Regulatory T cells in Allergic Diseases

Abstract

The pathogenesis of allergic diseases entails an ineffective tolerogenic immune response towards allergens. Regulatory T cells (T_Reg) cells play a key role in sustaining immune tolerance to allergens, yet mechanisms by which T_Reg cells fail to maintain tolerance in allergic diseases are not well understood. We review current concepts and established mechanisms regarding how T_Reg cells regulate different components of allergen-triggered immune responses to promote and maintain tolerance. We will also discuss more recent advances that emphasize the “dual” functionality of T_Reg cells in allergic diseases: how T_Reg cells are essential in promoting tolerance to allergens but also how a pro-allergic inflammatory environment can skew T_Reg cells towards a pathogenic phenotype that aggravates and perpetuates disease. These advances highlight opportunities for novel therapeutic strategies that aim to re-establish tolerance in chronic allergic diseases by promoting T_Reg cell and stability function.

Introduction

The increased prevalence in allergic diseases has become a major health problem in affluent and rapidly developing societies. Over the last 150 years, accelerated social and environmental changes augured by the industrial revolution, and which profoundly altered patterns of human activity, living arrangements, diet and infections, all came to influence the rise and severity of allergic disorders¹. In the USA, food allergy prevalence reaches up to 8% among children and 5% of the adult population whereas 8.6% of children and 7.4% of adults are affected by asthma^2,3. The dramatically increased burden of allergic diseases has exacted considerable morbidity on those suffering from those disorders and resulted in substantial financial costs incurred by affected individuals and their healthcare systems. While therapies for allergic diseases have improved over the years with the introduction of agents aimed at combating the inflammatory processes as well as providing symptomatic relief, those therapies have remained for the most non-curative.

Allergic diseases arise in response to normally innocuous environmental agents, including aeroallergens and foods. They involve the participation of components of the innate and adaptive immune responses, such innate lymphoid cells type 2 (ILC2) mast cells, basophils, eosinophils, as well as activated T helper type 2 (Th2) cells and B cells switched to the production of immunoglobulin type E (IgE)^4,5. Immune regulatory mechanisms normally operative to maintain tolerance to allergens breakdown for reasons that still remain obscure. The dramatic increase in prevalence of allergic disease during the past decades indicates a strong influence of environmental factors acting on genetically susceptible hosts to promote disease^6,7. Emerging studies emphasize the interaction of environmental factors, including diet, antibiotic usage and others, with components of the immune system, affecting their function and modify the outcome of the immune response⁸. They also support the idea that the commensal bacteria play a central role in the regulation of allergic diseases and that they dynamically interact with host genetic background and environmental factors to promote or disrupt oral tolerance^9-11. Genetic and immunological evidence also reinforce the idea of a pivotal role for regulatory T (T_Reg) cells in promoting tolerance to allergens and preventing allergic disorder^12-16. In this review, we will discuss recent advances demonstrating the “dual potential” of T_Reg cells in allergic diseases; how T_Reg cells are beneficial in promoting tolerance but also how the pro-allergic environment can derange the T_Reg cell response to aggravate and perpetuates disease.

Natural and induced Foxp3⁺ T_Reg cells

T_Reg cells were initially described as a population of CD4⁺ T cells expressing the IL-2 receptor α chain (CD25) and CD45RB, able to protect mice from developing autoimmune diseases^17,18. Afterwards, the establishment of T_Reg cells as a distinct CD4⁺ T cells sub-population was empowered by the identification of the Forkhead winged helix transcription factor FOXP3 (Forkhead Box 3) as a specific T_Reg cell maker essential to their function^19,20. FOXP3 is required for the differentiation of T_Reg cells, evidenced by the generation of aberrant T_Reg cells lacking in regulatory function in mice with loss-of-function mutations in Foxp3 ^21,22. FOXP3 deficiency results in the development of a multi-organ lymphoproliferative autoimmune disease, referred to as Immune Dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) in human subjects and scurfy in mice^12,23-26. Expression of FOXP3 into human and murine conventional CD4⁺ Foxp3⁻ non-T_Reg cells by means of retroviral gene transfer, converts naïve T cells into T_Reg cells¹⁹. It is now well established that T_Reg cells enforce tolerance to both self-antigens and to the “extended-self”, the latter encompassing commensal flora and innocuous environmental antigens such as allergens [Reviewed in ^27-30].

A major population of T_Reg cells arises in the thymus and is known as CD4⁺ FOXP3⁺ “natural” T_Reg (nT_Reg, also known as thymus-derived or tT_Reg) cells, which chiefly mediates tolerance to self-antigens³¹ (Fig 1). A second population of CD4⁺ FOXP3⁺ T_Reg cells arises extra-thymically in peripheral lymphoid tissues from a pool of naïve conventional CD4⁺ FOXP3⁻ T cells (T_conv) after exposure to antigens and in the presence of TGF-β [reviewed in³²]. These induced T_Reg (iT_Reg, also known as peripheral or pT_Reg) cells are particularly enriched in the gastro-intestinal tract and in the lungs during chronic inflammation, with specificities directed against microbial antigens or environmental allergens^33-35 (Fig 1). The generation of iT_Reg cells at the intestinal mucosa is facilitated by the large abundance of TGF-β and retinoic acid (RA), a vitamin A metabolite, both secreted by the CD103⁺ CD11c⁺ dendritic cells (DCs)^36-38. In lung tissues, resident macrophages (CD45⁺ CD11c⁺ MHC class-II^low F4/80⁺) constitutively expressing TGF-β and RA are the main subset of cells driving iT_Reg cells induction from naïve CD4⁺ T_conv cells³⁹ (Fig 1). Both FOXP3⁺ nT_Reg and iT_Reg cells subsets play a key function in the maintenance of peripheral tolerance by suppressing reactivity to self-antigens and by containing the amplitude of immune responses to foreign antigens.

Fig 1. Natural and inuced Foxp3⁺ T_Reg cells subsets.

The T_Reg cell pool is composed by two different sub-populations, nT_Reg and iT_Reg cells, both expressing the transcription factor Foxp3 crucial for their development and regulatory functions. Foxp3⁺ Nrp-1^high Helios^high nT_Reg cells arise in the thymus and mediate tolerance to self- antigens. Foxp3⁺ Nrp-1^low Helios^low iT_Reg cells, which mediate tolerance to foreign antigens, are induced extra-thymically from naïve CD4⁺ Foxp3⁻ T_conv cells in the presence of TCR stimulation, TGF-β and RA by either CD103⁺ DCs at the intestinal mucosa or F4/80⁺ CD11c⁺ macrophages at the airways epithelial surfaces.

Because of their different origins, the TCR repertoires of thymic nT_Reg and peripheral iT_Reg cells are largely non-overlapping and biased towards self and non-self antigens, respectively ⁴⁰. However, iT_Reg cells are known to be less stable than nT_Reg cells and under inflammatory conditions can lose FOXP3 expression (ex-T_Reg) and produce cytokines such as IFN-γ and IL-17^41,42. This lack of stability can be explained by the methylation status of the conserved non-coding region 2 (CNS2) of the Foxp3 gene. The FOXP3 CNS2 locus, which acts to maintain T_Reg cell lineage identity under inflammatory conditions, is known to be stably hypomethylated in nT_Reg whereas it is incompletely demethylated in iT_Reg cells^43-46 .One difficulty for the functional and genetic study of iT_Reg and nT_Reg cells is the lack of unique and specific markers allowing the distinction between those two populations and their identification in vivo. nT_Reg and iT_Reg cells express similar levels of shared T_Reg cell markers such FOXP3, CTLA-4, GITR, ICOS, CD103 and CD25. However, many of those markers are also up-regulated by activated CD4⁺ T cells under inflammatory conditions and their level of expression do not allow the distinction between nT_Reg and iT_Reg cells⁴⁷. The use of Helios and Neuropilin-1 (Nrp-1) has been proposed to specifically discriminate nT_Reg from iT_Reg cells, since the expression of those markers is higher in nT_Reg compared to iT_Reg cells^48-50. While Nrp1 may be upregulated in the context of an inflammatory environment, Helios^low expression has been extensively used as an in vivo marker that distinguishes iT_Reg from nT_Reg cells^50-52.

In addition to FOXP3⁺ T_Reg cells, CD4⁺ type 1 T regulatory cells (Tr1) represent another subset of T_Reg cells defined by the expression of IL-10 and the surface marker LAG-3 and CD49b in the face of absent FOXP3 and CD25 expression⁵³. The relationship between FOXP3⁺ T_Reg cells and Tr1 cells remains obscure, with both subsets employing common effector pathways including IL-10, TGF-β and CTLA-4⁵⁴. Unlike FOXP3⁺ T_Reg cells, Tr1 cells are not uniquely defined by one transcription factor such as FOXP3, but express a number of transcription factors common to other T cell populations including c-MAF, Ahr (Aryl hydrocarbon receptor), and others⁵⁴ . Many studies that have referred to IL-10 producing T_Reg cells as Tr1 cells did not discriminate between the two populations by appropriate staining for differentiating markers including FOXP3. In this review, we will focus on FOXP3⁺ T_Reg cells as their role in the regulation of allergic disease is far more well defined.

Mechanisms of T_Reg cells suppression

The suppressive functions of T_Reg cells are essential to control autoimmunity, allergic and inflammatory reactions and responses to infectious agents and tumors. Foxp3⁺ nT_Reg and iT_Reg cells are characterized by a non-overlapping TCR repertoire, resulting in a division of labour where nT_Reg and iT_Reg cells regulate immune responses targeting “self” antigens and “non-self” infectious or innocuous agents respectively^40,55. T_Reg cells suppressive function are mediated by multiple mechanisms that involve either the release of inhibitory cytokines (IL-10, TGF-β and IL-35)^56-59 and cytolytic molecules (granzymes A and B)^60-62, or the down-modulation of antigen presenting cells (Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) and Lymphocyte-activation Gene 3 (LAG-3)^63,64, deprivation of trophic cytokines (IL-2 via CD25)⁶⁵ and modulation metabolic pathways (CD73 and CD39)⁶⁶ (Fig 2). Expression of select transcription factors and receptors enables T_Reg cells suppressive functions under inflammatory conditions. GATA-3 expression by T_Reg cells is triggered by TCR activation and is required to maintain FOXP3 expression and to allow accumulation of T_Reg cells at inflamed sites⁶⁷. More recently, it has been demonstrated that Helios expression by T_Reg cells is key to support their suppressive functions and phenotypic stability during inflammation⁶⁸. T_Reg cells functions can also be regulated by endogenous danger signals, or alarmins, released by epithelial cells at the mucosal barrier. Colonic T_Reg cells express the IL-33 receptor (ST2), allowing them to respond to epithelial cell IL-33 production resulting from tissue damage by amplifying their regulatory functions and restraining intestinal inflammation⁶⁹.

Fig 2. Mechanisms of Foxp3⁺ T_Reg cell-mediated suppression.

Foxp3⁺ T_Reg cells mediate tolerance to allergens by diverse suppressive mechanisms. These include T cell cytolysis by a granzyme dependent mechanism, IL-2 deprivation, production of inhibitory cytokines including IL-10, IL-35 and TGF-β capable of blocking the proliferation of T_eff cells and down-modulation of antigen presenting cells (APCs) via LAG-3-MHC II and CTLA-4-CD80/CD86 interactions.

T_Reg cell role in tissue repair

In addition to their immunosuppressive functions and their capacities to restrict the intensity of immune responses, T_Reg cells can also control non-immunological process such as tissue repair resulting from extensive inflammation. Studies have identified the presence of T_Reg cells in a wide variety of non-lymphoid organs such as the skin, the intestinal mucosa, the lungs and visceral adipose tissues⁷⁰. In mice, T_Reg cells accumulate and remain in skeletal muscles after acute injury and their depletion results in increased muscle damage⁷¹. T_Reg cell production of Amphiregulin (Areg), an epidermal growth factor family member known to promote healing and tissue regeneration, in injured lung and muscle tissues appears to be prevent the tissue damages^71,72.

T_Reg cells and allergic diseases

Allergic diseases reflect a failure to develop tolerance toward a specific allergen, provoking the emergence of an allergen-specific CD4⁺ T helper type 2 (Th2) cell response, the generation of allergen-specific immunoglobulin E (IgE) and the recruitment of effector cells to the GI tract or lung tissues^4,73. In humans, loss-of-function mutations affecting FOXP3 result in the development of IPEX, characterized not only by autoimmunity but also severe allergic inflammation including atopic dermatitis, food allergy, asthma, elevated serum IgE levels and peripheral eosinophilia^12,13,74. Foxp3 mutant mice spontaneously exhibit allergic airways inflammation, atopic-dermatitis skin-like disease and elevated serum IgE levels independently of their genetic background⁷⁵. By using another genetic murine model, the ‘DEREG’ mice which express the diphtheria toxin (DT) receptor under the control of the Foxp3 gene, Hadis et al. have demonstrated that T_Reg cells depletion in OVA-tolerant DEREG mice was sufficient to break oral tolerance⁷⁶. Furthermore, in vivo depletion of CD4⁺ CD25⁺ T cells in peanut sensitized mice by means of anti-CD25 monoclonal antibody results in impaired oral tolerance development and leads to an heightened allergic⁷⁷. The central role of T_Reg cells in oral tolerance development to food allergen has been confirmed in human studies where children who had outgrown milk allergy exhibited higher frequencies of milk protein-specific CD4⁺ CD25⁺ T_Reg cells and where the emergence of allergen-specific T_Reg cells is highly correlated with a favourable disease outcome^78,79.

Among the T_Reg cell populations, Foxp3⁺ iT_Reg cells play an essential role in maintaining tolerance at environmental interfaces, including small and large intestinal and the lung respiratory mucosa³². Allergen-specific iT_Reg cells are involved in controlling inflammation severity and the IL-4 Th2 cell immune response³⁵. Accordingly, the development of allergic reactions may result from decreased induction and/or impaired function of allergen-specific iT_Reg cells in genetically allergy-prone subjects. This proposition is supported by a study that took advantage of mice lacking CNS1, an intronic Foxp3 enhancer which contains binding sites for multiple transcription factors such as NFAT and Smad3 and is required for the differentiation of iT_Reg cells in vivo^80,81. Despite decreased iT_Reg cells expansion, CNS1-deficient mice do not develop a fatal autoimmune lymphoproliferative disease. Nonetheless, with time CNS1-deficient mice develop a pro-allergic phenotype associated with Th2 cell-induced pathologies at mucosal surfaces⁸¹. We have recently demonstrated using mice with a gain of function mutation in the IL-4Rα chain (Il4raF709 mice) that heightens susceptibility to oral sensitization that food allergy development is associated with impaired generation and function of allergen-specific T_Reg cells⁸². In peanut allergic patients undergoing successful oral immunotherapy (OIT) leading to peanut tolerance induction demonstrated increased numbers of circulating allergen-specific iT_Reg cells with heighten suppressive capacities and augmented stability, as evidenced by increased demethylation of CpG islands within the FOXP3 gene⁸³. Evidences also point towards a reduced frequency of T_Reg cells associated with allergic asthma^84,85. Compared to healthy controls, frequencies of pulmonary CD4⁺ CD25^high T_Reg cells in the bronchoalveolar lavage fluid (BALF) were significantly decreased in untreated asthmatic children⁸⁶. Four weeks of inhaled corticosteroid treatment were sufficient to restore the T_Reg cells compartment in the blood and the BALF⁸⁶.

A specific requirement for the cytokines IL-10 and TGF-β1 expressed by T_Reg cells in the control of allergic responses has emerged. Kearley et al. have demonstrated that adoptively transferred allergen-specific T_Reg cell suppressive functions during allergic airway inflammation rely on their capacity to induce IL-10 production by CD4⁺ T cells⁸⁷. However, subsequent studies by Rubstov et al. employed a genetic approach to show that T_Reg cell-specific deletion of Il10 promoted allergic airway inflammation; thereby suggesting that T_Reg cell-derived IL-10 plays a “privileged”, non-redundant role in the induction of immune tolerance in allergic airway inflammation⁸⁸. IL-10 has immunosuppressive functions and can modulate the activity of key cell subset involved in the allergic reaction such as mast cells^82,89, Th2 T cells⁹⁰, eosinophils and DCs⁹¹. The Similar to IL-10, TGF-β1 specifically expressed by T_Reg cells also appears to play a privileged role in the regulation of allergic responses. In mice, T_Reg cell-specific deletion of Tgfb1 heightens susceptibility to food allergy (M. NR and T. A. C; unpublished data). The respective role of T_Reg cell IL-10 and TGF-β1 in the regulation of different allergic responses remains to be fully mapped.

In addition to the defect in allergen-specific iT_Reg cell induction, aberrations of the T_Reg cell compartment during allergic disease can also be attributed to a decreased or failure of their suppressive functions. In vitro studies with peripheral T_Reg (CD4⁺CD25⁺) cells isolated from the blood of atopic and non-atopic patients demonstrated that atopic T_Reg cells can be distinguished from non-atopic T_Reg cells by decreased capacities to suppress allergen-driven proliferation of effector CD4⁺ T cells as well as their Th2 cell cytokines secretion⁹².

T_Reg cells regulation of the innate immune response in allergic diseases

T_Reg cells can exert their immunosuppressive functions on a broad variety of different cell types, including innate immune cells. Accumulating evidences demonstrate that T_Reg cells control the immediate hypersensitivity response by acting directly on mast cells and blocking their degranulation (Fig 3)^82,89,93. After allergen-sensitization, triggering of mucosal mast cells via the high affinity receptor for IgE (FcεRI) will induce the release of preformed mediators and elicit an IgE-mediated hypersensitity response⁹⁴. One mechanism by which T_Reg cells modulate IgE-mediated mucosal mast cells degranulation and decrease mast cells effector mediators release is through the OX40-OX40L pathway⁹³. Direct cell-to-cell contact between OX40 expressed on T_Reg cells and OX40L on mast cells will lead to increased intracellular levels of cyclic adenosine monophosphate (cAMP) and result in a blockage of extracellular Ca2⁺ influx and mast cell mediator release inhibition⁹³.

Fig 3. Regulation and suppression of allergic innate immune responses by T_Reg cells.

T_Reg cells control innate immune cell subsets involved in promoting allergy. T_Reg cells block mast cell activation and the release of pre-formed anaphylactic mediators through OX40-OX40L mediated interactions. T_Reg cells also impede the IL-33-driven ILC2 expansion in the intestinal mucosa and their subsequent IL-4 production. Adapted version from the graphical abstract of ref. 107.

IL-4 production by mast cells is critical in food allergic reaction. IgE interaction with the FcεRI expressed on mast cells act as amplifier of the Th2 cell and IgE responses during allergic sensitization⁸⁹. Importantly, dysregulated IgE mast cell activation and their subsequent IL-4 production profoundly inhibits allergen iT_Reg cells generation during allergic processes⁸². This IL-4 inhibition of iT_Reg cells generation is mediated through increased intracellular levels of GATA-3 which acts as a FOXP3 inhibitor in early T cell differentiation ⁹⁵. In contrast, food allergy prone mice that lack the α chain of the high affinity IgE receptor FcεRI (Il4raF709 Fcer1a^−/−) were protected from anaphylaxis⁸². This protection was reflected in decreased mast cell expansion and degranulation and inhibition of the conventional CD4⁺ cell response, consistent with the key function of IgE/FcεRI axis in mediating not only anaphylaxis but also driving the food allergen-associated cell response ^82,89. Moreover, FcεRI deficiency completely corrected the impaired allergen-specific iTreg cell generation⁸². Similar results were obtained by targeting IgE production (IgE^−/− mice or anti-IgE treatment) or by using mast cells ablation genetic murine models ⁸⁹. The key role of T_Reg cells in inhibiting mast cell degranulation and their Th2 cell cytokine secretion is thus critical to the prevention of food allergy (Fig 3).

Innate lymphoid cells type 2 (ILC2), a population of mucosal innate cells, are simultaneously characterized by a lack of antigen specificity (absence of T and B cell receptor) and lymphoid traits, as demonstrated by a shared development origin and phenotypic traits with T cells⁹⁶. ILC2 produce large amounts of Th2 cell cytokines and are linked to allergic disorders such as asthma, chronic rhinosinusitis and atopic dermatitis^97-100. In mice, ILC2 can be identified based on the expression of CD25 (IL-2Rα), IL-33R (ST2) and CD127 (IL-7Rα)¹⁰¹ . ILC2 are located in the blood and various organs such as the spleen, the gastrointestinal tract, the liver, the lungs and the lymph nodes^102,103. The transcription factor GATA-3 is required for ILC2 differentiation, their stability and Th2 cell cytokines production^104,105. Halim et al. demonstrated that ILC2 are required for the development of protease allergen papain-induced airway inflammation as ILC2 deficient mice (RORα^−/−) were incapable of mounting an effective Th2 cell immune response and had reduced type 2 lung inflammation¹⁰². The critical role of ILC2 in triggering Th2 cell adaptive immune responses involves their production of IL-13, which promotes migration of DC to the draining lymph nodes and enhance the conversion of naïve CD4⁺ T cells into Th2 cells¹⁰²

The role of ILC2 in food allergy has been less documented. It appears that IL-13 production by ILC2 enhances allergic mucosal inflammation and promotes IgE-mediated experimental food allergy¹⁰⁶. Food allergy development is associated with defective allergen-specific T_Reg cells induction, consequently resulting in disease promotion⁸². We have recently demonstrated that increased IL-33 production at the intestinal mucosa during food allergy promotes ILC2 expansion, which further enhances the IgE-mediated food allergic response through their IL-4 production¹⁰⁷. ILC2-derived IL-4 inhibits T_Reg cell response and promotes mast cells activation. Reciprocally, T_Reg cells block ILC2 expansion and suppress their IL-4 production¹⁰⁷(Fig 3). Together, these findings point to the disruption of T_Reg cell control of mast cells and ILC2 as a key mechanism in the pathogenesis of food allergy. At steady state, T_Reg cells control both mast cells and ILC2 by restricting their capacity to promote food allergy. Perturbation of this regulatory interaction will subsequently result in a dysregulated pro-allergic innate immune response skewing the immunological balance towards food allergy.

By processing and presenting antigens to naïve T cells, dendritic cells (DCs) are key initiators and master regulators of the allergen-specific immune response. T_Reg cells also directly act on DCs by down-modulating their surface expression of CD80/CD86 expression and subsequently blocking the generation of an allergen-specific Th2 cell immune response. T_Reg cell suppression of DCs appears to be mediated via CTLA-4, LAG-3 and Leukocyte Function-Associated antigen-1 (LFA-1)^63,64,108. DCs, mostly plasmacytoid DCs (pDCs) have the capacities to prime and naïve T cells and induced their differentiation into IL-10 secreting T_Reg cells upon ICOS-ICOS ligand interactions^109,110.

T_Reg cells regulation of the adaptive immune response in allergic diseases

Allergic disorders are characterized by increased dysregulated and aberrant immune responses mediated by the Th2 cell cytokines IL-5, IL-4 and IL-13. T_Reg cells have also the capacities to regulate allergen-induced adaptive T and B cells responses through diverse mechanisms, either soluble or membrane-bound suppressive molecules (Fig 4). T_Reg cells express constitutively CTLA-4, a negative co-stimulatory molecule which is essential to their suppressive functions. Mice deficient for CTLA-4 exhibit a lethal multi-organ lymphoproliferative disease¹¹¹. T_Reg cell-specific deletion of CTLA-4, by means of crossing Foxp3-Cre with Ctla4^fl/fl mice, leads to an autoimmune disease characterized by an increased Th2 cell immune response as evidenced by elevated IL-4 production by CD4⁺ Foxp3⁻ T_conv cells and increased serum IgE levels⁹¹. Ovalbumin (OVA)-specific nT_Reg cells are efficient in controlling in vitro Th2 cell immune responses and IL-4 production by inhibiting the polarization of naive CD4⁺ T cells into Th2 cells via a GITR-dependent suppressive mechanisms¹¹². Circulating CD4⁺ CD25⁺ T_Reg cells isolated form the blood of atopic human subjects were also less efficient in vitro than heathy controls CD4⁺ CD25⁺ T_Reg in controlling the Th2 cell cytokines production by effector CD4⁺ T cells⁹². Furthermore, frequencies of allergen-specific T_Reg cells secreting IL-10 with suppressive functions were predominant in peripheral blood mononuclear cells (PBMCs) from healthy subject whereas frequencies of Th2 CD4⁺ IL-4 secreting T cells frequencies were overrepresented in allergic subjects⁹⁰. Effector CD4⁺ IL4⁺ Th2 cells and suppressive T_Reg IL-10⁺ cells are present in both healthy and allergic patients, their ratio frequencies determine either tolerance induction or allergic response development⁹⁰.

Fig 4. T_Reg cell mediated suppression of the adaptive allergic immune response.

T_Reg cells regulate allergen-specific Th2 immune responses and B cell IgE production. GITR stimulation of T_Reg cells increase their suppressive functions, leading to their blockade of naïve CD4⁺ T_conv cells conversion into allergen-specific Th2 T cells. T_Reg cells are also able to control B cells and block their IgE production by a direct CTLA-4 and cell contact-dependent mechanism and through the production of immunosuppressive cytokines such as IL-10.

Through their production of IgE, B cells are essential in the development of allergic immune responses. IgE responses are highly dependent of the immune response Th polarization; the Th2 cell cytokines IL-4 and IL-13 and CD40-CD40L cognate interactions are two signals required to class switching and IgE production by B cells¹¹³. In vitro, peripheral allergen-specific T_Reg cells from healthy subjects repress B cells IgE production by inducing IgG4 class switching¹¹⁴. T_Reg cells can also exercise their suppressive functions through the release of immunosuppressive cytokines such as IL-10, B cell suppression by T_Reg cells appears to be cell-to-cell contact mediated and probably occur via CTLA-4 and and TGF-β1¹¹⁴.

Pathogenic T_Reg cell Th-reprogramming in allergic diseases

An important problem in chronic allergic diseases relates to the mechanisms that enable persistence of inflammation in the face of T_Reg cell responses¹¹⁵. In the course of regulating Th cell immune responses, T_Reg cells appropriate partial or “aborted” forms of the transcriptional programs of the target Th cells by expressing their master transcription factors, such as T-bet for Th1 cells and IRF-4 for Th2 cells, and co-opting their function ^116,117. Whereas under physiological conditions such partial Th cell programming remains restrained, such restraint is lost under the influence of chronic inflammation leading to pathogenic reprograming of T_Reg cells into Th cells^118,119. In the context of allergic diseases, emerging evidence indicates that a sharply skewed inflammatory environment can overcome the allergen-specific T_Reg cell regulatory response and redirect those cells towards a pathogenic and pro-inflammatory phenotype (Fig 5 and 6). Recent studies from our laboratory have provided two examples of how allergen-specific T_Reg cells may acquire T effector (T_Eff) cell programs and in the process contribute to disease pathogenesis. In the first set of studies, a tyrosine (Y) to phenylalanine (F) mutation at position 709 of the murine IL-4 receptor alpha chain (IL-4Rα) inactivated the receptor’s immunotyrosine inhibitory motif and resulted in augmented activation by IL-4 and IL-13 of the downstream transcription factor STAT6^120,121. This mutation, which models human polymorphisms that promote STAT6 activation via the IL-4Rα, imparted on mice heightened susceptibility to allergic diseases, including food allergy and allergic airway inflammation, and reproduced a Th2 cell-high disease “endotype” common in some subjects with those disorders. Importantly, allergen-specific T_Reg cells became reprogrammed to express a Th2 cell-like phenotype, including IL-4 production, all the while retaining their Foxp3 expression⁸². Whereas the Th2 cell master transcription factor GATA-3 normally plays a positive role in the accumulation of T_Reg cells at sites of inflammation and prevents their polarization into Th17 cells, its abnormally expression in T_Reg cells may contribute to their Th2 cell-like reprogramming under conditions of intense cell polarization^67,122,123. The significance of this reprogramming was underlined by the observation that T_Reg cell-specific deletion of the Il4/Il13 genes restrained the induction of food allergy and allergic airway inflammation in these mice⁸². Consistent with these results, human food allergic subjects manifest increase expression of Th2 cell cytokines in their circulating allergen-specific T_Reg cells, indicative of their acquisition of a “Th2-cell like” T_Eff phenotype ⁸². Oral immunotherapy was associated with reversal of TH2 cell–like reprogramming of allergen-specific T_reg cells, which is coincident with their improved suppression function (data not shown).

Fig 5. Pathogenic “Th2 cell-like” T_Reg cell reprogramming in food allergy.

Food allergy is characterized by a decreased induction of allergen-specific iT_Reg cells at the intestinal mucosa. Induced allergen-specific T_Reg cells in food allergic subjects are prone to acquire a pathogenic skewed “Th2-like” phenotype resulting in increased GATA-3 expression and IL-4 secretion. “Th2 cell-like” iT_Reg cells are dysfunctional and lacking in suppressor function. They are not able to control the T_eff Th2 cell immune response and mast cells expansion, perpetuating in the process the allergic phenotype.

Fig 6. Pathogenic “Th17 cell-like” T_Reg cell reprogramming by the IL-4Rα-Q576R allele.

Human IL-4Rα-Q576R is associated with increased asthma severity. Signalling through the IL4Rα-Q576R on iT_Reg cells induces dual activation of STAT6 and STAT3, the latter through an autocrine IL-6 production loop. The IL-6-STAT3 axis promotes pathogenic “Th17 cell-like” T_Reg cell reprogramming resulting in ROR-γt expression and IL-17 secretion by the reprogrammed T_Reg cells.

A second example of allergen-specific T_Reg cell reprogramming came from studies on a human IL-4Rα allele that bears a glutamine (Q) to an arginine (R) substitution at position 576 (IL-4Rα-Q576R)¹²⁴. This allele is associated with asthma severity, while introduction of the Q576R substitution into the murine IL-4Rα results in exaggerated allergic airway inflammation when the mice are sensitized then challenged in their airways with allergens^125-127 (Fig 6). Signalling via IL-4Rα-Q576R allele does not impact the activation by the IL-4R of its dedicated transcription factor STAT6, and Th2 cell responses promoted via the IL-4R are preserved. Nevertheless, in both humans and mice, the Q576R substitution acts to create a novel branch of signalling via IL-4Rα that activates microtubule-associated protein kinases (MAPK), leading to the induction by IL-4 of IL-6 production¹²⁸ (Fig 6). The newly produced IL-6 destabilizes newly formed allergen-specific T_Reg cells towards the Th17 cell lineage, thus giving rise to mixed Th2-Th17 cell responses in the context of allergic inflammation¹²⁸. Inhibition of the capacity of allergen-specific T_Reg cells to differentiate into Th17 cells, whether by neutralization of IL-6 or by T_Reg cell-specific deletion of genes encoding IL-6 receptor alpha chain (Il6ra) or the Th17 cell master transcription factor RORγt, reversed the exaggerated allergic inflammation induced by IL-4Rα-Q576Rmice¹²⁸ (Fig 6).

Microbiome – T_Reg cell interactions in allergic diseases

Altered environmental exposures early in life may play a critical role in setting in motion the atopic diseases of childhood¹²⁹. The “hygiene hypothesis” stipulates that increased allergic rates observed over the years result from reduced microbial exposures arising from lifestyle changes, such as family size reduction, use of antibiotics and improved hygiene¹³⁰. The influence of the intestinal microbiome in tolerance induction and allergies development is becoming more appreciated. The intestinal colonization of neonates starts at birth from the mother’s vaginal flora, as the microbiota composition of vaginally delivered infants is similar to the maternal vagina¹³¹. Infants born by cesarian section have a different microbiota composition, mostly derived from maternal skin, and are at increased risks of developing asthma and allergy¹³². The first months of life are a critical period for the intestinal flora to settle and stabilize as children exhibiting intestinal dysbiosis in this “time-window” are at increased risks of developing asthma¹³³. Exposure to a farm environment and the associated subsequent large diversity of environmental microbial signals reduce the risk of developing allergies^133,134. The importance of the microbial flora for allergic diseases development is further emphasized by the observation that Germ Free (GF) mice cannot be tolerized to oral antigens and develop a Th2 cell-biased immune response¹³⁵. In humans, a polymorphism in the promoter of CD14, a high affinity receptor for bacterial lipopolysaccharide (LPS) and co-receptor of TLR-4, has been associated with the development of atopic disease¹³⁶. Food allergic responses are also aggravated in TLR4^−/− mice or WT mice treated with antibiotics and repopulation of commensal flora in antibiotics treated mice result in reduced allergen-specific IgE and Th2 cell cytokines responses¹³⁷. These observations highlighted an important function of the intestinal microbial flora and microbial exposure in maintaining and shaping the immune response and inducing protection against the development of atopy^138,139.

By using Il4raF709 food allergy-prone mice, we have demonstrated that food allergy is associated with the emergence of an altered intestinal microbial flora¹⁰. The microbiome of allergic Il4raF709 mice exhibits decreased relative abundances of members from the Firmicutes phylum and increased abundance of bacteria belonging to the Proteobacteria phylum. Adoptive transfer of allergen-specific T_Reg cells prevented the development of food allergy in allergic-prone mice as well as the emergence of the food-allergy associated microbial dysbiosis¹⁰. Importantly, disease susceptibility can be transferred from allergic Il4raF709 mice to GF mice via transplantation of commensal flora from allergic donors. Allergy susceptibility transfer was associated with increase allergen-specific IgE production and expansion of IL-4 secreting Th2 CD4⁺ T cells in GF mice reconstituted with the allergic microbial flora¹⁰.

The commensal flora can target different immune cell subsets belonging to either the innate or/and adaptive allergic effector responses (Fig 7). In the steady-state, the microbial flora promotes intestinal IgA production by a T_Reg cell intrinsic MyD88-dependent mechanism that enables the generation of iT_Reg cells in the gut and their differentiation into T follicular helper cells (T_FH)¹⁴⁰. Depletion of the commensal flora by antibiotic treatment¹⁴¹ or the use of GF mice¹¹ is associated with the development of Th2 cell-type allergic responses and higher serum IgE levels. Th2 cell-type allergic immune responses were held in check by MyD88-dependent microbial sensing by B cells, which suppressed the IgE responses¹⁴¹. The commensal microbiota also influence the outcome of the allergic response modulating the ILC (Fig 7). Mono-colonization of GF mice with anaerobic bacteria belonging to the Clostridia class blocks and protects from oral allergen sensitization by inducing IL22⁺ ROR-γt⁺ ILC at the intestinal mucosa¹¹. ILC2 are expanding in the course of allergic disorders and have a pathogenic function in further promoting disease through their Th2 cell type cytokines secretion^102,107. However, how the intestinal or the upper airways microbial flora could affect, regulate or promote the ILC2 immune response needs still to be further investigated.

Fig 7. Microbiota-immune cell interactions shape oral tolerance.

Metabolites, such as SFCAs produced by bacterial fermentation of dietary fibers promote the proliferation and de novo induction of iT_Reg cells through FFAR2 (GPR43) receptor and HDAC inhibition. Clostridial bacterial species promote the production of IL-22 by ROR-γt ILC, reinforcing oral tolerance by decreasing gut permeability and oral allergen uptake. Bacteroides Fragilis production of PSA promotes de novo iT_Reg cells generation via TLR2 signalling. MyD88/STAT3-sensing by T_Reg cells enforces oral tolerance by inducing and directing the T_FH - T_FReg and IgA axis. The microbiota also promote the emergence of ROR-γt expressing iT_Reg cells. ROR-γt deficiency in T_Reg cells promotes a Th2 environment and oral allergen sensitization possibly by inducing iT_Reg cell reprograming into “Th2 cell-like” cells expressing GATA-3.

The microbiota promotes either a tolerant or a pro-Th2 cell type allergic immune response by interacting directly with immune cells and their TLRs or indirectly through the release of microbial products. Polysaccharide A (PSA), produced by the commensal bacterium Bacteroides fragilis, acts by a TLR2-dependent mechanisms to induce the conversion of CD4⁺ T cells into functional iT_Reg cells with enhanced suppressive activities and increased IL-10 production^142,143. In mice, Clostridial species are among the most abundant Gram-positive bacteria present at the intestinal mucosa. Colonization of GF mice with a mix of Clostridium isolated from either murine or human faeces resulted in a strong induction of iT_Reg cells in the colonic lamina propia of the reconstituted mice^144,145. Through their production of IL-10, Clostridium-induced T_Reg cells also controlled the systemic IgE production by reducing in vitro the IL-4 production by splenic CD4⁺ T cells¹⁴⁴.

Short chain fatty acids (SCFAs) produced by bacterial fermentation of dietary fibers act on T cells via a G protein coupled receptor (GPR43) and protect mice from intestinal inflammation by expanding the pool of the colonic T_Reg cells¹⁴⁶. SCFAs also promote the generation of intestinal iT_Reg cells from naïve CD4⁺ T cells by T-cell intrinsic epigenetic mechanisms^147,148. Butyrate, a SCFA known as an histone deacetylase (HDAC) inhibitor, increases Foxp3 protein acetylation conferring thereby increased stability and enhanced suppressive function to de novo generated intestinal iT_Reg cells¹⁴⁸. Accordingly, a high-fiber diet results in modulation of the intestinal flora composition characterized by increased Bacteroidetes and decreased Firmicutes abundances, resulting in increased circulating levels of SCFAs and allergic airways inflammation protection¹⁴⁹. More recently, it has been reported that the microbiome and oral antigen promote the induction of iT_Reg cells expressing RORγt at the intestinal mucosal surfaces¹⁵⁰. Specific ablation of RORγt in T_Reg cells resulted in increased frequencies of GATA-3⁺ Foxp3⁺ T_Reg cells and increased production of IL-4 and IL-13 by CD4⁺ Foxp3⁻ T_conv cells, leading to the conclusion that the microbiome control the Th2 cell immune response through the expansion of T_Reg RORγt⁺ cells and the regulation of DC activation¹⁵⁰. Whether “pro-allergic” or “pro-tolerant” bacterial species are linked to and directly affect the generation of those GATA3⁺ or RORγt⁺ T_Reg cells needs to be further investigated. Since atopic disease are associated with a defect in the generation of allergen-specific iT_Reg cells, it will be of interest to pursue investigation of these pathways to identify potential therapeutic targets to promote allergen-specific tolerance.

Conclusion

A dynamic view of T_Reg cells in allergic disease is emerging in which they play a central, determining role not only in tolerance induction but also, when destabilized and reprogrammed, in mediating disease pathogenesis, severity and chronicity. Novel approaches to the re-establishment of tolerance are suggested by the results of preclinical models in which reinforcement of iT_Reg cell stability by interrupting their pathogenic programming may be of therapeutic benefit in these disorders.

What do we know?

• T_Reg cells have a key role in promoting and maintaining tolerance to allergens by regulating both innate and adaptive allergen-triggered immune response.

• Allergic diseases are associated with a failure to develop tolerance towards a specific allergen leading to the emergence of a pathogenic Th2 immune response.

• A pro-allergic inflammatory environment may skew allergen-specific T_Reg cells towards a pathogenic phenotype that perpetuates and aggravates disease.

• Allergic responses are influenced by the commensal flora, acting in part via T_Reg cells.

What is still unknown?

• Mechanisms by which T_Reg cells fail to maintain tolerance in allergic diseases are not well understood and require further investigation.

Abbreviations

T_Reg

Regulatory T cells

BALF

Bronchoalveolar Lavage Fluid

ILC

innate lymphoid cells

Th

T helper

IgE

Immunoglobulin type E

Areg

Amphiregulin

Ahr

Aryl hydrocarbon receptor

SCFA

Short Chain Fatty Acids

HDAC

Histone Deacetylase

T_Eff

T effector cells

T_Conv

Conventional T cells

DCs

dendritic cells

APCs

antigen presenting cells

CTLA-4

Cytotoxic T Lymphocyte Antigen 4

LAG3

Lymphocyte-Activation Gene 3

TCR

T cell receptor

Tr1

Type 1 T regulatory cells