukkan perpindahan pasif aktivitas
penekan untuk sensitivitas IgE ragweed segera di
kulit dengan menggunakan serum yang diperoleh
dari pasien yang telah menjalani imunoterapi
subkutan ragweed. Aktivitas penghambatan serum
dan nasal untuk IgE setelah imunoterapi subkutan
kemudian ditunjukkan berada dalam fraksi IgG,
IgG 4 , dan IgA dalam serum . 47 - 51 Penelitian
telah menunjukkan peningkatan konsentrasi IgG
serum 10 sampai 100 kali lipat,khususnya
IgG 4 . 7,52 - 55 Imunoterapi sublingual juga telah
terbukti menginduksi antibodi IgG 1 , 56 IgG 4 ,
dan IgA alergen spesifik . 10.49,57 - 59 Peningkatan
kadar antibodi imunoreaktif ini
telah diamati setelah imunoterapi pada keduanya
yaitu serbuk sari musiman dan alergen abadi,
seperti tungau debu
rumah. 60,61 kadar IgG 4 spesifik serum telah
terbukti meningkat dalam waktu dan tergantung
dosis selama imunoterapi serbuk sari rumput. 59
Beberapa penelitian telah menyoroti
kapasitas penghambatan IgG 4 untuk kejadian
yang bergantung pada IgE. Antibodi IgG 4 bersifat
bispesifik dan memiliki kemampuan untuk
menukar lengan F (ab) dengan menukar pasangan
rantai ringan antara molekul IgG 4 dengan
spesifisitas yang beragam. 62 IgG dapat bersaing
dengan IgE untuk alergen, 63 sehingga
menghalangi alergen-IgE pembentukan
kompleks. Hal ini mencegah cross-linking reseptor
IgE afinitas tinggi (Fc ε RI) pada sel basofil dan
mast, menghambat pelepasan histamin.Kompetisi
IgG / IgG 4 untuk IgE juga dapat memblokir
pengikatan kompleks IgE alergen ke reseptor
dengan afinitas rendah (Fc g RIIb) pada sel B,
sehingga menghambat presentasi antigen yang
difasilitasi IgE ke sel T, pendorong utama T
spesifik alergen H 2 tanggapan.16.64 - 66
Paradoksnya, meskipun tingkat IgG /
IgG 4 imunoreaktif turun 80% sampai 90% dalam
waktu 1 tahun menghentikan imunoterapi alergen,
aktivitas penghambatan IgE terkait IgG bertahan
selama beberapa tahun dan disertai efikasi klinis
jangka panjang. 49 Hal ini menunjukkan bahwa
meskipun kadar yang lebih rendah, antibodi IgG
yang bertahan setelah penghentian imunoterapi
dapat memiliki aviditas tinggi atau afinitas yang
lebih tinggi. Data ini meningkatkan kemungkinan
bahwa sel memori B jangka panjang yang
disebabkan oleh imunoterapi dapat bertahan
sebagai akibat stimulasi alergen lingkungan
tingkat rendah, sehingga berkontribusi pada
toleransi jangka panjang.
Antibodi IgG telah terdeteksi secara lokal
baik dalam cairan hidung dan serum setelah
imunoterapi. 53 Kedua tingkat spesifik IgG 4 dan
aktivitas penghambatan terkait untuk IgE-
difasilitasi antigen mengikat (IgE-FAB)
meningkat pada cairan hidung dari pasien yang
menjalani imunoterapi sublingual dibandingkan
dengan peserta yang tidak
43
diobati. Ketergantungan IgG 4 dalam aktivitas
inhibitor IgE telah ditunjukkan dalam percobaan
penipisan menggunakan IgG 4 afinitas
kromatografi. Besarnya penekanan IgE lebih besar
dengan cairan hidung dibandingkan dengan serum,
sehingga menyoroti potensi antibodi IgG lokal. 52
Alergi imunoterapi dan sel efektor
Pengaruh imunoterapi alergen pada sel
efektor telah dipelajari setelah provokasi alergen
hidung dan selama paparan serbuk sari musiman
alami. Imunoterapi subkutan dan sublingual
menghambat respons fase awal dan akhir setelah
tantangan alergen. 57,67 Supresi disertai dengan
pengurangan kenaikan awal histamin nasal
lokal, tosyl L -arginine methyl ester -esterase, dan
konsentrasi tryptase dalam cairan
hidung. Penghambatan tanggapan terlambat
dikaitkan dengan penurunan angka eosinofil 68 dan
kadar sitokin T H 2, termasuk IL-4, IL-5, IL-9, dan
IL-13. 69,70 Sebuah pengurangan setelah
imunoterapi juga dicatat dalam kadar cairan
hidung dari chemokine eotaxin CC, yang
berkontribusi pada perekrutan eosinofil. Uji coba
double blind untuk imunoterapi serbuk sari rumput
subkutan menghasilkan penurunan jumlah sel
efektor, termasuk sel mast CD117 1 ( c-
kit 1 ), 71 basofil, 72 dan eosinofil, 73 pada mukosa
hidung dibandingkan dengan jumlah pretreatment
yang signifikan dibandingkan dengan peserta yang
diobati dengan plasebo. 72,74 Perubahan lokal ini
terdeteksi pada spesimen biopsi hidung disertai
dengan perbaikan gejala musiman dan penurunan
persyaratan untuk pengobatan
penyelamatan. Sebuah korelasi langsung dicatat
antara penurunan kadar IL-5 dan nomor eosinofil
mukosa hidung dan juga antara angka eosinofil
dan tingkat keparahan gejala musiman. Pada
tungau debu rumah - pasien sensitif imunoterapi
sublingal dengan ekstrak tungau menghambat
morfin interceptor adhesi mukosa lokal 1 dan juga
menurunkan eosinofilia lokal. 72 Data ini
menggambarkan bahwa kedua sublingual dan
hasilnya imunoterapi subkutan di penurunan
perekrutan, aktivasi, atau keduanya sel efektor di
situs jaringan alergi.
Alergi imunoterapi dan respons T-
limfosit
Turun dalam jumlah
sel T H 2. Penekanan respons alergen
yang disebabkan alergen nasal selama subkutan
imunoterapi serbuk sari rumput dikaitkan dengan
penurunan jumlah sel CD4 1 dan sel T
positif IL4 mRNA di selaput hidung. 74 Temuan
ini didukung oleh pengamatan penurunan
T H tingkat 2 sitokin dalam cairan lapisan hidung
setelah tantangan hidung. 69 Teknik terbaru yang
mencakup ex vivo tetramer analisis 75-78 telah
memungkinkan fenotip sel T alergen spesifik
beredar perifer. 79-82 ini telah diizinkan identifikasi
penanda permukaan T-sel kunci, seperti
CD27, molekul -homologous reseptor
chemoattractant diekspresikan pada T H 2 limfosit
(CRTH2), CD161, dan kemokin reseptor 4
(CCR4), terkait dengan jenis 2 tanggapan
proallergic . Pada pasien dengan alergi serbuk sari
rumput, sel T spesifik tetramer yang tidak
mengekspresikan CD27 sebagian besar
mengekspresikan penanda permukaan CRTH2 dan
CCR4. 75 - 78 Hal ini berbeda dengan subyek non-
alergi, yang sel T-nya menunjukkan kadar CRTH2
dan CCR4 rendah dan kadar CD27 yang tinggi.
Pasien dengan alergen serbuk sari mengungkapkan
frekuensi tinggi sel CD27 2 T H 2 yang menurun
setelah imunoterapi subkutan. Demikian pula,
dalam Responses Mengukur di Rhinitis alergi
untuk SCIT dibandingkan SLIT Trial
45,70
(GRASS), baik imunoterapi subkutan dan
sublingual mengakibatkan perbaikan klinis selama
2 tahun yang disejajarkan dengan penurunan
perifer tetramer-positif
1 1 2
CRTH2 CCR4 CD27 Jumlah
CD4 1 T H 2. Perubahan ini disejajarkan dengan
penurunan tingkat sitokin T H 2 lokal, termasuk
IL-4, IL-5, dan IL-13, pada cairan hidung setelah
provokasi alergen hidung. Kedua nomor beredar
tetramer-positif T H 2 sel dan tingkat hidung lokal
T H 2 sitokin pulih selama 3 tahun , bersama
dengan penurunan gejala musiman 1 tahun setelah
penghentian imunoterapi. Kegagalan 2 tahun
imunoterapi (berbeda dengan 3 tahun pengobatan
terus menerus) 9,10 untuk menginduksi toleransi
tahan lama mungkin terkait dengan munculnya
kembali kekebalan T H 2 spesifik antigen ini .
Meningkatnya jumlah sel T 21, dan IL-6 . Sel T FH berada di zona marjinal
regulasi. Imun Toleransi selama imunoterapi telah
terbukti terkait dengan induksi sel T (Rule
regulatory) spesifik alergen. 83-88 sel Treg dapat
dikelompokkan menjadi 2 subset: peraturan T
alam (nTreg) sel, yang mengungkapkan faktor
transkripsi kotak forkhead P3 (FOXP3), dan
diinduksi peraturan T (iTreg) sel, yang
memproduksi sitokin peraturan, seperti IL- 10, IL-
35 , danTGF - b . 59,79,83
Sel nTreg. Sel nTreg pertama kali
dideskripsikan oleh Sakaguchi et al. 80 Selain
faktor transkripsi FOXP3, sel nTreg telah
meningkatkan ekspresi reseptor IL-2 (CD25) dan
menurunkan ekspresi reseptor IL-7 (CD127). sel
nTreg mengerahkan kapasitas mereka untuk
menekan dalam kontak langsung sel-
sel . 81,82 Peran fungsional telah diusulkan untuk
membran sitotoksik T protein yang berhubungan
dengan limfosit 4 , TGF- b terikat permukaan ,
reseptor TNF yang diinduksi glukokortikoid,
dan kematian sel terprogram 1 . Sel nTreg juga
telah ditunjukkan untuk memodulasi respons sel T
spesifik alergen pada subjek nonatik yang
sehat. 89 imunoterapi subkutan dikaitkan dengan
peningkatan lokal di FOXP3 1 CD25 1 nomor T-
sel 84 di mukosa hidung dibandingkan dengan
mereka pada subyek kontrol yang tidak
diobati. Setelah imunoterapi serbuk sari
sublingual, penelitian imunofluoresensi pada
spesimen biopsi sublingual menunjukkan
peningkatan jumlah sel FOXP3 1 CD3 1 pada
mukosa sublingual. 87 Di studi in vitro manusia
spesimen biopsi mukosa bukal manusia dan terkait
amandel lingual dan kelenjar gondok
mengidentifikasi mukosa oropharyngeal sebagai
lingkungan yang kaya dengan DC protoperogen
dan sel Treg. 90,91 Sel nTreg yang diubah fungsi
telah dikaitkan dengan modifikasi epigenetik pada
daerah promotor FOXP3. Dalam penelitian
terkontrol secara acak terhadap imunoterapi
sublingal ganda pada peserta yang alergi terhadap
tungau debu rumah dan serbuk sari rumput, situs
CpG yang termodifikasi dalam lokus FOXP3 dari
memori perifer yang diperkaya sel Treg menurun
setelah 12 bulan pengobatan. 92
Sel T FH / T FR . T FH sel ditandai dengan
permukaan CXCR5, faktor transkripsi protein B-
sel limfoma 6, dan peningkatan ekspresi IL-4, IL-
folikel germinal dalam kelenjar getah bening
regional, di mana mereka memberikan bantuan
penting untuk pematangan sel B dan pengalihan
kelas. Pada tahun 2004, populasi yang berbeda
dari CXCR5-mengekspresikan sel FoxP3 1 Treg
diidentifikasi, yang
memiliki kemampuan untuk bermigrasi ke pusat
germinal dan menekan respons sel T dan
B. 93,94 Namun, tidak sampai 2011 bahwa populasi
sel ini dikenali sebagai subset sel CD4 1 yang
berbeda
dengan kapasitas pengatur, yaitu sel T
regulasi folikuler (T FR ). Satu studi telah
menunjukkan bahwa sel T FH memori berkurang
secara signifikan setelah imunoterapi. 95 Selain
itu,sel-sel T FR dari pasien imunoterapi diobati
yang terbukti memiliki kapasitas yang lebih tinggi
untuk menghasilkan IL-10 dibandingkan
dengan sel T FH. Ketika sel
CXCR5 1 T FH diperkaya oleh donor yang diobati
dengan imunoterapi dan dikultur dengan adanya
stimulasi reseptor sel T dan IL-2 selama 5 hari,
analisis flow cytometric menunjukkan
peningkatan jumlah sel T FR . Temuan ini
menyoroti plastisitas sel T FR dan perannya yang
mungkin terjadi dalam menekan respons T H 2 dan
produksi antibodi IgE selama imunoterapi
alergen. 95
Gambar 3. Waktu untuk efek imunoterapi
pada tanda klinis pengganti (tanggapan
kutaneous awal dan akhir) dan kejadian
imunologis terkait selama tahap induksi dan
perawatan imunoterapi (desensitisasi), serta
persistensi mereka setelah penarikan
pengobatan (fase toleransi) . Abs, Antibodi;
BAT, Uji Aktivasi Basofil; BHR, respon
pelepasan histamin basofil; EPR, respon fase
awal; LPR, respon fase akhir.
Sel iTreg Sel iTreg menghasilkan IL-10
(T R1 ) atau TGF- b (T H 3) dan telah ditunjukkan
untuk memodulasi respons proliferasi sel T-sel
alergen dan pelepasan sitokin T H 2. 83 Studi
spesimen biopsi hidung diperoleh sebelum dan
pada 2 tahun setelah imunoterapi serbuk sari
rumput diidentifikasi pergeseran dalam
mendukung respon sel iTreg lokal pada mukosa
hidung. Ada peningkatan jumlah IL-10 sel T -
expressing selama musim serbuk sari yang
dikaitkan dengan peningkatan tingkat serum
IgG 4. 96meningkat musiman dalam jumlah dari
TGF b 1 T sel di mukosa hidung berkorelasi
dengan peningkatan perifer beredar konsentrasi
IgA. 51
Induksi IL-10 perifer 1 sel Treg dilaporkan
setelah imunoterapi sublingual rumput dan birch
serbuk sari. 56,85
Sebuah studi waktu-kursus selama subkutan
serbuk sari rumput imunoterapi menunjukkan
bahwa PBMC diperoleh sedini 2 sampai 4 minggu
selama updosing awal, ketika cocultured dengan
serbuk sari rumput alergen selama 6 hari,
menghasilkan tingkat tinggi IL-10 di
supernatan. 57 awal sinyal IL-10 ini disejajarkan
erat dengan menekan respon akhir-fase alergen-
diinduksi ( Gambar 3 ). Peningkatan IL-10
produksi dan penindasan respon akhir yang diikuti
secara berurutan oleh peningkatan serum
IgG 4 tingkat pada 6 sampai 8 minggu yang
memuncak pada 16 minggu, bersama dengan
penekanan paralel tanggapan kulit segera. Setelah
imunoterapi serum terbukti memiliki IgG terkait
aktivitas IgE-blocking untuk kedua aktivasi basofil
(alergen meningkat dirangsang basofil CD63) dan
IgE-FAB penghambatan yang sejajar peningkatan
IgG 4 tingkat. Dalam waktu in vivo saja dari
PBMC IL-10 produksi dan perubahan terkait
dalam serum memblokir antibodi, respon kulit
alergen-diinduksi dan perubahan hipotetis dalam
limfosit Th2 selama updosing, 57 dan pemeliharaan
serbuk sari rumput imunoterapi selama 3 tahun
dan selama imunoterapi penarikan 49 ditampilkan
di Gambar 3 .
Regulasi sel B. Regulasi sel B adalah
subset sel B yang memproduksi IL-10 dan
memiliki kapasitas untuk menghambat sel T - dan
respon inflamasi DC-dimediasi dan memelihara
toleransi imunologi alami. 97 populasi dimurnikan
dari IL-10 -producing sel Breg di racun
lebah pelajaran -tolerant dipamerkan ekspresi
permukaan tinggi CD25 dan CD71 dan ekspresi
rendah CD73. Sel-sel ini memiliki kemampuan
untuk menekan racun lebah proliferasi sel-T -
specific. 98 Selain itu, asal dari alergen spesifik
IgG 4 antibodi setelah racun lebah imunoterapi itu
terbukti dari fosfolipase A 2 -specific IL-10 1 Sel
Breg. Selain IL-10, sel Breg telah ditunjukkan
untuk mengerahkan kemampuan penekan mereka
melalui produksi TGF b dan IL-35. 97 Sangat
mungkin bahwa respon sel Breg yang sama dapat
diperoleh selama imunoterapi dengan serbuk sari
rumput atau tungau debu rumah alergen. Apakah
fenotipe yang sama diungkapkan oleh sel B
setelah imunoterapi dengan alergen inhalan masih
harus ditentukan.
Deviasi imun T H 1. Penindasan kekebalan
T H 2 selama kedua imunoterapi subkutan dan
sublingual juga telah dikaitkan dengan
penyimpangan kekebalan tubuh dan induksi sel
T H 1. 9,99 Dalan studi in situ hibridisasi mukosa
hidung setelah imunoterapi subkutan sukses
meningkat ditunjukkan dalam IFNG mRNA 1 sel T
setelah tantangan alergen yang berkorelasi dengan
penurunan gejala hidung selama musim serbuk
sari. 9 Pollen imunoterapi dikaitkan dengan
penurunan rasio IL5 / IFNG mRNA 1sel di
mukosa dan peningkatan hidung IFN- g tingkat
protein dalam cairan hidung saat terpapar alergen
musiman alami. 52 Demikian pula, subkutan serbuk
sari rumput imunoterapi mengakibatkan
1
kenaikan IL12 mRNA makrofag pada kulit yang
disertai penekanan respon kulit an dan berkorelasi
positif dengan IFN- lokal g 1 T sel dan berbanding
terbalik dengan IL-4 sel T -expressing. 100
Bukti untuk / terhadap deviasi T H 1 dalam studi
darah perifer telah lebih kontroversial. 101.102 Satu
studi menyarankan bahwa bergeser dari T H 2
untuk T H 1 tanggapan mungkin telah berhubungan
dengan kematian aktivasi sel yang diinduksi
alergen-responder T H 2 sel. 103 Selama birch
serbuk sari imunoterapi subkutan, peningkatan
sementara Bet v 1 -specific IL-10 -secreting sel
pada 3 bulan diikuti pada 12 bulan oleh penurunan
rasio Bet v 1 -specific IL-5 / IFN- g sel -secreting
T. 104.105 Bahkan,kelangsungan hidup sel T H 1
telah dilaporkan setelah penghapusan sel T H 2. 106
Imunoterapi alergen dan ILCs
Pengaruh imunoterapi pada sel ILC2 telah
dipelajari dalam darah perifer tetapi tidak pada
organ target, sebagian karena kesulitan dalam
mengidentifikasi sel-sel yang tidak
mengekspresikan garis keturunan sel penanda
diakses lokalisasi imunohistokimia pada jaringan.
Setelah serbuk sari rumput imunoterapi subkutan,
ada penghambatan ditandai kenaikan musiman di
garis keturunan-negatif CRTH2 1 CD127 1 ILC2s
yang berkorelasi dengan keparahan gejala yang
dilaporkan sendiri selama musim serbuk
sari. 26 Hasil ini sangat signifikan bila
dibandingkan dengan kenaikan musiman di nomor
ILC2 diamati pada subyek kontrol cocok yang
tidak diobati dengan rhinitis alergi musiman. Data
ini didukung oleh penghambatan kenaikan
musiman dalam jumlah dari CD117 1 (c-kit 1 )
ILC2s dan dalam proporsi IL-13 1 ILC2s,
sebagaimana ditentukan oleh sarana pewarnaan
intraseluler sitokin. Dalam sebuah studi dari
imunoterapi pada peserta dengan asma
musiman, 27 tidak ada perubahan dalam jumlah
ILC2s, meskipun hal ini mungkin dijelaskan
dengan pengukuran telah dilakukan di luar musim
ketika peserta tanpa gejala. Untuk pengetahuan
kita, belum ada laporan dari pengaruh imunoterapi
pada bawaan sitokin epitel berasal, yang diketahui
terlibat erat dalam regulasi kedua peristiwa TH 2
dan ILCs.
Imunoterapi alergen dan DCs
Mukosa bukal terkena terus-menerus untuk
protein asing dalam makanan dan mewakili
lingkungan protolerogenic yang berbeda. Studi ex
vivo bukal spesimen biopsi mukosa dari
pasien dengan alergi serbuk sari rumput telah
menunjukkan bahwa sel-sel Langerhans mukosa
mulut mengikat utama alergen serbuk sari rumput
Phl p 5 dalam dosis-dan tergantung waktu dengan
cara yang dataran tinggi pada 5 menit dan
mengarah ke pematangan melambat sel
Langerhans lisan secara paralel dengan kapasitas
migrasi ditingkatkan dan peningkatan pro-duksi
sitokin tolerogenic yang mencakup IL-10 dan
TGF b . 107
Dalam uji coba terkontrol secara acak dari
imunoterapi sublingual, meskipun kenaikan lokal
dalam jumlah dari FOXP3 1 sel Treg di spesimen
biopsi mukosa sublingual, tidak ada perubahan di
daerah DC monosit yang diturunkan, meskipun
CD1a 1 sel Langerhans tidak diperiksa secara
khusus. 87 Namun, pengaruh imunoterapi alergen
pada subtipe dari DC dalam sirkulasi darah telah
dipelajari. Studi PCR dari sampel darah perifer
diambil sebelum dan setelah 4 bulan sublingual
serbuk sari rumput imunoterapi digunakan untuk
mengkarakterisasi perubahan DC fenotipe. Sebuah
peningkatan yang signifikan dalam jumlah DC
dengan fenotipe DCreg diamati. 32 DCreg
tercermin dengan peningkatan ekspresi mRNA untuk IgE
untuk stabilin-1 dan melengkapi komponen 1Q ELIFAB 43
(C1q), seperti yang diperkirakan dari studi in aktivasi 111,
vitro . 32 Menariknya, tanda tangan DCreg ini basofil CD63 115-117
diamati hanya pada mereka ' 'penanggap ' ' untuk CD203c 112, 118
imunoterapi, yang tercermin dari penurunan yang 16, 112,
signifikan dalam gejala DAO 113
rhinoconjunctivitis. 32 Untuk mendukung temuan pelepasan
ini, pengobatan 1 tahun dengan imunoterapi histamin 16, 112,
sublingual pada anak-anak dengan alergi tungau basofil 113
mengakibatkan DC perifer yang menunjukkan T H 2: IL-4, IL-
fenotipe belum matang dan peningkatan kapasitas Sitokin 13, IL-9, IL-17,
untuk memproduksi IL-10 dan penurunan tingkat dan eotaksin, 99, 119-
IL-12. 102 kemokin TNF a 122
T H 1: IFN- g ,
IL-12 99
Biomarker terhadap respon imunoterapi Peraturan: IL-
alergen 10, TGF b 83
biomarker
Pedoman internasional menyoroti kebutuhan seluler sel Treg 83-88
untuk pengukuran kuantitatif dan divalidasi untuk sel Breg 123, 124
biomarker potensial. 32, 33,
DC 102, 114
TABEL I. Enam domain biomarker: (1) antibodi, tes alergen
(2) aktivitas penghambatan serum untuk IgE, (3) In provokasi
aktivasi basofil, (4) sitokin dan kemokin, (5) vivo biom (SPT, ID, NPT, 9, 72,
biomarker seluler, dan (6) in vivo biomarker arker dan EEC) 125-127
studi Chamber 128, 129

b
EEC, ruang eksposur lingkungan; ID, uji
i
intradermal; IgE-BF, IgE faktor
o
menghalangi; NPT, tes provokasi hidung; SPT, uji
m
tusuk kulit.
a
r
k Ref
Sebuah Eropa Academy of Allergy and Clinical
e ere
Immunology Task Force melaporkan pernyataan
Domain r nsi
konsensus tentang biomarker potensi imunoterapi
alergen. 101 ini diklasifikasikan menjadi 7 domain
IgE (SiGe, IgE 44, 51, ( Tabel I ) * : (1) IgE (IgE total, Sige, dan SiGe /
total, Sige / 59, 96, rasio total IgE), (2) subclass IgG (alergen spesifik
Antibodi total IgE) 109, 110 IgG [IgG] 1 dan Sigg 4 , termasuk yang Sige /
83, 95, IgG 4 ratio), (3) aktivitas penghambatan serum
Sigg 4 106 untuk IgE (IgE-FAB), (4) aktivasi basofil, (5)
IgA 51, 87 sitokin dan kemokin, (6) penanda selular (sel
Kegiatan Treg, sel Breg, danDC), dan (7) in
Hambat vivo biomarker, yang meliputi tes provokasi. 108
serum IgE-FAB 59

IgE (rasio total IgE total, sIgE, dan sIgE / IgE)
Kriteria inklusi untuk inisiasi dari
imunoterapi mengandalkan riwayat gejala pada
paparan alergen 1.130.131 dan peningkatan kadar
serum SigE ke alergen yang relevan secara klinis,
yang diukur dengan menggunakan sistem
ImmunoCAP. Pemilihan pasien telah
disempurnakan oleh ketersediaan teknologi
alergen rekombinan untuk mengidentifikasi IgE
spesifik terhadap alergen penentu utama dan
mengenali relevan alergen bereaksi
silang. 132 Misalnya, seorang pasien dengan kadar
IgE tinggi untuk Phl p 1 dan Phl p 5 mungkin
calon yang baik untuk imunoterapi serbuk sari
rumput. Sebaliknya, sensitivitas IgE ke serbuk sari
rumput profilin Phl p 12 mungkin mengakibatkan
peningkatan kadar IgE tinggi dan respon tes kulit
positif palsu untuk seluruh ekstrak birch serbuk
sari karena reaktivitas silang dengan birch profilin
Bet v 2.
Peningkatan awal di tingkat SigE selama
kedua subcutane-ous 59 dan sublingual 109
imunoterapi serbuk sari telah terbukti diikuti oleh
menumpulkan kenaikan musiman di tingkat
SigE. Dalam studi jangka panjang dari
imunoterapi subkutan, penurunan bertahap dalam
tingkat SigeE selama beberapa tahun 44 diamati,
meskipun tidak ada hubungan yang jelas antara
perubahan tingkat SigE dan besarnya respon
klinis. 51,96 Rasio tertentu IgE / total serum IgE
pada awal dilaporkan berkorelasi dengan respons
klinis terhadap imunoterapi, 110.133 meskipun orang
lain 53,60,134 belum mereplikasi temuan ini.
IgG subklass ( Rasio sIgG1 , sIgG4 , dan sIgE /
IgG 4 )
Immune-reaktif IgG 1 dan IgG 4 antibodi
dapat diukur dengan menggunakan ImmunoCAP
( Gambar 4 , A ) dan dengan menggunakan
microarray alergen (misalnya, ImmunoSolid
Allergen Chip Assay [ISAC]). Alergen spesifik
subtipe IgG, termasuk IgG 1 dan khususnya
IgG 4 telah terbukti ditingkatkan di kisaran 10
sampai 100 kali lipat dibandingkan dengan nilai-
nilai dasar selama imunoterapi, meskipun dengan
tidak ada korelasi yang konsisten dengan respon
klinis terhadap pengobatan. 83,95,106
ISAC dapat dilakukan dengan
menggunakan volume yang sangat kecil dari
serum atau cairan hidung. Sebuah komponen besar
peningkatan yang diamati dalam serum alergen
spesifik IgG atau IgG 4 tingkat setelah imunoterapi
alergen mungkin mencerminkan paparan alergen
tinggi dan dapat digunakan berpotensi untuk
memantau kepatuhan pasien 'untuk rejimen
imunoterapi. 108 Penurunan rasioSigE / IgG 4 telah
dilaporkan setelah imunoterapi subkutan dan
dikaitkan dengan penurunan reaksi kulit
terlambat. 101 Namun, temuan ini belum
direproduksi dalam penelitian lain. 101
Aktivitas serum IgE inhibisi (IgE-FAB
dan enzyme-linked immunosorbent -
facilitated assay antigen-binding)
IgG-terkait aktivitas IgE-hambat dapat
dinilai dengan menggunakan aliran
cytometry assay berbasis (IgE-FAB) yang telah
divalidasi sesuai dengan Konferensi Internasional
tentang pedoman Harmonisasi. 135 Assay ini
mengukur kemampuan IgG yang mengandung
serum diperoleh setelah imunoterapi alergen untuk
menghambat Fc ε RII tergantung pengikatan
kompleks alergen-IgE pada sel-sel B, pengganti
untuk presentasi antigen IgE-difasilitasi untuk sel
T ( Gambar 4 , B ) . Sebuah pendekatan alternatif
adalah enzim-linked immunosorbent -facilitated
antigen-binding assay (ELIFAB). 43 IgE-FAB
assay adalah direproduksi tetapi secara teknis
lebih kompleks dan saat ini terbatas pada
laboratorium khusus. 59 Data yang terbatas yang
tersedia menunjukkan korelasi sederhana antara
IgE-FAB dan hasil ELIFAB dan respon klinis
untuk imunoterapi lebih dan di atas yang diamati
ketika hanya mengukur tingkat imunreaktif
IgG. 43,59 ini kemungkinan terkait dengan IgE-FAB
dan ELIFAB, memberikan ukuran lebih
fungsional dari afinitas dan / atau aviditas antibodi
yang mengikat.

Gambar 4. Induksi antibodi IgG4 dan aktivitas
penghambatan IgE terkait selama imunoterapi.
A, kadar IgG4 Spesifik. B, alergen IgE yang
difasilitasi mengikat sel B. C, pelepasan
histamin pada tingkat sel tunggal dengan
menggunakan DAO berlabel (peningkatan
DAO intraseluler menunjukkan penghambatan
pelepasan histamin). D, Histamin ELISA.
Pengukuran dilakukan dari pasien yang tidak
diobati dengan alergi rumput (SAR), pasien
imunoterapi subkutan (SCIT) - dan pasien sub-
taloterapi (SLIT), dan mereka yang telah
menyelesaikan SLIT 3 tahun, diikuti dengan
penghentian hingga 3 tahun (SLIT-TOL ). Data
dinyatakan sebagai data individual (kotak
kuintil dengan kontur). Tes P <.05 dan *** P
<.001, Mann-Whitney U.16
Aktivasi basofil
Dalam aliran tes cytometry berbasis
menggunakan seluruh darah, aktivasi basofil dapat
dipelajari dengan memantau ekspresi penanda
permukaan, seperti CD63 dan CD203c. Meskipun
ekspresi CD63 mengukur basofil
111
degranulasi, CD203c merupakan penanda
basofil tertentu yang juga mengukur IL-3 aktivasi
-tergantung dari basofil. Sebuah uji fungsional
novel yang mendeteksi pewarnaan intraseluler
phycoerythrin-terkonjugasi diamin oksidase
(DAO) juga telah divalidasi. DAO mengikat erat
histamin substrat, sehingga hasil rangsangan
alergen dalam penurunan tingkat DAO intraseluler
basofil sebanding dengan jumlah histamin
intraseluler dirilis. Penurunan ini telah terdeteksi
selama immunotherapy baik subkutan dan
sublingual ( Gambar 4 , C dan D ). 112.113
Sitokin dan kemokin
Kemajuan terbaru dalam miniatur analisis
multipleks sitokin dengan penemuan skala meso
dan Luminex platform telah memungkinkan
pengukuran sitokin dan kemokin dalam cairan
hidung yang meningkatkan respon terhadap
alergen provokasi dan dimodifikasi oleh
imunoterapi. 69,70
Penanda seluler dan molekuler
Penanda seluler potensi penggunaan untuk
menilai atau memprediksi respon terhadap
imunoterapi meliputi penanda fenotip sel T (T H 2,
Treg, T FH / T FR , dan T H 1 sel) dan sub-populasi
sel Breg, yang semuanya telah ditunjukkan untuk
diubah selama immunotherapy, terutama dengan
menggunakan aliran cytometry. Meskipun dalam
uji klinis tanda tersebut telah mampu membedakan
antara kelompok perlakuan dan berkorelasi secara
keseluruhan dengan hasil klinis dari
khasiat, 69,83,88,136,137 mereka tidak mampu
membedakan responden dari nonresponders atau
memprediksi respon pada subyek individu. Selain
itu, ada kebutuhan untuk pengolahan sel yang
optimal dan pengalihan dan penyimpanan sampel
sehingga cytometry aliran kompleks untuk
beberapa T dan sel B -associated penanda adalah
di luar lingkup laboratorium klinis rutin.
DC mengekspresikan penanda molekuler
yang berbeda sesuai dengan sel T
mereka kapasitas -differentiating. DCregs
istimewa mengungkapkan C1q dan Fc g RIII,
yang mendukung perkembangan sel Treg
preferensial, 32 sedangkan DC2s mengungkapkan
CD141, gata-3, OX40 ligan, dan RIPK4, yang
mendukung polarisasi sel T naif menjadi T H 2
sel. 33 Ekspresi tanda tersebut di PBMC dievaluasi
sebelum dan pada 2 dan 4 bulan setelah sublingual
serbuk sari rumput imunoterapi. RT-PCR ini
berbasis metode kuantitatif berkorelasi dengan
hasil klinis. 33.114 Hebatnya, kombinasi optimal
dari 5 penanda molekuler yang termasuk 3 DC2
penanda (CD141, gata-3, dan RIPK4) dan 2
penanda DCreg (C1QA dan Fc g RIIIA) mampu
membedakan responden klinis dari nonresponders
dengan sensitivitas 90,48% dan spesifisitas
61,9%. Hasil ini menarik menuntut evaluasi lebih
lanjut dalam uji klinis dan akhirnya dalam praktek
klinis.
In vivo biomarker
In vivo biomarker mengacu pada penggunaan
tes provokasi alergen untuk mengevaluasi
pasien reaktivitas alergen spesifik 'sebelum dan
setelah pengobatan. Tes provokasi meliputi tes
kulit tusuk, tes intradermal, dan hidung,
konjungtiva, dan tes provokasi
101
bronkial. Misalnya, besaran respon awal dan
akhir setelah serbuk sari rumput tantangan hidung
dan setelah pengujian intradermal yang dihambat
oleh kedua imunoterapi subkutan dan sublingual.
Sebuah korelasi sederhana yang diamati antara
skor total gejala nasal dicatat setelah tantangan,
respon kulit akhir, dan peserta penilaian subjektif
'demam keparahan selama musim serbuk
sari. 69,70 European Medicines Agency telah
menguraikan penggunaan pengujian provokasi
untuk bukti konsep untuk pendekatan baru,
temuan dosis alergen, dan digunakan sebagai
mendukung poin akhir kemanjuran sekunder
selama uji klinis dari imunoterapi alergen. 138
Kesimpulan
Dalam konteks sejarah klinis dari gejala
yang paparannya relevan pada alergen hirup,
tingkat serum sIgE adalah satu-satunya biomarker
yang paling relevan bagi pasien yang telah dipilih
untuk imunoterapi alergen, dan ini telah
disempurnakan oleh ketersediaan teknologi
rekombinan alergen. 132 Namun, pada saat ini,
tingkat sIgE tidak dapat dipercaya untuk
memprediksi atau memantau respon klinis untuk
imunoterapi. Saat ini, rasio IgE / IgE total pada
awal tetap sedang dalam evaluasi sebagai sebuah
prediktor respon yang lebih memungkinkan. Tes
fungsional dari aktivitas penghambatan IgG terkait
untuk IgE (IgE-FAB dan ELIFAB) memiliki
korelasi yang lebih baik dengan respon klinis
dalam hasil uji klinis dari tingkat immunoreactive
IgG / IgG 4 tetapi tidak dapat memprediksi
keberhasilan dalam subyek individu. Tes berbasis
serum memiliki keuntungan dari kemudahan
penanganan dan penyimpanan sampel, dan
tampaknya mungkin bahwa tingkat IgG /
IgG 4 cenderung lebih efektif sebagai pengganti
untuk kepatuhan dalam pengobatan, yang bisa
menjadi nilai khusus untuk memantau pasien yang
menerima immunotherapy sublingual. Dilaporkan
bahwa berbagai tes mengenai sel telah dibatasi
pada pusat-pusat spesialis. Tes mengenai daya
tanggap Basophil dan fenotipik T / B-celltes
membutuhkan aliran cytometry dan melibatkan
masing-masing, baik pengolahan darah segar atau
pemisahan sel kompleks dan protokol
penyimpanan. Mereka sangat informatif sebagai
bukti bagi konsep dalam uji klinis, tetapi tidak
layak untuk praktek klinis rutin.
Studi dari DC fenotipe dengan
menggunakan RT-PCR pada darah secara
keseluruhan atau PBMC telah ditunjukkan untuk
memisahkan responden klinis dan nonresponders
terhadap menyerbuki sublingual immunotherapy,
dan studi lebih lanjut diperlukan untuk mereplikasi
temuan ini dan menguji nilai mereka pada pasien
individu.
Pendekatan Novel untuk imunoterapi
Pemahaman yang lebih baik dalam
mekanisme harus diterjemahkan secara ideal
dalam pendekatan imunoterapi
1.139
baru. Tujuannya adalah untuk meningkatkan
efektivitas lebih terhadap standarekstrak alergen -
ekstrakberbasisketika memperkenankan strategi
yang lebih pendek, lebih aman, dan lebih nyaman
untuk pasien. Rute alternatif, seperti
epicutaneous 140 dan pendekatan
intralymphatic 141.142 , telah terbukti lebih aman
daripada imunoterapi subkutan konvensional,
meskipun tidak ada uji coba secara langsung untuk
menilai komparasi kemujarabannya. Rute
intradermal tidak efektif untuk penyerbukan
alergen dan mungkin telah memperburuk gejala
musiman. 143 Menarget kepada penyimpangan
kekebalan tubuh menggunakan Toll-like receptor
4 agonis monofosforil.A dalam kombinasi dengan
penyerbukan allergoid imunoterapi subkutan telah
efektif dengan 4 suntikan preseasonal tanpa
peningkatan efek samping. 144 Sebuah percobaan
oligonukleotida DNA bakteri yang kaya akan
urutan CpG secara kovalen terhubung dengan
ragweed alergen utama Amb 1, telah efektif dalam
uji coba fase II, mungkin dengan menginduksi sel
Treg dan / atau penyimpangan kekebalan
tubuh, 145 meskipun pendekatan ini gagal pada
tahap III. 94 Demgan menargetkan IgE atau tipe 2
sitokin (IL-4 dan IL-5) telah berhasil dalam
mengurangi eksaserbasi pada pasien asma,
meskipun tanpa efek yang bertahan lama setelah
penghentian. 146 Anti-IgE dalam kombinasi dengan
imunoterapi alergen sangat efektif dalam
mengurangi risiko reaksi alergi
147
sistemik. Kombinasi dari anti -iL-4 dengan
subkutan imunoterapi alergen adalah efektif dalam
menekan beredar T H 2 sel dan alergen-diinduksi
yang terlambat merespon tetapi tidak
menunjukkan keuntungan lebih dari ekstrak
alergen itusendiri. 148 Pendekatan Novel saat ini
adalah untuk mengurangi efek samping sistemik
termasuk penggunaan rekayasa molekul
hypoallergenic rekombinan 132 dan alergen
peptida berbasis pendekatan yang secara khusus
menargetkan baik T-sel epitop 149.150 atau strategi
yang berbasispadasel B epitop yang secara
selektif menonjolkan respon IgG alergen
spesifik. 151.152 Untuk kedepannya, menargetkan
respon imun bawaan menggunakan antibodi
terhadap sitokin epitel IL-33, IL-25, atau thymus
stroma lymphopoietin dalam kombinasi dengan
imunoterapi alergen akan menjadi strategi
kombinasi yang menarik untuk memungkinkan
pengurangan peradangan, menekan ILC2s, dan
menonjolkantolerogenic DC fenotipe yang lebih.