Mechanisms of allergen-specific immunotherapy.

Abstract

Allergen-specific immunotherapy (allergen-SIT) is a potentially curative treatment approach in allergic diseases. It has been used for almost 100 years as a desensitizing therapy. The induction of peripheral T cell tolerance and promotion of the formation of regulatory T-cells are key mechanisms in allergen-SIT. Both FOXP3+CD4+CD25+ regulatory T (Treg) cells and inducible IL-10- and TGF-β-producing type 1 Treg (Tr1) cells may prevent the development of allergic diseases and play a role in successful allergen-SIT and healthy immune response via several mechanisms. The mechanisms of suppression of different pro-inflammatory cells, such as eosinophils, mast cells and basophils and the development of allergen tolerance also directly or indirectly involves Treg cells. Furthermore, the formation of non-inflammatory antibodies particularly IgG4 is induced by IL-10. Knowledge of these molecular basis is crucial in the understanding the regulation of immune responses and their possible therapeutic targets in allergic diseases.

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REVIEWOpen Access
Mechanisms of allergen-specific immunotherapy
Hiroyuki Fujita1,2,3, Michael B Soyka1, Mübeccel Akdis1and Cezmi A Akdis1*
Abstract
Allergen-specific immunotherapy (allergen-SIT) is a potentially curative treatment approach in allergic diseases. It
has been used for almost 100 years as a desensitizing therapy. The induction of peripheral T cell tolerance and
promotion of the formation of regulatory T-cells are key mechanisms in allergen-SIT. Both FOXP3+CD4+CD25+
regulatory T (Treg) cells and inducible IL-10- and TGF-b-producing type 1 Treg (Tr1) cells may prevent the
development of allergic diseases and play a role in successful allergen-SIT and healthy immune response via
several mechanisms. The mechanisms of suppression of different pro-inflammatory cells, such as eosinophils, mast
cells and basophils and the development of allergen tolerance also directly or indirectly involves Treg cells.
Furthermore, the formation of non-inflammatory antibodies particularly IgG4 is induced by IL-10. Knowledge of
these molecular basis is crucial in the understanding the regulation of immune responses and their possible
therapeutic targets in allergic diseases.
Background
The immune system is a complex interactive network
with the capacity of protecting the host from a number
of pathogens while keeping a state of tolerance to self
and innocuous non-self antigens. Allergy is one of the
immune tolerance-related diseases that arises as a direct
consequence of a dysregulated immune response. Cur-
rently, allergen-specific immunotherapy (allergen-SIT)
by the administration of increasing doses of allergen
extracts remains the single curative approach to allergic
diseases with the potential to modify its course [1,2].
The aim of this review is to discuss the mechanism of
allergen-SIT and the current clinical and experimental
evidence in the field of immune tolerance induction in
allergic diseases.
Pathogenesis of allergic diseases
Allergic diseases represent complex innate and adaptive
immune responses to environmental antigens leading to
inflammatory reactions with a T-helper-2-type cell and
allergen-specific IgE predominance [3,4]. CD4+Naïve T
cells differentiate into distinct T cell subsets such as
Th1, Th2, Th9, Th17 and Th22 type memory and effec-
tor cells depending on the cytokines, other molecules
and cells present in the microenvironment [5]. Once a
Th2 shift is established, the mechanism of allergic dis-
eases consists of two main phases. In the early phase
sensitization and the development of memory cells takes
place. The late phase is characterized by inflammation
and tissue injury caused by effector cell action. During
the sensitization phase, the differentiation and clonal
expansion of allergen-specific CD4+Th2 cells, with the
capability of producing IL-4 and IL-13, are essential in
the induction of class switching to the ε immunoglobu-
lin heavy chain in B cells and the production of aller-
gen-specific IgE antibodies. Allergen-specific IgE binds
to the high affinity receptor FcεRI, on the surface of
mast cells and basophils as well as to antigen presenting
cells (APCs), which in turn allows for an increased
uptake of allergens [6]. The engagement of IgE on effec-
tor cells leads to the sensitization of the patients to a
specific allergen [7]. Upon re-exposure receptor-bound
IgE molecules are crosslinked, which in turn results in
the activation and release of mediators that cause[8] the
development of type I hypersensitivity reactions [9,10].
During the development of allergic diseases, effector
Th2 cells not only produce traditional Th2 cytokines
such as IL-4, IL-5, IL-9 and IL-13 [11,12], but also novel
cytokines with proinflammatory functions, such as IL-
25, IL-31 and IL-33 [13-19]. These cytokines induce
allergen-specific IgE, eosinophilia, mucus production
and the recruitment of inflammatory cells to inflamed
tissues. Predominance of Th2 cells might be caused by
an increased tendency to activation-induced cell death
* Correspondence: akdisac@siaf.uzh.ch
1Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich,
Davos, Switzerland
Full list of author information is available at the end of the article
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© 2012 Fujita et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
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of high IFN-g-producing Th1 cells as it is commonly
observed in patients with atopic disorders [20]. Th1
cells also play a role in the effector phase of allergic dis-
eases by inducing apoptosis of epithelial cells and/or
smooth muscle cells in asthma and keratinocytes in ato-
pic dermatitis [21-25]. In vitro, the suppressive capacity
of CD4+CD25+T-regulatory (Treg) cells from hay fever
patients is decreased during the pollen season [26].
Allergen-specific IL-10 secreting Treg cells were shown
to be decreased in blood obtained from patients with
persistent allergic rhinitis although the number and
function of CD4+CD25+Treg cells were normal [27].
Different symptomatic treatments like antihistamines,
leukotriene receptor antagonists and glucocorticoids are
used in allergic diseases, however do not provide the
possibility of cure [6]. Glucocorticoids, systemically
applied, increases the frequency of CD25+memory CD4
+T cells and FOXP3 messenger RNA [28].
Mechanisms of allergen-specific immunotherapy
T cell regulation
Since allergic diseases are not only Th2 driven, but
much rather form complex immune disorders, the aim
of allergen SIT is to induce the peripheral T cell toler-
ance, modulate the thresholds for mast cell and basophil
activation and decrease IgE-mediated histamine release
[29] (Figure 1 and 2). The induction of peripheral T cell
tolerance represents an essential step in allergen-SIT.
Peripheral T cell tolerance is characterized by the gen-
eration of allergen-specific Treg cells that are able to
produce anti-inflammatory cytokines such as IL-10 and
TGF-b. Multiple mechanisms are involved in the sup-
pression and/or control of allergic inflammation. Treg
cells not only diminish Th2 immune responses, but also
target other cell types such as DCs, mast cells, basophils
and eosinophils. Treg cells regulate allergen-specific-IgE
and are capable of inducing IgG4 and IgA production
[30-33]. Treg cells are able to directly inhibit mast cell
degranulation by OX40-OX40Ligand interaction [30].
There are two main Treg cells subsets with distinct phe-
notypes and mechanisms of action. One is the naturally
occurring, thymic selected FOXP3+CD4+CD25+Treg
cells. The other subset is referred to as the inducible
Treg cells, generated in the periphery under tolerogenic
conditions. The two subsets of inducible Treg cells,
namely the FOXP3+and the IL-10-positive Tr1 cells
play a key role in allergen tolerance and they can be
induced by allergen SIT in humans [34,35].
It is well established that FOXP3 acts as the main
transcription factor for Treg cells development and
function [36]. FOXP3 mutations in humans lead to the
X-linked immune dysregulation polyendocrinopathy
enteropathy syndrome [37]. Patients affected by this
defect suffer from allergic and autoimmune diseases due
to non-functional Treg cells. FOXP3 mutations in mice
leadtothespontaneous developmentof
Figure 1 Immune regulation during the time course of allergen-SIT. Specific immune responses are observed during the course of allergen-
SIT. 1. An early desensitization effect including decrease in mast cell and basophil degranulation soon after the first administration of allergens. 2.
Generation of allergen-specific Treg cells and suppression of effector cells. 3. An early increase and a late decrease in specific IgE levels. 4. A
relatively early increase in specific IgG4. 5. A late decrease in type I skin test reactivity. 6. A decrease in tissue mast cell and eosinophil numbers
and a release of their mediators after a few months.
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lymphoproliferative disease, allergic airway inflamma-
tion, hyper IgE syndrome, eosinophilia and autoimmune
diseases [38]. It has been demonstrated that FOXP3
directly interacts with the Runt-related transcription fac-
tor 1 (RUNX1), which reduces IL-2 and IFN-g expres-
sions and exerts suppressive functions [39]. A recent
study in mice has also shown that the RUNX transcrip-
tion factors are essential for maintaining high FOXP3
expression and confirm Treg lineage identity [40].
Moreover, a novel molecular mechanism defining Runx
transcription factors as a linking molecule in TGF-beta
induced Foxp3 expression in Treg differentiation and
function was shown [41].
Several studies have demonstrated that allergen-speci-
fic Tr1 cells are highly present in healthy individuals to
prevent unwanted immune response to nonpathogenic
environmental antigens [42-45]. The three different
allergen-specific T cell subsets Th1, Th2 and Tr1 that
recognize the same T-cell epitopes co-exist in both
healthy and allergic individuals in different proportions.
Persons with high numbers of Th2-cells are prone to
develop an allergic phenotype, whereas Tr1 predomi-
nance seems rather protective in this perspective [43].
High dose-allergen exposure and the induction of
tolerance have been well investigated for bee venom and
cat allergens [45]. Beekeepers face high levels of bee
venom antigens during the beekeeping season. Repeated
exposure to the venom allergens results in a reduction
in T-cell-related cutaneous late-phase reactions and an
impaired capacity of allergen-specific T cells to prolifer-
ate and produce Th1 and Th2 cytokines. This reaction
persists as long as bee venom exposure continues.
Venom-specific T cell proliferation, which is suppressed
at the time of exposure returns to initial levels within
several months after the end of the beekeeping season.
This phenomenon correlates with a clonal switch of
venom antigen-specific Th1 and Th2 cells toward IL-10-
secreting Tr1 cells. In this model, histamine receptor 2
is up-regulated on specific Th2 cells and plays a dual
role in the suppression of allergen-stimulated T cells
and in the induction of IL-10 production. Allergen-spe-
cific IgG4/IgE ratios are about thousand times higher in
non-allergic beekeepers compared with bee venom aller-
gic individuals [46]. In another high-dose allergen expo-
sure model with cat allergens, an increase of allergen-
specific IgG4 and IL-10-producing Tr1 cells were
demonstrated [47]. IL-10 reveals multiple ways of action
in allergen tolerance. It leads to the downregulation of
Figure 2 Mechanisms of allergen-specific immunotherapy and the role of regulatory T cells in allergic diseases. An allergen is taken up
by regional dendritic cells leading to the induction of regulatory T cells. These cells suppress allergic responses directly and indirectly by the
following mechanisms. 1. Suppression of mast cells, basophils and eosinophils. 2. Suppression of effector T cells. 3. Suppression of inflammatory
cell migration to tissues and tissue inflammation. 4. Suppression of mucus production. 5. Suppression of inflammatory dendritic cells and
induction of tolerogenic dendritic cells. 6. Suppression of allergen-specific IgE and induction of IgG4 from B cells.
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MHC-II molecules on APCs and inhibits a wide range
of proinflammatory cytokines and cytokine receptors
[48]. IL-10 reduces IL-5 production by Th0 and Th2
and downregulates eosinophil activity [49]. Thus,
reduced levels of eosinophilic cationic protein were also
found during SIT [50]. Treg cells derived TGF-b bears a
great potential in allergen tolerance. This cytokine not
only inhibits B-cell proliferation and differentiation, but
also decreases immunoglobulins with the exception of
mucosal IgA [51,52]. TGF-b is able to promote further
CD4+CD25+T cell conversion from naïve CD4+CD25-
T cells [53]. SIT is able to increase TGF-b production
and is therefore associated with higher amounts of spe-
cific IgA [44]. Furthermore Treg cells are capable of
downregulating costimulatory molecules on DCs and
compete with naïve T cells by creating aggregates
around DCs, thus inhibiting their maturation [54].
Apart from the two mentioned main subsets of Treg
cells, several other T cells with regulatory function have
been demonstrated. CD8+CD28-T cells showed sup-
pressor capacity in vitro. They are able to prevent upre-
gulation of B7 molecules induced by helper T cells on
professional APCs and play role in oral tolerance
[55,56]. TCRab+CD4-CD8-double-negative Treg cells
have been shown to suppress antigen-specific immune
responses mediated by CD4+T and CD8+T cells in
humans and mice [57]. NKreg cells have the capacity to
abort antigen-specific T cell responses [58]. A certain
subset of invariant NKT cells also possesses control
functions. The combination of IL-27 and IFN-g pro-
duced by invariant natural killer T (iNKT) cells sup-
presses the established Th2 functions in mice [59].
Antigen-containing liposomal a-galactosylceramide,
which is a representative ligand for iNKT cells, lowers
antigen-specific IgE via the induction of tolerogenic DCs
and Treg cells [60].
Regulation of allergen-specific antibodies
IgG4 is a non-inflammatory isotype protecting from
allergic reaction. It is thought to capture the allergen
before reaching the effector cell-bound IgE and thus to
prevent the activation of mast cells and basophils [29].
IgG4 is unable to bind complement efficiently and con-
tains two different antigen-binding sites on one mole-
cule. The bi-specificity turns the antibody functionally
monovalent, thus preventing it from forming complexes
[61]. Allergen-specific IgG4 might be directed against
different epitopes of the allergen than IgE, yet an inhibi-
tion of the IgE-allergen binding by certain IgG is
observed resulting in a blocking effect [62]. Successful
SIT is associated with an increase in IgG-blocking activ-
ity that is not solely dependent on the quantity of IgG
antibodies [63,64]. It seems to be relevant rather to
measure the blocking activity & affinity of specific IgG
and its subsets (i.e. IgG4, IgG1), instead of their levels in
sera. The induction of IgG also plays a role both by the
inhibition of IgE-facilitated antigen presentation and the
inhibition of IgE-mediated release of mediators from
mast cells and basophils [64]. Allergen-SIT induces a
transient increase of specific IgE levels in serum, fol-
lowed by a gradual decrease over months or years of
treatment [65]. Serum IgE levels cannot explain the
diminished responsiveness to a specific allergen, because
the decrease of serum IgE levels is relatively late and
does not correlate with clinical improvement after SIT.
The decrease in IgE/IgG4 ratio during allergen SIT
seems to be a feature of skewing from allergen-specific
Th2 to Treg cell predominance. Since the class switch-
ing of IgG4 is caused by the co-stimulation with IL-4
and IL-10, IL-10 decreases IL-4-induced IgE switching
but increases IL-4-induced IgG4 production. Thus, IL-
10 not only generates tolerance in T cells, but also regu-
lates the allergen-specific antibody isotype formation
toward a non-inflammatory direction.
Novel suppressive cell subsets and cytokines
Recently, several novel suppressive cell subsets and cyto-
kines have been demonstrated. They could form targets
for novel allergen-specific therapies and need to be
included in the future research on SIT.
Regulatory B cells
B cells are the only cell type that is capable of producing
antibodies and therefore are the central cellular compo-
nent of the humoral immune responses. In addition, B
cells can modulate CD4+T cell responses by presenting
antigens, expressing costimulatory molecules or produ-
cing cytokines [66]. Regulatory B (Breg) cells, which are
able to secrete IL-10, regulate the development, prolif-
eration and maintenance of CD4+T effector and mem-
ory T cells as well as Treg cells [67]. Recent studies
suggest that there are several phenotypically distinct
populations of IL-10-producing Breg cells [68-72]. Tran-
sitional 2-marginal zone precursor B cells which express
CD1dhiCD21hiCD23+IgM+, follicular B cells and B cells
expressing high levels of CD1d can produce IL-10 and
play a role as Breg cells [73-76]. Among several Breg
cell subsets, CD1dhiCD5+CD19hiBreg cells are well stu-
died [77]. The transfer of this subset prevents CD4+T
cell-dependent contact hypersensitivity in mice. Since
this suppressive function is antigen dependent, Breg
cells from mice primed with an antigen were not able to
suppress the T cell inflammation elicited by another
antigen. Moreover, this function requires their ability to
produce IL-10. The Breg cell subset is also characterized
in human blood as CD24hiCD27+B cells [78]. They can
negatively regulate monocyte cytokine production via
IL-10-dependent pathways. Taken together, the antigen-
specific Breg cell subset can be a potent candidate for
novel allergen-specific immunotherapies.
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IL-35
IL-35 is a heterodimeric cytokine consisting of EBI3 and
the p35 subunit of IL-12 [79]. In mice, IL-35 is constitu-
tively secreted by FOXP3+Treg cells [80]. The expres-
sion of EBI3 and p35 in FOXP3+Treg cells is higher
than in effector T cells and transcription analysis identi-
fies EBI3 as a downstream target of FOXP3 [81].
Although IL-35 does not affect the FOXP3 expression
on Treg cells, it can induce the IL-10 production in
CD4+CD25+Treg cells. Stimulation of CD4+CD25-
effector T cells by IL-35 and anti-CD3/anti-CD28 anti-
bodies induces proliferation of these, enhances IFN-g
production and up-regulates the transcription factor T-
bet on T cells [82]. CD4+CD25+T cells expanded in the
presence of IL-35 are able to suppress the proliferation
of CD4+CD25-T cells. IL-35, but not EBI3 alone, inhib-
ited the differentiation of CD4+T cells into Th17 cells.
IL-35 also increases serum levels of IL-10 and IFN-g
whereas it decreases IL-17 [82]. Furthermore, treatment
of naïve T cells with IL-35 induces a novel regulatory T
cell subset, which mediates suppression via IL-35 but
not via other Treg related cytokines such as IL-10 or
TGF-b [83].
Clinical use
Allergen-SIT has been used for more than 100 years in
the therapy of allergic diseases. Desensitization repre-
sents a potentially curative and specific approach to
allergies [29]. Although sublingual immunotherapy
(SLIT) and subcutaneous immunotherapy (SCIT) are
the two main routes of administration, SLIT seems to
be the more safe and favorable route of both. Several
large-scaled, randomized, double-blinded, placebo-con-
trolled trials demonstrated the long lasting and disease-
modifying effects of SLIT [84-88]. Oral SIT possesses a
high potential for the development of novel treatment
modalities. In fact such approaches including oral
immunotherapy for food allergy are under development
[89-93].
SLIT depends on anatomical and functional character-
istics of the oral mucosal tissue, which has a natural tol-
erogenic character. It possesses rapid wound healing
capabilities with little scar formation and defies inflam-
mation in spite of a high bacterial colonization. The lack
of inflammatory cells around mucosal tissue and a high
permeability for allergens enable efficient sublingual
immunotherapy [94]. The initial step in SLIT is the
uptake of an allergen by Langerhans cells within the
mucosa via the high affinity surface IgE receptors
[95,96]. This leads to the production of IL-10 and
induction of T cells with a regulatory phenotype in
vitro [97]. The mechanisms of action in SLIT have been
found to be similar as in injection immunotherapy: Sub-
lingual FOXP3-expressing cells are promoted, allergen-
specific IgG4 and IgA is increased and the inhibitory
activity on IgE was found to be enhanced in a time
dependent manner [98].
Although clinical trials of allergen-SIT demonstrate
treatment efficacy in various allergic diseases such as
allergic asthma, allergic rhinitis, stinging insect hyper-
sensitivity and aero-allergen-induced atopic dermatitis,
there is a risk of serious adverse reactions, which can be
classified in two categories: local reactions appearing as
erythema, pruritus and swelling at the injection site of
SCIT; and systemic reactions appearing as anaphylaxis
from mild to serious life-threatening severity [99-101].
Such side effects are one of the difficulties of SIT to
be overcome. Allergen extract-based SIT includes the
risk of anaphylactic side effects and the potential to
induce novel sensitization to proteins from vaccines
[102]. On the other hand, it might be difficult to stan-
dardize such vaccines leading to inconsistent results in
SIT. Current research focuses on these problems by
creating recombinant vaccines [102] and by altering the
route of administration. Modification of recombinant
vaccines by fusion with so called modular antigen trans-
ducer proteins may enhance specific antibody produc-
tion and is a potential way of reducing the amount of
vaccine needed and therefore limiting side effects [103].
The administration of a vaccine by intralymphatic injec-
tion is also a promising way of reducing the amount of
the allergen dose and therefore improving safety [104].
Systemic side effects are known to occur in the initial
phase of desensitization. Patients need to be monitored
during this period. Different time regimes are used for
the administration of the first vaccinations. Currently
ultra-rush procedures are efficiently used in hymenop-
tera allergy SIT [105]. An increase in Treg cells along
with a Th2 to Th1 switch has been shown to occur
already during the first 24 h [106]. This type of time
regime is now also being investigated in SLIT [107].
Conclusions
Recent developments on molecular mechanisms of
immune regulation in the area of allergy have provided
substantial knowledge on allergen-tolerance. The induc-
tion of peripheral T cell tolerance by Treg cells is a key
point in the suppression of allergic inflammation.
FOXP3+CD4+CD25+Treg cells and Tr1 cells secrete
suppressive cytokines such as IL-10 and TGF-b, and
lead to the production of non-inflammatory antibody
subtypes such as IgG4 and IgA. Novel suppressive cell
subsets and cytokines such as Breg cells and IL-35 may
form targets for new SIT approaches. The understand-
ing of the molecular processes enables us to better
understand the regulation of the immune response.
Novel vaccines are expected to shorten the duration,
decrease the side effects and increase the efficiency of
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