Foxp3-dependent and -independent functions of peripherally-induced Treg cells [08075]

Regulatory T (Treg) cells expressing the lineage-defining transcription factor Foxp3 are essential for the maintenance of immune tolerance. Foxp3 expression can be induced in a subset of developing self-reactive thymocytes to yield thymic Treg (tTreg) cells, critical for tolerance against self-antigens. Alternatively, activation of mature naïve T cells under non-inflammatory conditions can drive the differentiation of peripherally-induced Treg (pTreg) cells, thought to contribute to tolerance against commensal microbes and dietary antigens. While Foxp3 is indispensable for tTreg cell development and function, its role in pTreg cells has remained unknown. Here, we used a genetic fate mapping approach to characterize polyclonal pTreg cells induced by microbial colonization. We found that expression of a pTreg cell-specific gene expression program was initiated in the mesenteric lymph node (mLN) in a Foxp3-independent manner. Moreover, in contrast to Treg cells in secondary lymphoid tissues, colonic microbiota-dependent pTreg cells did not depend on Foxp3 for their fitness or lineage commitment and were capable of suppressing colonic effector T cell expansion in a Foxp3-independent manner. Rather, Foxp3 was required in a cell-intrinsic manner to limit IL-17 production and to prevent the expansion of intestinal mast cells. Our results suggest that, extrathymic Foxp3 induction likely acts as a mechanism to fine-tune the activity of Th17-like cells with Foxp3-independent regulatory functions. Foxp3DTR-GFP/Thy1.1CD4CreER/wtR26lsl-tdTomato/wt mice were bred and weaned on AVNM and an amino acid-defined diet. Extrathymic Foxp3- CD4 T cells were labeled by treating mice with tamoxifen and allowing ~3 weeks for thymocyte maturation while concomitantly treating mice with diphtheria toxin (DT). Following the labeling period, mice were switched to chow diet and colonized with SPF fecal microbiota. 12 days post-colonization tdTomato-GFP+, tdTomato-Thy1.1+, tdTomato+GFP+ and tdTomato+Thy1.1+ CD4 T cells cells were isolated from the mesenteric lymph nodes and double sorted into Trizol for RNA extraction, library preparation and sequencing.