Integrated in vivo and multiparameter in vitro screen uncovers NFIL3 as 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 a multi- parameter in vivo discovery screen of 400 transcription factors, which revealed NFIL3 as a driver of CAR T cell dysfunction. Genetic disruption of NFIL3 in CAR T cells sustained their expansion, increased 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 multi-parameter genetic screens conducted in vivo and reveal NFIL3 as a novel therapeutic target to enhance cancer immunotherapy. Overall design: CAR T cells from two donors at the pre-stimulation, after two rounds and four rounds of tumor stimulation were counted and assessed for scRNA-seq analysis