Nr4a transcription factors limit CAR T cell function in solid tumors

T cells expressing chimeric antigen receptors (CAR) targeting human CD19 (huCD19) have exhibited impressive clinical efficacy against B cell malignancies(1,2). CAR-T cells have been less effective against solid tumors(3–5), in part because they enter a hyporesponsive (“exhausted” or “dysfunctional”) state(6–9) triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR-T cells in solid tumors, we transferred huCD19-reactive CAR-T cells into huCD19(+) tumor-bearing mice. CD8(+) CAR(+) tumor-infiltrating lymphocytes (TILs) and endogenous TILs expressing inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors (TFs) Nr4a1 (Nur77), Nr4a2 (Nurr1) and Nr4a3 (Nor1) by the initiating TF NFAT (nuclear factor of activated T cells)(10–12). CD8(+) T cells from humans with cancer or chronic viral infections(13,14,15) expressed high levels of Nr4a TFs and displayed enrichment of Nr4a binding motifs in accessible chromatin regions. CAR-T cells lacking all three Nr4a TFs (Nr4aTKO) promoted tumor regression and prolonged the survival of tumor-bearing mice. Nr4aTKO CAR-TILs displayed phenotypes and gene expression profiles characteristic of CD8(+) effector T cells, and chromatin regions uniquely accessible in Nr4aTKO CAR-TILs compared to WT were enriched for binding motifs for NFκB and AP-1, TFs involved in T cell activation. Our data identify Nr4a TFs as major players in the cell-intrinsic program of T cell hyporesponsiveness and point to Nr4a inhibition as a promising strategy for cancer immunotherapy.