Multi-Dimensional Profiling Reveals a Molecularly Distinct Injury-Responsive Population of Regulatory T Cells [Bulk RNA-seq]

CD4+ regulatory T cells (Tregs) activate and expand in response to many different types of injuries, suggesting that they play a critical role in controlling the host response to tissue damage. Here we use multi-dimensional profiling techniques to comprehensively characterize injury responsive Tregs in mice. CD44high Tregs expand in response to injury and are more suppressive than CD44low Tregs. T cell receptor (TCR) analysis reveal that CD44high Tregs undergo TCRaß clonal expansion and increased CDR3 diversity in response to injury. Bulk and single-cell RNA sequencing with paired TCR clonotype analysis demonstrate that important Treg-associated genes were significantly differentially expressed in both CD44high and CD44low Tregs with expanded TCR clonotypes. CyTOF analysis verified protein expression of these genes on CD44high Tregs, with increases in Helios, Galectin-3 and PYCARD expression following injury. Collectively, these data provide new insights into CD44high Tregs, an important subset in the response to injury to tissue damage. Overall design: Lymph node CD4+CD44highFoxP3-GFP+ and CD4+CD44lowFoxP3-GFP+ T cells or FoxP3-GFP+ T cells were purified by FACS sorting at 7 days after injury from injured or uninjured Foxp3DTR mice