Molecular diversification of regulatory T cells in non-lymphoid tissues

Foxp3+CD4+ regulatory T (Treg) cells accumulate in certain non-lymphoid tissues, where they control diverse aspects of organ homeostasis, partly via a direct impact on neighboring non-immune cells. Populations of tissue-Tregs, as they have been termed, have transcriptomes distinct from those of their counterparts in lymphoid organs and other non-lymphoid tissues. Exploiting recent advances in profiling the chromatin accessibility and gene expression of rare cell populations, we examined the diversification of Tregs in visceral-adipose tissue, skeletal muscle and the colon vis-à-vis lymphoid organs from the same individuals. The unique transcriptomes of the various tissue-Treg populations reflected layering of tissue-restricted stretches of accessibility over a “primed” landscape of sites already open in the spleen, where they were tagged by super-enhancers and “bivalent” histone marks of transcriptional activation and repression. Tissue-restricted chromatin accessibility and gene expression correlated with the binding motifs of a small number of transcription-factor (TF) families repeatedly enriched across the various non-lymphoid tissues. However, a bioinformatically and experimentally validated transcriptional network constructed using a combination of chromatin-accessibility and single-cell transcriptomic data predicted usage of different TF-family members in the different tissues. The network analysis also revealed that tissue-restricted and ubiquitously acting TFs were integrated into feed-forward loops to enforce tissue-specific gene expression in non-lymphoid-tissue Treg cells. Overall, this study provides a framework for understanding the epigenetic dynamics of T cells operating in non-lymphoid tissues, which should inform strategies for specifically targeting them. Tregs were isolated from visceral-adipose tissue, injured skeletal muscle, and colonic lamina propria. For each tissue, Tregs were also isolated from the spleen at the time of harvest and ATAC-seq (Assay for Transposase-Accessible Chromatin) was performed. The experiment was done in duplicates for all populations. Each VAT, muscle, colon sample was paired with a spleen sample taken from the same animal. For example, Treg_ATAC_VAT_1 and Treg_ATAC_Spl_wVAT_1 come from the same animal. Mouse IDs are also provided so that samples can be easily matched by mouse.