Background T cell exhaustion is a state of T cell with reduced proliferation capacity, cytokine production, and lose of responsiveness to immune checkpoint blockade. Growing evidence demonstrates that the global epigenetic alterations are the critical events to drive T cell exhaustion under chronic infection or persistent antigen exposure. However, it is unclear how the epigenetic alteration is engaged.

Methods For tumor engraftment, tumor cell lines (YUMM1.7-OVA/gp33) were injected subcutaneously into C57BL/6 mice, followed by analysis on day 14 or 18 post tumor engraftment. Tumors were minced and digested using collagenase, and tumor-infiltrating immune cells were enriched by density gradient centrifugation. The enriched cells were used for subsequent in vitro culture, stimulation, flow cytometry and ATAC-seq analyses.

To examine mitochondrial activity, cells were stained with MitoTracker Deep Red and MitoTracker Green for mitochondrial membrane potential and mass, respectively, followed by regular surface staining. For cytokine detection, cells were stimulated with either OVA or gp33 peptide in the presence of brefeldin A and stained by intracellular staining procedure.

Results We found tumor-infiltrating CD8⁺ T cells (TILs) accumulated damaged mitochondria, characterized by increased mitochondrial mass but reduced mitochondrial membrane potential. The TILs with disturbed mitochondria exhibited more severe exhausted phenotype, including reduced cytokine production and upregulation of co-inhibitory receptors. In addition, the combination of glucose deprivation, hypoxia, and TCR signaling can induce the accumulation of damaged mitochondria in vitro and drastically weakened T cell immunity in vivo as seen in TILs previously. Intriguingly, ATAC-seq revealed that genes involved in DNA damage signaling were more accessible in TILs. Indeed, PD-1⁺ TIM-3⁺ TILs displayed highest expression of γH2AX, a marker of DNA damage responses, implying mitochondria dysfunction may be associated with DNA damage response, which may coordinately drive T cell exhaustion.

Conclusions Our study suggests that mitochondrial fitness is pivotal for T cell-mediated immunity and the accumulation of dysfunctional mitochondria could result in exhaustion phenotype in T cells. Moreover, we unveil that the accumulation of depolarized mitochondria in T cells associating with DNA damage responses. Together, these results point out the contribution of mitochondrial dynamics on guarding T cell functional capacity and engagement of DNA damage responses.