BLIMP1 and NR4A3 transcription factors reciprocally regulate antitumor CAR T cell stemness and exhaustion.

Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of , encoding the BLIMP1 transcription factor, defined highly potent [encoding T cell factor 1 (TCF1)]-expressing CD8 CAR T cells, whereas enrichment of [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]-expressing CD8 T cells with elevated was associated with poor outcomes. knockout promoted -dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)-driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of and skewed CAR T cell phenotypes away from TIM-3CD8 and toward TCF1CD8 to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with and single knockout alone. These data underscore dual targeting of and as a promising approach to advance adoptive cell immuno-oncotherapy.