Abstract 6039: Triads of dendritic cells, CD4+ T cells, and CD8+ T cells are found within PDA tumors and are associated with intrinsic anti-tumor immune responses

Abstract Cytotoxic T cells (CTLs) are the main effectors of anti-tumor immunity, and their activation is dependent on carefully coordinated interactions between antigen presenting cells (APCs) and CD4+ T cells (Th). In a pivotal model, synchronized interaction between a dendritic cell (DC), Th, and CTL were posited as requisite for effective CTL responses. Recent studies have resurrected this hypothesis in the context of anti-tumor immunity and demonstrated the function of 3 cell type clusters in immune checkpoint blockade (ICB) response in murine tumors and human cancer. We hypothesized intratumoral 3-cell type clusters are present in an “immune excluded tumor”, pancreatic adenocarcinoma (PDA), outside the context of immunotherapy. Analysis of single cell RNAseq and PDA tumors identified DC:Th:CTL clusters, defined as DC, Th, and CTL within 20µm radius, occurring with ∼15% of DCs. Analysis of multiplex proteomics data from fibrolamellar carcinoma, a rare stromal dominant liver tumor with poor immune infiltration, revealed the presence of DC:Th:CTL clusters. We sought to confirm our findings in a larger dataset using multiplex immunohistochemistry (mIHC) on a tissue microarray (TMA) of >500 patients with untreated resected PDA. Using automatic image analysis software, we identified cell types based on surface marker expression, specifically tumor cells, CTL, Th, macrophages, and non-macrophage APCs. 3-cell type clusters were found in 90% of tumor samples, occurred with ∼10% of APCs, located >50µm from tumor cells, and correlated strongly with APC:Th dyads. Weak correlation was found between 3-cell type clusters and isolated cell types, suggesting cluster formation is unlikely to be related to abundance of infiltrating immune cells. DC:Th:CTL clusters were also readily seen in scST of untreated non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Analysis of PDA, NSCLC, and CRC scST datasets revealed that CTLs within triads were more likely to have an effector-like phenotype. Th within triads were enriched in CXCL13+-like Th phenotype, which previously has been associated with immune triads. Using associated clinical data in the PDA TMA, we found that patients with the highest tertile of APC:Th:CTL clusters had improved overall survival compared to patients with the lowest tertile (HR=0.70, p=0.005). Similar findings were found for patients with the top tertile of APC:Th dyads (HR=0.63, p=6.x10-4), but not APC:CTL or CTL:Th dyads (HR=0.76 and 0.77 and p=0.16 and 0.07, respectively). Overall, our findings demonstrate 3-cell type clusters are readily found in an “immune excluded” tumor like PDA without immunotherapy and are associated with improved patient outcomes. These data are strong evidence for the role of 3-cell type clusters in endogenous anti-tumor immune responses and point toward future opportunities for immunotherapeutic intervention. Citation Format: Sheela R. Damle, Jason A. Carter, Jose M. Pineda, Kristin E. Goodsell, Lindsay K. Dickerson, Shreeram Akilesh, Ian N. Crispe, Venu G. Pillarisetty. Triads of dendritic cells, CD4+ T cells, and CD8+ T cells are found within PDA tumors and are associated with intrinsic anti-tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6039.