Mechanisms of allergen-specific immunotherapy

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 tolerance, 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 generation of allergen-specific Treg cells that are able to produce anti-inflammatory cytokines such as IL-10 and TGF-β. Multiple mechanisms are involved in the suppression 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 phenotypes 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 lead to the spontaneous development of lymphoproliferative disease, allergic airway inflammation, hyper IgE syndrome, eosinophilia and autoimmune diseases [38]. It has been demonstrated that FOXP3 directly interacts with the Runt-related transcription factor 1 (RUNX1), which reduces IL-2 and IFN-γ expressions and exerts suppressive functions [39]. A recent study in mice has also shown that the RUNX transcription 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-specific 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 predominance 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 proliferate 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-specific IgG4/IgE ratios are about thousand times higher in non-allergic beekeepers compared with bee venom allergic individuals [46]. In another high-dose allergen exposure 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 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-β 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-β is able to promote further CD4⁺CD25⁺ T cell conversion from naïve CD4⁺CD25^- T cells [53]. SIT is able to increase TGF-β production and is therefore associated with higher amounts of specific 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 suppressor capacity in vitro. They are able to prevent upregulation of B7 molecules induced by helper T cells on professional APCs and play role in oral tolerance [55, 56]. TCRαβ⁺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-γ produced by invariant natural killer T (iNKT) cells suppresses the established Th2 functions in mice [59]. Antigen-containing liposomal α-galactosylceramide, which is a representative ligand for iNKT cells, lowers antigen-specific IgE via the induction of tolerogenic DCs and Treg cells [60].

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 contains two different antigen-binding sites on one molecule. 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 inhibition 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 activity 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, followed 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 switching 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 regulates the allergen-specific antibody isotype formation toward a non-inflammatory direction.

B cells are the only cell type that is capable of producing antibodies and therefore are the central cellular component of the humoral immune responses. In addition, B cells can modulate CD4⁺ T cell responses by presenting antigens, expressing costimulatory molecules or producing cytokines [66]. Regulatory B (Breg) cells, which are able to secrete IL-10, regulate the development, proliferation and maintenance of CD4⁺ T effector and memory 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]. Transitional 2-marginal zone precursor B cells which express CD1d^hiCD21^hiCD23⁺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, CD1d^hiCD5⁺CD19^hi Breg cells are well studied [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 CD24^hiCD27⁺ 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.