RNA-seq profiling of human Tregs and conventional CD4+ cells that were left untransduced or transduced with a non-tonic signaling (CD19) or a tonic signaling (HA) CAR

Whether or not human Tregs are susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. We studied how a tonic-signaling CAR, which induce Tconv exhaustion, affect human Tregs. We thus sorted naïve Tregs and naïve conventional CD4s and either left them untransduced (UT) or transduced them with a non-TS CAR (CD19) or a TS-CAR (HA). After 12 days in culture, we isolated their RNA and did RNA sequencing. Human naive Tregs or naive conventional CD4s were flow-sorted from 3 independent donors (2 independent experiments) and were kept untransduced (UT) or were retrovirally transduced with a non-tonic signaling C AR (CD19) or a TS CAR (HA) at day 2 and 3. They were than cultured for another 4 days and live CAR positive cells (or live CD4s for UT) were flow sorted. They were then expanded for another 5 days using CD3/28 beads with 300U il-2.