High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFN?. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy. Overall design: CAR Tregs and CAR Teff cells were co-cultured with irradiated K562 (No Activation), CD19-K562 (CAR Activation) or CD64-CD80-K562 loaded with anti-CD3 antibody (TCR/CD28 Activation) at a 1:1 ratio in RPMI10 medium. CAR Treg co-cultures were supplemented with 1,000 IU/ml IL-2. After 24h, CD4+ cells were isolated using the EasySep Human CD4 Positive Selection Kit (STEMCELL Technologies), following the manufacturer's instructions. At least two independent blood donors per condition were analyzed. With this experimental design, we kept CAR lentivirus transduction and sorting for CAR+ cells constant across all samples and kept the number of irradiated K562 target cells constant across the three modes of activation to rigorously compare the impact of activation via endogenous CD3 and CD28 with activation via CAR intracellular domain CD3 and CD28.