Abstract PR007: Heterotypic CD8 T cell clusters isolated from clinical samples are distinct and enriched for antitumor activity

Abstract CD8+ T cell-tumor cell proximity correlates with immunotherapy response. However, it is unknown whether these cells can be captured as functional clusters from clinical samples. In defined co-cultures, tumor antigen-recognizing T cells outcompeted unmatched T cells in forming heterotypic clusters with tumor cells, showing >10-fold enrichment when comprising only 1% of the total T cell population, which was seen in across cancer cell lines. This prompted us to investigate whether this feature could be used to isolate tumor-reactive CD8+ T cells directly from cancer specimens. By regular and imaging flow cytometry, we could identify heterotypic clusters from 21/21 human melanoma metastases, comprising CD8+ T cells interacting with one or more tumor cells and/or antigen-presenting cells (APCs). These results prompted us to investigate whether these heterotypic T cell clusters could be used to better understand biology and whether they hold therapeutic potential. Regarding biology, we observed that CD8+ T cells from clusters isolated from clinical samples were significantly enriched for tumor-reactive and exhausted gene signatures. Integration with T cell receptor (TCR)-sequencing revealed increased clonality of clustered T cells, indicative of expansion. Whereas single CD8+ T cells showed enrichment of viral antigen-specific signatures, T cells from clusters were enriched for tumor antigen-reactivity signatures. In-depth single cell RNA analyses revealed conjugation of these T cells to tumor cells and different APC types. These were not random events, as both T cell-associated APCs and tumor cells were enriched for specific cell states and associated with differential cell-cell communication. CD8+ T cells carrying identical TCRs showed more exhaustion and co-inhibition when conjugated to APCs than to tumor cells. Regarding therapeutic opportunities, we found that upon rapid expansion (REP), CD8+ TIL from clusters exerted on average 9-fold increased melanoma-killing activity in ex vivo assays, accompanied by enhanced production of cytokines. Upon ACT, T cells from clusters showed superior in vivo killing of matched patient-derived xenografts (PDX), which was associated with more infiltration and activation than for ACT of single T cells. Together, these results demonstrate that tumor-reactive CD8+ T cells are enriched in functional clusters with tumor cells and/or APCs, and that they can be isolated and expanded from clinical samples. Being often excluded during sorting for single-cell studies, these distinct heterotypic CD8+ T cell clusters serve as a valuable source amenable to deciphering functional tumor-immune cell interactions, while they may also be therapeutically explored. Citation Format: Daniel S Peeper, Sofia Ibáñez-Molero, Johanna Veldman, Juan Simón-Nieto, Joleen Traets, Austin George, Kelly Hoefakker, Anita Karomi, Jonas Nilsson, John Haanen, Winan van Houdt. Heterotypic CD8 T cell clusters isolated from clinical samples are distinct and enriched for antitumor activity [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR007.