Background Although immunotherapy with immune checkpoint inhibitors (ICI) has revolutionized melanoma therapy, a substantial portion of patients fail to respond. While this refractoriness (lack of efficacy, failure to respond) is likely multifactorial, a growing body of evidence links effector T-cell failure to disruptions in mitochondrial bioenergetics. Repairing these disruptions is an attractive strategy for improving immunotherapy success; however, the consequential bioenergetic lesions, across the T-cell mitochondrial network, remain unidentified. To fill this gap in knowledge, this study leveraged an in-house diagnostic workflow to characterize intrinsic bioenergetic remodeling commensurate with melanoma T-cell activation/function.

Methods From freshly excised tumor from melanoma patients, CD8+ tumor-infiltrating lymphocytes (CD8+ TILs) were isolated and the remaining tumor tissue was used for TIL culture in the presence of IL-2. Freshly isolated melanoma CD8+ TILs, IL-2 expanded TILs and αCD3/CD28-activated CD8+ T cells isolated from the patients PBMCs were subjected to both comprehensive mitochondrial phenotyping and immunophenotyping using high-resolution respirometry and multi-parametric flow cytometry.

Results Relative to αCD3/CD28-activated CD8+ T cells isolated from patient PBMCs, fresh tumor-derived CD8+ TILs were characterized by specific deficiencies in respiratory complex I. Exposure of tumor-derived CD8+ TILs to IL-2 partially recovered complex I function, suggesting that complex I impairments in CD8+ TILs arise from the conditions within the tumor microenvironment. Interestingly, even in IL-2 expanded TIL cultures, mitochondrial respiratory capacity in CD8+ T cells failed to fully recover to match that of the patient‘s peripheral blood CD8+ T-cells.

Conclusions This persistence of bioenergetic stress in vitro suggests that at least a portion of the metabolic stress encountered by CD8+ T-cell mitochondria in the tumor microenvironment is sustained even upon removal and expansion ex vivo. Such insights could have implications for improving the efficacy of adoptive cell-based therapies in melanoma.

Acknowledgements This study was supported by Department of Defense, Melanoma Research Program - # W81XWH-22-1-0625.

Ethics Approval The study was approved by the instituitional review board at East Carolina University (UMCIRB 22-000852). PArticipants gave informed consent before taking part in the study.