Discussion
Although PFA is a widespread allergic disorder, the only available disease-modifying therapy for pollen allergy has a rather limited effect on the associated food allergy.25–27 Therefore in this study, human monocyte-derived IL-10 DC were investigated with regard to their potential to induce allergen-specific (birch) and cross-reactive (hazelnut) tolerance in birch pollen allergic patients with associated hazelnut allergy in vitro and in vivo . We found that IL-10 DC induce Bet-specific iTreg which show a regulatory phenotype and strong suppressive capacities to inhibit allergen-specific and cross-reactive immune responses in vitro . In addition, Bet-specific iTreg were able to ameliorate allergic symptoms in vivo in a humanized mouse model of allergic intestinal and airway inflammation.
PFA results from highly conserved protein structures of pollen (e.g. Bet) and food allergens (e.g. Cor), which was shown to facilitate cross-reactions on IgE level and in T cell clones.12,53,54 In a previous study, we confirmed the data of cross-reactivity between pollen and food allergens in primary T cells directly obtained from patients with allergies to birch pollen and associated food allergens.12 Here, we focused on the induction of allergen-specific and cross-reactive iTreg to modulate the primary and secondary allergic immune response in patients suffering from PFA.
Bet-specifically stimulated iTreg but not non-specific iTreg underwent vigorous proliferation towards Bet- and Cor-induced restimulation, suggesting activation as prerequisite for suppressive activity. In this context, Pellerin et al. investigated peanut-specific Tr1 cells induced in vitro by IL-10 DC from allergic subjects.55 They found a highly proliferative phenotype with TH2-cytokine profile upon peanut-specific restimulation and, in contrast to our data, suggested a functional impairment of the peanut-specific Tr1 subset.55 Our experiments revealed that IL-10 DC-induced Bet-specific iTreg did have the ability to suppress allergen-specific responder T cell proliferation, displayed an activated and suppressive phenotype, even though they were highly proliferative. This discrepancy might be due to (1) different protocols for IL-10 DC culture and Treg generation, (2) different allergen-specific immune responses and/or (3) lack of functional assays in the study by Pellerin et al .55 Anergy has been initially described as a fundamental characteristic of functional Treg, but this idea has hence been revised: although breaking Treg anergy can be accompanied by loss of suppressive function, this is not always the case.56 In fact, it was shown that proliferating Treg can suppress T cell responses in vivo 56,57 and Treg that have been stimulated to proliferate can even display an enhanced suppressive capacity.57–59
One very crucial aspect of therapeutic tolerance induction is the allergen-specificity. We are therefore thrilled to report that Bet-specific iTreg showed significantly greater abilities to suppress allergen-specific responder T cell proliferation than non-specific iTreg, as was seen in in vitro suppressor assays from up to 80% of allergic donors. These results were strongly supported by the T cell cytokine profile in suppressor assays. Here, we found that Bet-specific iTreg significantly decreased levels of the TH2 cytokine IL-13, which was not achieved with non-specific iTreg. IL-13 is an IgE-promoting TH2 cytokine, which contributes to airway inflammation and food-induced anaphylaxis in asthma and type 1 allergies.60,61 In line with these data, amounts of the immunosuppressive cytokine IL-10 were significantly increased in the presence of Bet-specifically primed iTreg, which was not the case for non-specific iTreg. Accordingly, IL-10 is well known for its suppressive function in regulatory T cell activity, and particularly in control of TH2-driven allergic diseases.62 As the described cytokine shift was observed for both, Bet- and Cor-specific responder T cells after coculture with Bet-stimulated iTreg - but not with non-specific iTreg0- these data underlined the induction of an allergen-specific (birch) and cross-reactive tolerance (hazelnut) through IL-10 DC-induced Bet-specific iTreg priming.
In addition, Bet-specific iTreg were able to ameliorate asthmatic and intestinal allergic symptoms provoked by challenge with birch extract and reduced birch-specific IgE in allergic mice in vivo . These combined pieces of evidence strongly suggest an allergen-specific tolerance induction in vitro and in vivo by iTreg which have been stimulated by allergen-loaded tolerogenic IL-10 DC. Intriguingly, Bet-specific iTreg also reduced the allergic gut inflammation and hazelnut-specific IgE levels in vivo after challenge with the hazelnut extract in mice engrafted with PBMC from birch pollen allergic patients with associated hazelnut allergy, facilitating cross-reactive tolerance.
Aiming to replace general immunosuppressive therapies, tolerogenic DC (tolDC) have been applied as antigen-specific immune-suppressors in numerous phase 1 clinical trials for multiple sclerosis, type I diabetes, rheumatoid arthritis and organ transplantation.32,34,63–65 In all studies, tolDC had negligible adverse effects and did not worsen disease symptoms. Clinical outcomes were only investigated in a few trials so far but preliminary evidence for antigen-specific tolerance induction was found.
In a comparative study by Boks et al. IL-10 modulation for human tolDC generation was identified as the protocol most suited for tolDC vaccination.66 We developed a protocol for IL-10 DC that resulted in a subpopulation of tolerogenic CD83highCCR7+ IL-10 DC that exhibit a high migratory activity, stability to pro-inflammatory stimuli and profound capacity to induce iTreg with a strong suppressive function.35 In our current study, we did show that human IL-10 DC through priming of allergen-stimulated iTreg are able to induce specific- and cross-reactive tolerance in vitro andin vivo and, therefore are promising candidates to modulate pollen as well as associated food allergies.
Combined with previous findings by us and other groups,35,51,66–68 our study results might further support the development of DC-based tolerance-inducing therapies for allergic and autoimmune diseases.