CD8+ T cell exhaustion or dysfunction is frequently observed in chronic viral infections and cancer due to persistent antigen exposure and chronic T cell receptor (TCR) signaling. The exhausted T cell state is associated with a decrease in proliferative capacity, reduced effector functions, changes in gene expression and metabolism, and elevated and sustained expression of multiple inhibitory receptors, including PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, and 2B4/CD244. Under steady state conditions, these inhibitory receptors, also known as immune checkpoint proteins, are transiently expressed on activated T cells and play a key role in maintaining immune homeostasis by regulating the duration and magnitude of T cell responses. In the exhausted T cell state, the expression of these receptors remains high and T cell activity is reduced, thereby preventing effective elimination of pathogens and tumors.
CTLA-4 and PD-1 Have Been Targeted to try to Reverse T Cell Exhaustion
Exhausted T cells were initially characterized in mice infected with chronic lymphocytic choriomeningitis virus (LCMV), but a similar T cell phenotype was also later reported in humans with hepatitis C virus (HCV) or human immunodeficiency virus (HIV) infections, and in human cancer. Subsequent studies showed that the severity of T cell exhaustion typically correlated with the number of inhibitory receptors that were co-expressed. As a result, researchers and clinicians have tried to improve anti-viral and anti-tumor immune responses by targeting one or more of these inhibitory receptors using antibodies that block their interactions with their respective ligands to try to reverse the exhausted phenotype and restore T cell functions. Some of these studies have shown remarkable promise. For example, monoclonal antibodies directed against the T cell co-inhibitory receptors, CTLA-4 or PD-1 have resulted in increased patient survival in a number of different cancer types including melanoma, renal cell carcinoma, squamous cell carcinoma, and non-small cell lung cancer. Unfortunately, however, clinicians have found that blocking CTLA-4 or PD-1 is only effective in a minority of patients and many of the patients that initially respond to treatment later become resistant or relapse likely due to an upregulation of other inhibitory receptors. Similarly, CAR T-cell therapy, which has been successful in the treatment of leukemias and lymphomas, has not been successfully used to treat solid tumors at least in part because the CAR T cells become exhausted. Consequently, researchers started searching for the specific factors that drive the development of the exhausted T cell phenotype with the hope of understanding how this phenotype is being generated and identifying additional potential targets for cancer immunotherapy.
The Transcription Factor NR4A1 is Involved in the Development of T Cell Exhaustion
In April 2019, two papers published in Nature reported that the nuclear receptor transcription factor (NR4A) family plays a key role in regulating the induction of T cell exhaustion.1,2 In the first paper by Liu, et al., the authors used an in vitro T cell tolerance induction system to analyze the gene expression and epigenetic features of mouse tolerant T (Ttol) cells. This analysis demonstrated that anergy-related genes and transcriptional repressors were up-regulated in Ttol cells, while effector genes and mitochondria-, ribosome-, and proteasome-related genes were suppressed.1 Additionally, the authors showed that both the gene loci for anergy-associated genes and exhaustion-related genes, including the genes encoding the inhibitory receptors, LAG-3, PD-1, and TIGIT, displayed histone H3 trimethylation on lysine 4 (H3K4me3), indicating that these genes were actively transcribed in Ttol cells, while those encoding transcription factors and effector cytokines associated with different T cell subsets had low levels of H3K4me3 and were not actively transcribed. Significantly, one particular transcription factor that was up-regulated in Ttol cells was Nr4a1, which the authors investigated further to try to understand the role that this factor was playing in these cells. Subsequent experiments demonstrated that overexpression of Nr4a1 in CD4+ T cells led to an up-regulation of anergy-related genes and a down-regulation of IL-2 and IFN-gamma expression, while deletion of Nr4a1 in either CD4+ or CD8+ T cells led to an increase in IL-2 and/or IFN-gamma expression, suggesting that Nr4a1 was involved in mediating the Ttol phenotype. Using either an oral tolerance or peptide-induced tolerance model in wild-type and Nr4a1-/- mice, the authors showed that Nr4a1 is required for the in vivo induction of T cell tolerance as development of the Ttol phenotype was blocked in the absence of Nr4a1. Having previously observed that Nr4a1 was up-regulated in chronic virus-associated T cell exhaustion, the authors next investigated the role of Nr4a1 in a mouse model of lymphoma and found that Nr4a1-/- CD8+ T cells adoptively transferred into tumor-bearing mice expressed lower levels of PD-1 and TIM-3, showed better tumor infiltration, and were significantly more efficient at eliminating tumors than wild-type CD8+ T cells. Using chromatin immunoprecipitation followed by sequencing to identify Nr4a1 binding sites, they found that a significant number of Nr4a1 binding sites overlapped with c-Jun binding sites and the top Nr4a1-binding sites were AP-1 consensus sequences, suggesting that Nr4a1 induces T cell tolerance by antagonizing AP-1-mediated gene expression.
Deletion of the NR4A Transcription Factors Improves CAR T Cell Functions
In the second paper, Chen, et al. found that two NR4A family members, NR4A1 and NR4A2, were up-regulated in CD8+ tumor-infiltrating lymphocytes (TILs) in human melanoma.2 They also showed that the expression of these proteins strongly correlated with the expression of PD-1 and TIM-3 and that the accessible chromatin regions in CD8+PD-1high TILs from human melanoma and non-small cell lung cancer were enriched for NR4A binding motifs, suggesting that the NR4A transcription factor family was involved in mediating T cell exhaustion. The authors further investigated the activities of the NR4A transcription factors by looking at the functions of wild-type CD8+ CAR T cells compared to Nr4a triple knockout (Nr4a1-/-Nr4a2-/-Nr4a3-/-) CD8+ CAR T cells in two mouse solid tumor models. This analysis demonstrated that tumor-bearing mice adoptively transferred with Nr4a triple knockout CD8+ CAR T cells displayed pronounced tumor regression and enhanced survival relative to those that received either wild-type CD8+ CAR T cells or CD8+ CAR T cells with a single knockout of Nr4a1, Nr4a2, or Nr4a3. Subsequent examination of the gene expression and chromatin accessibility profiles in Nr4a triple knockout CD8+ CAR TILs compared to wild-type CD8+ CAR TILs, revealed that the Nr4a triple knockout TILs showed reduced expression of inhibitory receptors, including PD-1, TIM-3, TIGIT, 2B4/CD244, and CD38, increased expression of effector genes, and a strong enrichment in open chromatin containing binding motifs for transcription factors involved in effector functions. As a result, this group concluded that the NR4A transcription factor family regulates the T cell exhaustion phenotype and may be potential next generation targets for cancer immunotherapy.
TOX and TOX2 Transcription Factors are also Involved in the Induction of CD8+ T Cell Exhaustion
These two papers were followed by a series of papers published in June and July of 2019 that suggested that the (HMG)-box transcription factors TOX and TOX2 are also involved in the induction of the exhausted T cell phenotype.3-8 Using mouse models of acute or chronic infection, Yao, et al., Khan, et al., Alfei, et al., and Scott, et al., all demonstrated that Tox expression was up-regulated in exhausted CD8+ T cells that developed in response to chronic viral infection.5-8 Additionally, Wang, et al., Khan, et al., and Scott, et al. showed that TOX is also highly expressed in tumor-infiltrating PD-1high CD8+ T cells in a number of different types of human cancers and that this expression correlates with high expression of CTLA-4, TIM-3, TIGIT, and PD-1.3, 6, 8 Furthermore, using a CAR T cell model, Seo, et al. found that like the NR4A transcription factors, Tox and Tox2 were highly induced in exhausted PD-1highTIM-3high CD8+ CAR T cells. The authors went on to show that tumor-bearing mice adoptively transferred with CD8+ T cells from Tox2-/- mice that also expressed a CAR retrovirus and four shRNAs targeting Tox displayed pronounced tumor regression and enhanced survival relative to those that received either wild-type CD8+ CAR T cells or CD8+ CAR T cells deficient in either Tox or Tox2 alone. Like some of the other groups, they also demonstrated the involvement of the NFAT transcription factor family in the induction of Tox, Tox2, Nr4a2, and Nr4a3 expression in exhausted CD8+ T cells in the absence of AP-1 binding. As a result, they suggested a model in which NFAT induces both the Tox and Nr4a family transcription factors, which together mediate the induction of the CD8+ T cell exhaustion program. Collectively, these observations demonstrate that inhibition of the NR4A and/or TOX family transcription factors may represent powerful new strategies for cancer immunotherapy.
- Liu, X. et al. (2019) Nature 567:525.
- Chen, J. et al. (2019) Nature 567:530.
- Wang, X. et al. (2019) J. Hepatol. [Epub ahead of print].
- Seo, H. et al. (2019) Proc. Natl. Acad. Sci. U.S.A. 116:12410.
- Yao, C. et al. (2019) Nat. Immunol. 20:890.
- Khan, O. et al. (2019) Nature 571:211.
- Alfei, F. et al. (2019) Nature 571:265.
- Scott, A.C. et al. (2019) Nature 571:270.
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