Genome-wide ATAC-seq maps in human Treg subsets and CD4+ T effector cells

We report the application of ATAC-seq to flow sorted Foxp3+Helios+ memory and naïve regulatory T cell subsets and memory CD4+ T effector cells in both resting condition and after in vitro stimulation (4 hours) with PMA/Ionomycin. CD4+ T cells were isolated from whole blood by negative selection using the RosetteSep human CD4+ T cell enrichment kit. CD4+ T cells (1–2 × 10^6 CD4s/well) were cultured in a 24-well flat-bottom cell culture plate at 37 °C + 5% CO2 resuspended in X-VIVO-15 (Lonza) + 5% heat-inactivated, filtered human AB serum in the presence or absence of phorbol myristate acetate (PMA), ionomycin, and protein transport inhibitors (brefeldin A and monensin). After 4 hours of culture, cells were harvested and stained with a panel of fluorochrome-conjugated antibodies against surface markers, fixed and permeabilised and stained with fluorochrome-conjugated antibodies against intracellular markers and the following CD4+ CD3+ve viable and single cell gated T cell subsets were sorted; HELIOS+FOXP3+CD25h iCD127lo CD45RA- (DP mTreg), HELIOS+FOXP3+CD25hiCD127lo CD45RA+ (nTreg) and CD25int CD127hiCD45RA- (mTeff).