Abstract Innate lymphoid cells (ILCs) represent critical mediators of tumor immunity, yet their response to checkpoint blockade remains poorly understood. This study shows profound ILC remodeling in HNSCC patients pre- and post-neoadjuvant anti-PD-1 (n=8) and anti-PD-1/IL-8 (n=7) treatments, marked by distinct metabolic, epigenetic, and functional signatures with potential therapeutic implications. Analyses were performed without response stratification due to the limited responders. Using functional-metabolic (MET) flow cytometry, we observed that CD294low ILC2s were numerically more abundant than CD294high ILC2s post-anti-PD-1 treatment. However, CD294hi ILC2 cells exhibited restricted cytokine production (high TNF, minimal IL-1b/IFN-g), elevated CCR8 and H3K27Me3, and dependence on fatty acid oxidation (FAO, increased CPT1A) and reduced mitochondrial metabolism (lower SDHA), indicating an immunosuppressive, epigenetically silenced phenotype. Although fewer in numbers, the CD294hi ILC2 subset became relatively enriched post-treatment, indicating selective survival of FAO-dependent CD294hi immunosuppressive ILC2s. Conversely, the CD294lo ILC2 showed greater plasticity, with increased IL-1b/IFN-g, supporting transition toward ILC1/ILC3-like states. These CD294lo cells displayed enhanced mitochondrial metabolism (increased SDHA) and reduced CPT1A, lower H3K27Me3, and elevated checkpoint inhibitors (LAYN/LAG3), indicating activation with concurrent checkpoint regulation. This metabolic dichotomy—FAO-dependent suppressive CD294hi ILC2 cells versus mitochondrially active, functionally plastic CD294lo ILC2 cells highlights metabolic fitness as a determinant of pro- versus anti-tumor ILC2 function. ILC1/ILC3 subsets demonstrated distinct CD39-defined patterns (high/medium/negative) correlating with their functional states. ILC1s were enriched in CD39med cells, and maintained TNF expression across all CD39 subsets, while CD39hi cells showed increased IFN-g. ILC3s were predominantly CD39- , post-treatment. Metabolically, ILC1s expressed high SDHA with minimal CPT1A, whereas ILC3s exhibited CD39-dependent metabolic heterogeneity, with higher SDHA in CD39hi cells. Notably, post-treatment, H3K27Me3 was significantly increased across all CD39-defined ILC1 subsets versus ILC3s, suggesting therapy-induced epigenetic constraints on ILC1 while preserving ILC3 flexibility. These results show metabolic fitness and epigenetic state as crucial factors in determining ILC function in cancer immunotherapy. The persistence of immunosuppressive CD294hi ILC2s despite treatment underscores a key resistance mechanism, while the emergence of metabolically active CD294lo cells and CD39neg ILC3s represents therapeutically targetable populations. Our data support precision strategies that combine checkpoint blockade with metabolic interventions to eliminate resistant ILCs and promote beneficial innate immune remodeling. Citation Format: Sujeetha A. Rajakumar, Namya Nanda, Chloe Kim, Dustin Dikeman, Tanguy Seiwart, Zubair Khan, Sewon Kang, Carole Fakhry, Martin P. Alphonse. Innate lymphoid cell reprogramming reveals immunometabolic and epigenetic signatures following checkpoint blockade in head and neck squamous cell carcinoma (HNSCC) 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 B075.
Rajakumar et al. (Wed,) studied this question.