Knocking out BATF improves CAR-T cell efficacy by resisting exhaustion and forming central memory cells

Chimeric antigen receptor modified T (CAR-T) cell therapy has limited efficacy against solid tumor, one major challenge is T cell exhaustion. To address this challenge, we performed a candidate gene screen using a hypofunction CAR-T cell model, and found that knocking out BATF improved the performance of CAR-T cells. In different types of CAR-T cells and mouse OT-1 cells, knocking out BATF endows T cells with improved resistance to exhaustion and better tumor eradication efficacy. We find that BATF binds to and up-regulates a subset of exhaustion genes in human CAR-T cells. Furthermore, BATF regulates the expression of genes involved in the development of effector and memory cells, and knocking out BATF shifts the population towards more central memory subset. Therefore, we conclude that BATF is a key factor limiting CAR-T cell function, and its depletion improves CAR-T cells efficacy against solid tumor. Overall design: We established an in vitro CAR-T cell hypofunction model and identify that BATF drives exhaustion via binding to and upregulating related genes in CAR-T cells, as well as regulates CAR-T cell subtypes. Knocking out BATF improves CAR-T cells efficacy against solid tumor. We sorted GFP-positive cells co-cultured with tumor at 0.1:1 E:T ratio at day 7, named as hypofunction M28Z. After being co-cultured with tumor cells for 24h at E: T=2:1 (achieved 90% tumor lysis), GFP-positive CAR-T cells were sorted and named as activated M28Z; CAR-T cells thawed from the cryopreserved stock were sorted as control (fresh M28Z). To further characterize these hypofunction CAR-T cells, we performed RNA-seq on CD8-positive cells isolated from these three groups mentioned above. In addition, to control for the influence of long-term cultivation on CAR-T cells, we sequenced CAR-T cells cultured alone for 7 days (named unstimulated M28Z). To investigate the role of BATF and IRF4 in CAR-T cell exhaustion, we performed RNA-seq analysis of M28Z, M28Z-BKO and IKO cells isolated from the hypofunction model. To further validate the function of BATF in human CAR-T cells, we over-expressed BATF in CAR-T cells (M28Z-BOE) by lentiviral transduction and RNA-seq was performed using M28Z, M28Z-BKO, M28Z-BOE after three rounds of tumor cell challenges. To further dissect the molecular mechanism, we performed ChIP sequencing (ChIP-seq) in M28Z CAR-T cells. To better dissect the role of Batf in primary T cells, RNA-seq was performed using OT-1 cells upon repetitive tumor challenge.