Integrated chronic in vivo and in vitro screens uncover NFIL3 as a driver of T cell dysfunction

CAR therapy has transformed the treatment landscape for hematological malignancies but its efficacy in solid tumors is limited, owing in part to insufficient functional persistence of the engineered T cells. To elucidate the basis for their functional decline, we conducted integrated chronic in vivo and in vitro screens of 400 transcription factors, which revealed NFIL3 as a driver of CAR T cell dysfunction. Genetic disruption of NFIL3 in CAR T cells sustains their expansion, increases cytokine production, overall restraining terminal differentiation. Loss of NFIL3 enhances CAR T cell efficacy, improving tumor control and prolonging survival in xenograft and syngeneic mouse tumor models across different CAR designs. Under chronic stimulation, disruption of NFIL3 establishes a transcriptional state predictive of favorable clinical outcomes. Our findings underscore the power of comprehensive in vivo genetic screens integrated with multi-parameter in vitro assessment and identify NFIL3 as a novel therapeutic target to enhance cancer immunotherapy.