The tissue-resident regulatory T cell pool is shaped by transient multi-tissue migration and a conserved residency program. scRNA-Seq profiling of mouse tissue Tregs with TCR sequencing and CITE-Seq

The tissues are the site of many important immunological reactions, yet how the immune system is controlled at these sites remains opaque. Recent studies have identified Foxp3+ regulatory T cells (Tregs) in non-lymphoid tissues, with unique characteristics compared to lymphoid Tregs. However, tissue Tregs have not been considered holistically across tissues. Here we performed a systematic analysis of the Treg population residing in non-lymphoid organs throughout the body, revealing shared phenotypes, transient residency and common molecular dependencies. Tissue Tregs from different non-lymphoid organs shared T cell receptor (TCR) sequences, with functional capacity to drive multi-tissue Treg entry, and were tissue-agnostic on tissue homing. Together these results demonstrate that the tissue-resident Treg pool in most non-lymphoid organs, other than the gut, is largely constituted by broadly self-reactive Tregs, characterised by transient multi-tissue migration. This work suggests common regulatory mechanisms may allow pan-tissue Tregs to safeguard homeostasis across the body. scRNA-Seq was performed using 10x Genomics 5’ VDJ Single Cell Immune Profiling. Tregs were flow sorted on a BD Influx, Aria, Jazz or Fusion on the basis of CD4+Foxp3Thy1.1+ i.v.CD45- CD19-CD11b-CD8-F4/80-. Cells were labeled with Hashtag TotalSeq regents and loaded onto the 10x Chromium Controller. Sequencing was performed on an Illumina HiSeq. Data was processed in R using scripts as detailed below.