ATAC-seq profiling of in vitro FRC-conditioned, IL6-conditioned or control (anti-CD3/CD28)-stimulated CD8+ T cells

To broadly assess which pathways FRCs regulate in CD8+ T cells, we sorted CD8+ T cells for ATAC-seq following activation in whole splenocyte mixtures via soluble anti-CD3/CD28 (0.25µg/ml) for 48 hours (Stim), activated with anti-CD3/CD28 in the presence of Nos2–/– FRCs (FRC) or activated with anti-CD3/CD28 plus 100 ng/ml recombinant IL-6 (IL-6). Here we demonstrate that FRC-derived signals, including IL-6, act in concert with TCR signaling and co-stimulation to remodel chromatin and render essential transcription factor binding motifs accessible in activated CD8+ T cells. Signals from FRCs led to increased binding regions for MYC, HIF-1a and HIF-1ß, which are known to promote metabolic pathways following T cell activation. Additionally, FRC-derived signals promoted activity of transcription factors that regulate survival and memory programs in T lymphocytes such as BATF and BACH2. Overall design: CD8+ T cells were sorted for ATAC-seq following activation in whole splenocyte mixtures via soluble anti-CD3/CD28 for 48 hours (Stim), activated with anti-CD3/CD28 in the presence of Nos2–/– FRCs (FRC) or activated with anti-CD3/CD28 plus 100 ng/ml recombinant IL-6 (IL-6). Three biological replicates were used for each condition.