Role of Gut-Specific Factors in the Control of Regulatory T Cells

Belkaid, Yasmine

Abstract

Although a role for Foxp3 expressing regulatory T cells (Treg) in the maintenance of immune tolerance has been demonstrated in both humans and mice, the origin of these cells is still not completely understood. Aside from evidence that natural Foxp3+ Treg arise and mature in the thymus, there is mounting evidence that Foxp3+ Treg can also develop extra-thymically under certain conditions. In vivo, delivery of sub-immunogenic doses of antigen as well as endogenous expression of foreign antigen in a lymphopenic environment can also induce peripheral Foxp3+ Treg development. While results from these studies have provided promising therapeutic approaches, the occurrence of such conversion in an un-manipulated setting remained controversial. Previous work suggests that peripheral conversion does not occur under steady state conditions or during infections. However, these studies failed to evaluate peripheral conversion at sites that require high levels of control such as mucosal environments. The gastrointestinal tract is in constant contact with food proteins, commensals and potentially pathogenic microorganisms. In order to maintain immune homeostasis in this environment, the intestinal immune system has evolved redundant regulatory strategies. In this regard, the gut is home to a large number of regulatory T cells, including Foxp3+ Treg playing a central role in the control of intestinal homeostasis. Additionally, several subsets of dendritic cells (DCs) with regulatory properties have been described in this environment. We hypothesized that the gut environment and in particular small intestine lamina propria dendritic cells (LpDCs), could potentially mediate extrathymic Treg development. Our hypothesis that cells from the gut environment could mediate Foxp3+ Treg differentiation is supported by our adoptive transfer studies, which indicates that peripheral conversion occurs preferentially in the gut and/or associated tissue . Remarkably, the intestinal immune system tolerates repeated exposure to resident microflora and food antigens, while maintaining the capacity to mount powerful immune responses against pathogens. Our data support the idea that the induction of Foxp3+T cells in the gut also contributes to oral tolerance. Considering the amount of antigens persistently present in the lumen of the intestine, our data suggest that peripheral conversion may represent a significant pathway for the generation of Foxp3+ T cells. We next wanted to assess how Treg conversion could be affected by oral infection and potentially be associated with pathogenesis. To this end we adoptively transferred congenic transgenic T cells into recipient that were fed with the cognate antigen and orally infected or not with a T.gondii. After 5 days of antigen administration, Ag-specific T cells had expanded and were readily detectable in the GALT, spleen, and in distal lymph nodes (LNs) but Foxp3+ expressing T cells were only appreciably detected in the GALT. In contrast, following infection with T.gondii, the frequency of cells expressing Foxp3 de novo was dramatically reduced . . Our findings raises the possibility that exposure to neoantigen at a time of acute infection may prevent the acquisition of tolerance against innocuous antigens and potentially leads to the development inflammatory disorders. We next examined whether LpDCs could induce Treg conversion and found that in vitro, CD103+ DCs from the small intestine but not from other compartments can efficiently induce Treg from conventional T cells It is becoming clear that nutrient status can impact an individuals susceptibility to intestinal pathologies. In the case of vitamin A, and in particular, its active metabolite, RA, prolonged insufficiency not only disrupts the integrity of the intestinal epithelial barrier but also prevents the proper deployment of effector lymphocytes into the GALT following priming. Indeed, the GALT is a significant producer of RA in the body and some effects of GALT DCs on mucosal immunity are associated with their capacity to synthesize this metabolite. For instance, RA production by GALT DCs can selectively induce molecules, such as CCR9 and α4β7, on conventional T cells and Treg involved in directing gut tropism. The observation that Treg converted in the presence of LpDCs expressed high levels of α4β7 suggested that RA might be involved in the conversion process. Consistent with this observation, we found that the mechanism by which LpDCs induce Foxp3 de novo on T cells is associated with their capacity to release RA since blockade of RA signaling abrogate Treg conversion. Conversely, the addition of RA to culture of T cells in the presence of SpDCs dramatically enhances the conversion process . We showed that RA acts to enhance signals delivered by TGF-β. In collaboration with the group of Fiona Powrie we also showed that CD103+DCs from the mesenteric LN were able to induce conversion using a similar mechanism. Recent findings from other groups also support a role for RA in Treg conversion. These results newly identify a food metabolite, retinoic acid as a co-factor in Treg cell generation, providing a mechanism via which functionally specialized gut APCs can extend the repertoire of Treg focused on the intestine. To establish a model system in which RA mediated signals were absent we generated mice with vitamin A insufficiency (VAI) through diet depletion. As we previously described, transferred transgenic T cells acquire Foxp3 expression in the GI tract when mice were fed with the cognate antigen. In contrast, no conversion was observed in VAI mice (Fig 4). Importantly transfer of DCs from the Lp of wild type mice recapitulated the capacity of VAI to express Foxp3.Thus, these data confirm that Lp DCs are sufficient to induce Treg cell in the GI tract via their capacity to release RA. The actions of RA are mediated by nuclear receptors which are ligand-inducible transcriptional regulators and belong to two distinct families: RAR and RXR. Each family consists of the three genetic isotypes (RAR- or RXRα, βand γ). Taken together they display many aspects of the postnatal defects associated with vitamin A deficiency. However the phenotype of these RAR null mutants are confined to a small subset of these tissues suggesting a functional redundancy between RAR. We have obtained mice deficient for each receptor. Purified Foxp3- T cells from each deficient mouse strain were exposed to DCs in the presence of TGF-βand RA. Normal conversion occurred with RARβand RARγdeficient T cells but was abolished with RARαdeficient T cells. Conversely, these cells produced elevated levels of both IL-17 and IFN-γ. Thus, in contrast with the functional redundancy observed for other roles associated with RA, only one receptor, RARα, is involved in the generation of Treg de novo. The identification of the receptor involved in this pathway will allow us to selectively target this receptor in situations where Treg conversion has to be limited or enhanced. Despite reduced conversion in VAI mice we detected increased Treg frequencies in the MLN, Lp, and spleen of these mice at 8 weeks of age correlating with dramatic increase of Treg proliferation and significantly decreased of effector T cells (Teff) proliferation. Cumulatively, these differences account for the dysregulated Treg/Teff ratio that emerges in VAI mice. Thus, VAI results in a homeostatic dysregulation of the Treg/Teff homeostasis.