Abstract SY12-02: Tumor-reactive heterotypic T cell clusters from clinical samples to improve immunotherapy

Abstract Different types of immunotherapies have delivered unprecedented clinical benefit, yet their impact remains limited to a subset of cancer patients. Our view is that increasing the effectiveness of immunotherapy will require a better mechanistic insight into the dialogue between cancer and the immune system. We focus on three common challenges in this context: tumor-intrinsic resistance, T cell dysfunction and the scarcity of tumor-reactive T cells in the microenvironment. To investigate this, we use multi-omic strategies including genome-wide function-based screens. This has led to the identification of several genes whose inactivation overcomes tumor-intrinsic resistance to T cell killing, as well as T cell genes whose perturbation prevents dysfunction (1-7).In these studies, we also observed that in vitro, TCR/antigen-matched T and tumor cells engage to form tight heterotypic cell-cell conjugates. Given that emerging evidence links proximity between CD8 T cells and tumor cells to antitumor immunity, we investigated the specificity, features, in vivo relevance and any therapeutic capacity of these T cell clusters in detail. Using conventional and imaging flow cytometry, we identified similar heterotypic clusters in human melanoma metastases, containing CD8 T cells interacting with tumor cells and/or antigen-presenting cells. Of note, such clusters are typically discarded by standard flow cytometry gating for single-cell analyses. Cluster-derived T cells have several characteristics consistent with enriched tumor reactivity, including exhausted cell states and increased TCR clonality. They also show strongly enhanced tumor killing ex vivo and in tumor-bearing mice. We propose that heterotypic clusters may serve as a functional and therapeutically relevant source of tumor-reactive T cells (8). The details and progress of this study, as well as its therapeutic opportunities, will be discussed. References 1. Vredevoogd*, Kuilman* et al. Augmenting immunotherapy impact by lowering tumor TNF cytotoxicity threshold. Cell 2019. 2. Zhang*, Kong*, Ligtenberg* et al. RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells. Cell Rep Med 2022. 3. Kenski*, Huang* et al. An adverse tumor-protective effect of IDO1 inhibition. Cell Rep Med 2023. 4. Lin*, Levy*, Alflen*, Apriamashvili* et al. Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness. Cancer Cell 2024. 5. Ibáñez-Molero et al. Phosphoprotein dynamics of interacting tumor and T cells by HySic. Cell Rep 2024. 6. Lin et al, TSC2 regulates tumor susceptibility to TRAIL-mediated T-cell killing by orchestrating mTOR signaling. EMBO J 2023. 7. Vredevoogd et al. TMED inhibition suppresses cell surface PD-1 expression and overcomes T cell dysfunction. J Immunother Cancer 2024. 8. Ibáñez-Molero*, Veldman*, et al. Tumour-reactive heterotypic CD8 T cell clusters from clinical samples. Nature 2025. Citation Format: Daniel S. Peeper. Tumor-reactive heterotypic T cell clusters from clinical samples to improve immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr SY12-02.