Transient mTOR inhibition rescues 4-1BB-CAR-Tregs from tonic signal-induced dysfunction

RNAseq analysis of human CAR-Tregs, bearing CD28 or 4-1BB as a costimulatory domains, initially isolated (before engineering) from peripheral blood of healthy donors Overall design: CD25+ naive Tregs from healthy donors were sorted, activated, transduced with lentiviral vector for CAR+CD28/4-1BB expression and expanded for 16 days, with a CAR-negative sorting (CD25-NT) or CAR-positive sorting (CD25+28/25+BB) at D11 of culture. At D16 cells were stained and sorted by flow cytometry as DAPI– CD4+CD25+ FVD-EGFR+ or FVD-EGFR- (CD25-NT). 1,000 cells were double-sorted directly into lysis buffer for RNA-seq (Smart-seq2) library preparation following the standard ImmGen low-input protocol (www.immgen.org). Samples were collected in independent experiments and sequenced in the same RNAseq batch.