Single cell transcriptomic analysis (InDrops) of mouse CD4+ T cells isolated from colon

RORg+ and Helios+ Tregs in the colon are phenotypically and functionally distinct, but their origins and relationships are poorly understood. In monocolonized and normal mice, single-cell RNAseq revealed sharing of TCR clonotypes between these Treg populations, potentially denoting a common progenitor. In a polyclonal Treg replacement system, naïve conventional CD4+ (Tconv) cells, but not pre-existing tTregs, could differentiate into RORg+ pTregs upon interaction with gut microbiota. A smaller proportion of Tconv converted into Helios+ pTregs, but these dominated when the Tconv originated from pre-weaning mice. T cells from infant mice were predominantly immature, insensitive to RORg-inducing bacterial cues and to IL6, and showed evidence of higher TCR-transmitted signals, also characteristics of recent thymic emigrants (RTE). Correspondingly, transfer of adult RTE or Nur77high Tconv mainly yielded Helios+ pTregs, recapitulating the infant/adult difference. Thus, CD4+ Tconv can differentiate into both RORg+ and Helios+ pTregs, providing a physiological adaptation of colonic Tregs as a function of the age of the cell or of the individual. Overall design: Single cells were sorted by flow cytometry for single cell RNAseq library preparation (InDrops)