Single-cell RNA-seq uncovers unique gene expression profiles in the spleen of hypoxic mice

The stability of FOXP3 expression in Tregs influences the balance between tolerance and autoimmunity. Treg cell development and function are regulated by IL-2, which binds to IL-2Rα (also called CD25). Decreased expression of CD25 (Il2ra gene) characterizes Tregs that lose FOXP3 expression (exTregs) and undergo transdifferentiation into TH17 cells. This has been established under arthritic conditions but whether this Treg transdifferenciation into exTregs occur following hypoxia exposure, it is unknown. Therefore, single-cell RNA-seq (10x Genomics) was performed using a 10X Genomics Chromium system to test the hypothesis that hypothesis that chronic hypoxia initiates Treg transcriptional reprogramming. We used mice (Foxp3EGFP-cre-ERT2, Ai14-tdTomato) in which tamoxifen administration uncoveres 4 populations of CD4+ T cells; EGFP+, EGFP+ tdTomato+, EGFP-tdtomato+, and EGFP-tdtomato-. We performed single-cell RNA sequencing (scRNA seq) in isolated splenocytes from tamoxifen-treated mice following 5 days of CH or normoxia; tdTomato (WPRE sequence) predominated in the CD4+ memory T cell cluster. Treg marker Il2ra (CD25) was used to identify the Treg cluster and comparative gene expression profiling analysis of RNA-seq data was performed for normoxic and CH samples.