T cell dysfunction – in the form of exhaustion, anergy, or tolerance – is known to limit the effector functions of T cells, but the molecular mechanisms that induce dysfunctional programs are not fully understood. Two recent studies, published back-to-back in Nature, investigated the mechanisms underlying T cell dysfunction, and through different strategies, both studies identified NR4A transcription factors as essential to induction of dysfunction.
Focused on studying T cell exhaustion, particularly in the context of CAR T cells, Chen et al. transferred CD19-specific CAR T cells into mice bearing CD19-expressing cancers. After a period of time, the transferred CAR T cells developed hallmarks of exhaustion including high PD-1 and TIM3 expression, and decreased effector function. Analysis of chromatin accessibility profiles revealed that regions selectively accessible in PD-1hi T cells were enriched for NR4A, NFAT, and NFκB motifs. RNAseq data further supported these observations, pointing to a role for NR4A family transcription factors (NR4A1, NR4A2, and NR4A3) as transcriptional effectors of T cell dysfunction.
Liu, Wang, and Lu et al. similarly identified upregulation of NR4A1 after a genome-wide genetic and epigenetic analysis of in vitro-induced tolerant T cells. When they induced overexpression of NR4A1 in naive T cells, they observed upregulation of genes related to anergy and exhaustion, downregulation of genes related to effector programs, reduced TH1 and TH17 differentiation in CD4+ T cells, and reduced IFNγ production by CD8+ T cells. Similarly, ablation of NR4A1 enhanced effector functions of CD4+ and CD8+ T cells, increased expansion, and blocked the formation of tolerance.
To study the effective role of NR4A transcription factors in vivo, Liu, Wang, and Lu et al. treated tumor-bearing mice with tumor-specific CD8+ T cells with NR4A1 knocked out. NR4A1-/- T cells outperformed their wild-type counterparts, expressing less PD-1 and TIM3, producing more IFNγ and TNF, infiltrating tumors more readily, and mediating more pronounced tumor regression. In the context of both acute and chronic viral infection, NR4A1-/-CD8+ T cells showed enhanced effector function, Tbet expression, and proliferation, and reduced expression of exhaustion markers.
In a similar series of adoptive transfer experiments, Chen et al. compared the antitumor efficacy of wild-type CAR T cells to NR4A triple knockout (TKO; NR4A1, NR4A2, and NR4A3) CAR T cells. In both immunodeficient and immunocompetent tumor-bearing mice, wild-type CAR T cells showed limited efficacy, while NR4A TKO CAR T cells induced pronounced tumor regression and enhanced survival. NR4A TKO CAR T cells isolated from tumors 8 days after cell transfer showed reduced expression of PD-1 and TIM3, and the percentage of cells producing IFNγ and TNF upon restimulation was higher compared to wild-type CAR TILs. Compared to the TKO cells, CAR T cells with individual knockouts of NR4A1, NR4A2, or NR4A3 showed weaker antitumor efficacy, increased expression of inhibitory receptors PD-1 and TIM3, and decreased ability to produce cytokines upon restimulation.
Transcriptional and chromatin accessibility comparisons performed by Chen et al. showed that NR4A-expressing cells promoted the expression of dysfunction programs by favoring the accessibility and expression of exhaustion-related genes, and that each individual NR4A transcription factor, functioning downstream of NFAT, played a similar role in maintaining the accessibility of an enhancer for the Pdcd1 transcription start site. Similarly, Liu, Wang, and Lu et al. showed that NR4A1 expression correlated with HeK4me3 (a histone modification marking active transcription) near tolerance-related genes.
In addition to activating dysfunctional programs, NR4A was also found to repress effector programs. Chen et al. noted that in chromatin selectively accessible in NR4A TKO CAR T cells, regions near certain chemokine and cytokine genes were enriched for binding motifs of transcription factors including bZIP and Rel/NFκB, and the activity of these transcription factors was associated with the increased effector functions of NR4A TKO CAR T cells in vivo. Meanwhile, Liu, Wang, and Lu et al. found that the top NR4A1-binding motifs were consensus sequences containing AP-1 – a transcription factor involved in effector function programming. They determined that mechanistically, NR4A1 was recruited to the binding sites of AP-1, inhibiting its function and ultimately repressing T cell effector programs. Both research teams found that PD-1 blockade reduced NR4A expression in CD8+ T cells by reducing the accessibility of NR4A binding motifs.
Understanding how exhaustion is regulated on the epigenetic and transcriptional level is critical to designing immunotherapies which depend on T cells’ ability to maintain effector functions. As observed here, knocking out NR4A in T cells prior to adoptive transfer enhances their effector function and prevents exhaustion – an effect similar to that of checkpoint blockade. These studies thus identify NR4A as a new “checkpoint”, and novel strategies of targeting NR4A in immunotherapy may be effective in preventing or controlling T cell dysfunction.
by Lauren Hitchings
