Background Cancer immunotherapy has revolutionized treatment options for patients. However, many patients do not respond to immunotherapy, requiring a better understanding of the T cell response to cancer.¹ Immunotherapy functions in part by improving CD8 TIL (Tumor Infiltrating Lymphocytes) functionality. CD8 TIL progress from a progenitor state into a more terminal state of dysfunction termed exhaustion, with diminished cytolytic capacity and an inability to clear tumors.² Understanding the mechanisms regulating the development of exhausted T cells has clinical implications for improving current and development of new immunotherapies.

Terminally exhausted CD8 TIL are transcriptionally distinct from progenitor exhausted CD8 TIL and our previous study identified epigenetic mechanisms that contributed to T cell dysfunction.³ We found that terminally exhausted CD8 TIL had an increased proportion of histone modifications corresponding to active enhancers and active promoters that did not correspond with expected gene transcription. Intriguingly, we also found decreased expression of AP-1 family members in exhausted CD8 TIL, which are key transcription factors that play roles in transcriptional regulation by mediating enhancer-promoter looping. Intriguingly, enhancers in terminally exhausted CD8 TIL were enriched for AP-1 (bZIP) binding motifs. The physical interaction of enhancers to promoters are essential to their function to promote optimal transcription and recruitment of key transcription factors to these genes. Recent work has shown the importance of chromatin structure for T cell activation and differentiation from naïve states to effector states, highlighting the importance of chromatin interactions in T cell fate and function.^{4 5} However, the role of 3D chromatin structure in the development of T cell exhaustion has not been explored.

Methods We hypothesized that exhausted CD8 T cells may have alterations in 3D chromatin looping due to a loss of AP-1 factor expression that regulates their transcriptional potential and decreases their functionality. To investigate changes in chromatin interactions, we performed low-input Hi-C on terminally exhausted and progenitor CD8 T cells from B16 melanoma. We performed CUT&Tag for CCCTC-binding factor (CTCF) which regulates chromatin looping.

Results We observed a loss of long-range chromatin interactions in terminally exhausted CD8 TIL compared to progenitor CD8 TIL and differential topological associating domains (TADs). We found loss of CTCF binding between precursor and terminally exhausted CD8 T cells at TAD boundaries.

Conclusions Our findings indicate that chromatin conformation changes may be associated with T cell exhaustion and poor tumor control. Immunotherapies that aim to restore enhancer-promoter looping via AP-1 family binding may promote anti-tumor immunity.

References

1. Wei SC, Duffy CR, Allison JP. Fundamental Mechanisms of Immune Checkpoint Blockade Therapy. Cancer Discov. 2018;8(9):1069–86.

2. Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol. 2015;15(8):486–99.

3. Ford BR, Vignali PDA, Rittenhouse NL, Scharping NE, Peralta R, Lontos K, Frisch AT, Delgoffe GM, Poholek AC. Tumor microenvironmental signals reshape chromatin landscapes to limit the functional potential of exhausted T cells. Sci Immunol. 2022 Aug 5;7(74)

4. Bediaga NG, Coughlan HD, Johanson TM, Garnham AL, Naselli G, Schröder J, Fearnley LG, Bandala-Sanchez E, Allan RS, Smyth GK, Harrison LC. Multi-level remodelling of chromatin underlying activation of human T cells. Sci Rep. 2021 Jan 12;11(1):528.

5. Quon S, Yu B, Russ BE, Tsyganov K, Nguyen H, Toma C, Heeg M, Hocker JD, Milner JJ, Crotty S, Pipkin ME, Turner SJ, Goldrath AW. DNA architectural protein CTCF facilitates subset-specific chromatin interactions to limit the formation of memory CD8+ T cells. Immunity. 2023 May 9;56(5):959–978.e10.