Foxp3 Dependent and Independent Functions of Peripherally Induced Regulatory T Cells: colonic scRNA+TCR-seq

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are an essential suppressive CD4 T cell lineage of dual origin: Foxp3 induction in thymocytes and mature CD4 T cells gives rise to thymic (tTreg) and peripheral (pTreg) Treg cells, respectively. While tTreg cells primarily suppress autoimmunity, pTreg cells are thought to enforce tolerance to food and commensal microbiota. However, the role of Foxp3 in pTreg cells and the mechanisms underlying their differentiation and function remain poorly understood. Here, we used genetic lineage tracing to unambiguously identify microbiota-induced pTreg cells and found that many of their distinct features were Foxp3-independent. Lineage-committed, microbiota-dependent pTreg-like cells persisted in the colon in the absence of Foxp3 protein. While Foxp3 was critical for the suppression of a Th17 cell program, colitis and rampant mastocytosis, pTreg cells suppressed colonic effector T cell expansion in a Foxp3-independent manner. Thus, Foxp3 and the tolerogenic signals that precede and promote its expression independently confer distinct facets of pTreg functionality. Overall design: Approximately 5,000 sorted tdTomato+GFP+ and tdTomato-GFP- colonic lamina propria CD4 T cells were used as input for each replicate. Biological triplicates from the same group were labeled with TotalSeq-C reagents and multiplexed on one lane of 10X Chromium, aiming for a total of 15,000 cells per sample.