UC San Diego

CD8+ T cell exhaustion is a state of progressive functional decline that develops under conditions of chronic antigen stimulation, such as in the tumor microenvironment, and represents a major barrier to effective cancer immunotherapy. The transcription factor NFAT, acting without its cooperative partner AP-1, drives a transcriptional program in CD8+ T cells that closely mirrors exhaustion, including upregulation of co-inhibitory receptors PD-1 and TIM3. Among the downstream targets of this NFAT program are the NR4A family of nuclear receptors, which have been shown to drive exhaustion-associated gene expression and limit anti-tumor T cell function. NR4A3 has emerged as a functionally significant family member, with Nr4a3-deficient CAR T cells showing superior tumor control relative to Nr4a1 or Nr4a2 single knockouts. However, the specific relationship between NFAT and NR4A3 in shaping the exhaustion gene program remains poorly understood. We used constitutively active NFAT (CA-RIT-NFAT) in combination with acute Cas9-mediated Nr4a3 deletion and germline Nr4a3-/- mice to characterize how NR4A3 modulates NFAT-driven gene programs in primary mouse CD8+ T cells. Flow cytometric analysis revealed that Nr4a3 loss selectively amplified NFAT-driven PD-1 upregulation without consistently affecting TIM3. Transcriptome-wide RNA-seq analysis identified eight co-regulated gene clusters, among which two were most informative: a cluster of NFAT target genes whose full induction required NR4A3, and a cluster whose induction was normally constrained by NR4A3, including inhibitory receptor genes. These findings suggest that NR4A3 modulates the NFAT transcriptional response in a selective, gene-program-specific manner, providing new insight into the regulatory architecture of CD8+ T cell exhaustion.