Single cell transcriptomic analysis (10x Genomics) of mouse colon LP CD4+ Tcells cells isolated from mouse under different infection conditions

To determine the states that Teff actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analysis on colonic Teff cells, in germ-free or conventional mice, or after challenge with a range of Teff subset-biasing microbes. Subsets were marked by expression of interferon-signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete Th clusters. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as Teff markers distributed in a polarized continuum, which was also functionally validated. Clones derived from single progenitors gave rise to both IFN-? and IL17-producing cells. Most transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activity of AP1 and IRF transcription factor families, not the canonical subset master regulators T-bet, GATA3, ROR?. Overall design: Single cells were hashed and sorted by flow cytometry for single cell RNAseq library preparation (10x Genomics)