Pre-existing T Cells Drive Durable Anti-Tumor Immunity after Oncolytic Virus Therapy in Glioblastoma

Abstract Background: Recurrent glioblastoma (rGBM) remains refractory to immunotherapy due to sparse and suppressed T cell infiltration. We recently reported that survival correlated with immune activation signatures in rGBM patients receiving the oncolytic HSV-1 (oHSV) rQNestin34.5v.2 (CAN-3110). Here, we provide in-situ and molecular evidence that a single oncolytic virus injection can induce durable, tumor-reactive T cell immunity in rGBM. Methods: We integrated highly multiplexed spatial proteomics (CODEX), spatial transcriptomics (Xenium) with custom probes for viral, immune and TCR targets, and bulk TCR-sequencing on paired pre- and post-treatment specimens from a phase 1 clinical trial (NCT03152318). Results: T cell densities strongly increased after treatment, with deep infiltration into viable tumor regions persisting up to two years after a single intratumoral oHSV injection. Cytotoxic GZMB+ T cells were located in close proximity with cleaved-caspase 3+ apoptotic tumor cells, and shorter T cell-tumor distance correlated with longer-progression free survival, demonstrating ongoing anti-tumor immunity. Spatial transcriptomics identified CD8+ T cells states expressing early TCR activation (NR4A1, CD69) and tissue residency (ZNF683 [HOBIT], ITGAE [CD103]) programs enriched in the tumor bed while stem-like T cells localized within lymphoid aggregates. Bulk and spatial TCR analyses revealed in-situ expansion of pre-existing tumoral T cell clones whose amplification correlated with survival. Expanded clones featured tissue resident phenotypes and were positioned closer to tumor cells than non-expanded T cells. Viral remnants were limited to necrotic regions and did not co-localize with T cells, suggesting persistent tumor recognition rather than viral antigen. Conclusion: These results provide in-situ evidence that a single intratumoral oncolytic virus injection can amplify pre-existing T cells clones and induce sustained T cell mediated tumor cytotoxicity even after virus clearance. This suggests that oncolytic virotherapy is as potent T cell activating strategy in rGBM. Citation Format: Maxime Meylan, Ye Tian, Lijian Wu, Alexander L. Ling, Daniel Kovarsky, Graham L. Barlow, Linh D. Nguyen, Jason Pyrdol, Lucas Westphal, Michel Julius, Nicolas L. Gonzalez Castro, Sydney D. Dumont, Andres Santos, Itay Tirosh, Mario L. Suva, E Antonio Chiocca, Kai W. Wucherpfennig. Pre-existing T cells drive durable anti-tumor immunity after oncolytic virus therapy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7743.