Foxp3-dependent and -independent functions of peripherally-induced regulatory T cells: scRNA-seq

Regulatory T (Treg) cells expressing the X-linked lineage-defining transcription factor (TF) Foxp3 are essential for preventing fatal autoimmunity. During thymic differentiation, Foxp3 expression is induced in a subset of developing self-reactive thymocytes yielding thymic Treg (tTreg) cells, critical for enforcing tolerance to “self”. In the periphery, Foxp3 induction in mature naïve CD4 T cells activated under particular conditions results in extrathymic generation of Treg (pTreg) cells, thought to contribute to tolerance against commensal microbiota and dietary antigens. While Foxp3 is indispensable for tTreg cell differentiation and function, its role in pTreg cells has remained unknown. Here, we used complementary genetic approaches to characterize pTreg cells induced by microbial colonization and elucidate a role for Foxp3 in these cells. We found that a pTreg cell-specific gene expression program was installed in the gut-draining mesenteric lymph nodes (mLN) in a Foxp3-independent manner. In contrast to tTreg cells residing in the secondary lymphoid tissues, colonic microbiota-dependent pTreg cells did not depend on Foxp3 for their fitness or lineage commitment and were capable of preventing colonic effector T cell expansion in a Foxp3-independent manner. Rather, Foxp3 acted in a cell intrinsic manner to limit IL-17 production and was critical for the suppression of rampant intestinal mastocytosis. Thus, in contrast to tTreg cells, whose functionality is entirely Foxp3-dependent, Foxp3 expression plays a notably nuanced role in pTreg cells and acts to fine-tune the activity of IL-17 producing cells with Foxp3-independent regulatory functions. Germ-free male Foxp3GFP and Foxp3CNS1- mice were colonized with SPF fecal microbiota or kept germ-free. 9 days post-colonization, a 50%-50% mix of GFP+ and GFP- CD4 T cells was sorted from pooled mLN of 3 mice per group and used as input for scRNA-seq analysis.