Single-cell clonotype tracing identifies a stem-like intestinal Th17 population that gives rise to pathogenic effector T cells during autoimmunity

While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies in both humans and mice have implicated their roles in the development of extra-intestinal autoimmune diseases such as multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combining fate mapping with single-cell RNA sequencing to profile the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready-reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFNγ+ CXCR6+ T cells. Our study defines a direct relationship in vivo between IL-17+ non-pathogenic and GM-CSF+ and IFNγ+ pathogenic Th17 populations and provides, for the first time, a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease. We generated droplet-based single-cell RNA-seq or scRNA/TCR-seq profiles for over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation (EAE). A total of 80 samples were collected from multiple tissue sites and mice in 9 batches (32 samples were sequenced in individual channels, 48 samples were hashed per mouse and sequenced as 8 channels).