RNA-seq of donor matched freshly FACS-sorted Tregs (CD4+CD127loCD25hi) and Tconvs (CD4+CD127hiCD25lo) to compare both the protein-coding and non-coding transcriptomes from three different healthy donors (HD)

The major goal of this study was to perform an in depth characterization of the “gene signature” of human FoxP3+ T regulatory cells (Tregs). Highly purified Tregs and T conventional cells (Tconvs) from multiple healthy donors (HD), either freshly explanted or activated in vitro, were analyzed via RNA sequencing (RNA-seq) and gene expression changes validated using the nCounter system. Additionally, we analyzed microRNA (miRNA) expression using TaqMan low-density arrays. Our results confirm previous studies demonstrating selective gene expression of FoxP3, IKZF2, and CTLA4 in Tregs. Notably, a number of yet uncharacterized genes (RTKN2, LAYN, UTS2, CSF2RB, TRIB1, F5, CECAM4, CD70, ENC1 and NKG7) were identified and validated as being differentially expressed in human Tregs. We further characterize the functional roles of RTKN2 and LAYN by analyzing their roles in vitro human Treg suppression assays by knocking them down in Tregs and overexpressing them in Tconvs. In order to facilitate a better understanding of the human Treg gene expression signature, we have generated from our results a hypothetical interactome of genes and miRNAs in Tregs and Tconvs Conventional T cells (Tconvs) or Regulatory T cells (Tregs) isolated from peripheral blood of three healthy donors: 1HD (Healthy Donor 1, Male), 2HD (Healthy Donor 2, Female), 3HD (Healthy Donor 3, Male). Samples were recovered either upon fresh explantation (0hr) or after further in vitro activation at longer timepoints (6hr, 24hr, 7d) for Healthy Donor 3HD. Please note that each *Tconvs_vs_Tregs_DEGseq_merged_score.tsv contains both Tconvs_count and Tregs_count data from each donor (as indicated in the file name) and is linked to the corresponding *Tconvs sample records.