Abstract Immunometabolic reprogramming is a key determinant of anti-tumor immunity. This study examines immunometabolic signatures in Mucosal-Associated Invariant T cells (MAIT) and conventional CD8+ tumor-infiltrating lymphocytes (TILs) from head and neck squamous cell carcinoma (HNSCC) patients treated with neoadjuvant anti-PD-1 (n=8) and anti-PD-1/IL-8 (n=7). Analyses were performed without response stratification due to the limited number of responders. Using functional-metabolic (MET) flow cytometry, we identified three CD39 expression levels (high, medium, negative) that inversely correlate with OX40 expression and define metabolic states. Strikingly, OX40, a co-stimulatory receptor linked to T-cell activation, was significantly upregulated on CD39neg MAIT cells, but not on CD39hi exhausted cells. OX40 expression was further enriched on CD39neg MAIT cells following anti-PD-1 treatment, suggesting selective activation of the non-exhausted MAIT compartment. The CD39neg/med OX40+ activated MAIT subset, compared to the OX40- subset, showed unique metabolic features, with decreased glucose-6-phosphate dehydrogenase (G6PD) and acetyl-CoA carboxylase 1 (ACC1) expression, indicating reduced pentose phosphate pathway and fatty acid synthesis capacity in both pre- and post-treatment samples. Conversely, the rate-limiting enzyme for fatty acid oxidation (FAO), CPT1A, and the electron transport chain component, Cytochrome-C, increased in the CD39neg/med OX40+ MAIT subset compared to the OX40- subset, suggesting enhanced FAO and oxidative phosphorylation. These findings indicate that the OX40+ subset transitions from biosynthetic (anabolic) to energy-generating (catabolic) metabolism. Phenograph clustering revealed similar metabolic patterns in CD39neg/med CD8+ TILs, indicating a conserved program across MAIT and CD8+ TIL subsets. Additionally, the CD39hi CD8+ TILs displayed lower OX40 expression than the CD39neg/med subsets, consistent with impaired co-stimulation and exhaustion. Furthermore, in both MAIT and CD8+ TILs, CD39neg/med OX40+ subsets display reduced H3K27Me3 expression relative to the OX40- subset, indicating that OX40+ cells may be less epigenetically restrained than OX40- cells. Therefore, this CD39neg/med OX40+ subset represents a metabolically reprogrammed subset capable of maintaining anti-tumor immunity. Functionally, CD39neg MAIT/CD8+ TILs exhibited higher Granzyme-K levels than CD39med/hi cells, with a significant increase post-treatment. In contrast, CD39hi cells showed elevated cleaved caspase-3, indicating increased apoptosis. This suggests an inverse relationship between CD39 and cytotoxic function, and a positive correlation with apoptotic susceptibility. Overall, CD39/OX40 co-expression defines metabolically fit MAIT and CD8+ T cells with enhanced anti-tumor potential. The preferential expansion of metabolically fit CD39neg/med OX40+ cells following checkpoint blockade highlights this population as critical for immunotherapy response, emphasizing CD39/OX40 immunometabolic profiling as a biomarker approach for HNSCC immunotherapy. Citation Format: Sujeetha A. Rajakumar, Namya Nanda, Chloe Kim, Dustin Dikeman, Tanguy Seiwart, Zubair Khan, Sewon Kang, Carole Fakhry, Martin P. Alphonse. Metabolic fitness determines T cell fate: CD39/OX40 axis controls anabolic-to-catabolic reprogramming in head and neck squamous cell carcinoma abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A055.
Rajakumar et al. (Wed,) studied this question.
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