Integrated Chronic In Vivo and In Vitro Screens Uncover NFIL3 as a Driver of T-cell Dysfunction

Chimeric antigen receptor (CAR) therapy has transformed the treatment landscape for hematologic 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 and 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 multiparameter in vitro assessment and identify NFIL3 as a novel therapeutic target to enhance cancer immunotherapy. SIGNIFICANCE: This study presents a two-step screening framework, integrating an in vivo pooled guide RNA screen with a multiparameter, in vitro arrayed screen. NFIL3 emerged as the top candidate, and its disruption enhanced CAR T-cell antitumor efficacy in both hematologic malignancies and solid tumors across diverse CAR architectures.