FIGURE 1.

Single cell and bulk sample analysis workflow. We adopted a multifaceted approach to assess differences between CB and APB derived Tregs. Fresh CB Tregs, CB Tconv, APB Tregs, and APB Tconv were fluorescence activated cell sorting (FACS) isolated. Sorted CB Tregs and APB Tregs were directly analyzed by single cell RNA sequencing (scRNA-seq) on the 10x Genomics platform. We assessed single cell gene expression and T cell receptor (TCR) repertoire differences. In addition, freshly sorted CB Tregs, CB Tconv, APB Tregs, and APB Tconv were expanded in vitro for 14 days, after which we performed scRNAseq, as well as bulk transcriptional analysis by microarray, flow cytometry and cytokine secretion analysis by Luminex assay.