320 - Hypoxanthine Enforces Cytotoxic CD8⁺ T Cells via Purine Salvage Synthesis and Epigenetic Modification to Potentiate Immunotherapy

Purpose/Objective(s): While nucleotide metabolism within tumor microenvironment (TME) is recognized as a determinant of immunotherapy efficacy, the metabolic competition between tumor cells and immunocytes for metabolite in this pathway remained poorly investigated. This study delineates how hypoxanthine, a precursor in purine salvage synthesis, dictates antitumor immunity, and pioneers novel strategies to augment immunotherapy responsiveness.

Materials/Methods: C57BL/6 mice bearing B16-F10 subcutaneously underwent paired plasma and tumor interstitial fluid (TIF) sampling for metabolomic profiling. CD3/CD28/IL2-driven activation was used to induce effector T cells (T_e) from splenic naive CD8⁺T cells(T_n). ¹⁵N₄-hypoxanthine and ¹⁵N-glutamine isotope tracing quantified purine salvage and de novo dynamics. Hypoxanthine-modulated T cell responses were systematically assessed through CFSE proliferation assays, IFN-?/Granzyme B secretion tests, and cytotoxicity assays. Integrated ATAC-seq and mRNA-seq mapped chromatin accessibility and immune related signaling networks. Therapeutic validation of 3-dose anti-PD-1 (10mg/kg) alongside daily hypoxanthine oral gavage (20mg/kg) spanned three subcutaneous models: B16-F10/LLC/KPC. TME immune remodeling were analyzed by flow cytometry. The efficacy of SLC29A1-overexpressing NKG2D-CAR T cells was examined in A549-bearing NSG mice.

Results: Unlike guanosine, adenosine, etc., hypoxanthine reduced in TIF compared to paired plasma samples. In vitro hypoxanthine supplementation enhanced T_e proliferation, increased IFN-? and Granzyme B secretion, and promoted tumor cell killing. T_e exhibited elevated purine salvage synthesis activity relative to T_n, with a greater proportion of AMP/GMP derived from the salvage pathway than de novo route. Moreover, hypoxanthine increased chromatin accessibility at RUNX2-binding motifs, which regulates stemness-related genes, while activating PI3K/AKT pathway to upregulate RUNX2 expression, thereby enduing stem-like phenotypes. Tumor cells competitively acquired hypoxanthine through SLC29A1, impairing CD8⁺ T cell antitumoral function. Hypoxanthine supplementation enhanced the efficacy of anti-PD-1 therapy. SLC29A1-overexpressing NKG2D-CAR T cells effectively suppressed subcutaneously A549 tumor progression in NSG mice.

Conclusion: Hypoxanthine sustains effector function and endows CTLs with stem-like plasticity by maintaining purine synthesis and mediating RUNX2 related epigenetic modifications. Tumor cells impair CTLs function via SLC29A1-dominated hypoxanthine scavenging. These results uncover a novel metabolic mechanism of immune evasion and propose promising optimized approaches for immunotherapies.