Immunometabolic Profiling of T Cells and Innate Lymphoid Cells Uncovers Therapeutic Targets to Enhance Immunotherapy in Head and Neck Cancer

Abstract Immunometabolic reprogramming governs antitumor immunity, yet its role in checkpoint blockade responses remains poorly defined. Using functional-metabolic flow cytometry, we characterized tumor-infiltrating mucosal-associated invariant T (MAIT) cells, CD8 T cells, and innate lymphoid cells (ILCs) from head and neck squamous cell carcinoma (HNSCC) patients receiving neoadjuvant anti–programmed cell death protein 1 (PD-1) monotherapy or combined anti–PD-1/anti–interleukin-8 (IL-8) therapy. We identified CD39-negative OX40 MAIT and CD8 tumor-infiltrating lymphocytes (TILs) as metabolically fit, less-exhausted populations. In contrast, OX40− cells exhibited elevated glucose-6-phosphate dehydrogenase (G6PD) expression, identifying the pentose phosphate pathway as a targetable metabolic vulnerability. Ex vivo transcriptomic analysis of CD8 TILs from untreated patients revealed CD39-stratified responses to anti–PD-1/polydatin (G6PD inhibitor) combination treatment: CD39-negative cells amplified effector programs; CD39-medium cells shifted toward lipid catabolism; and CD39-high exhausted cells showed downregulation of STAT3 and mTOR-pathway genes only under combination treatment, consistent with a shift in metabolic regulation. Secretome profiling confirmed the functional translation of this reprogramming, with combination treatment significantly increasing soluble CD27 secretion in CD39-high TILs—indicating enhanced activation in terminally exhausted populations. ILC profiling identified immunosuppressive CD294-high ILC2s, marked by elevated C-C-motif chemokine receptor 8 (CCR8) and carnitine palmitoyltransferase 1A (CPT1A) protein expression, as a potential barrier to checkpoint blockade efficacy. Conversely, CD39-negative ILC3s emerged as metabolically active populations amenable to therapeutic reprogramming. These findings establish immunometabolic profiling as a framework for guiding precision checkpoint blockade combinations in HNSCC.