Harnessing Dendritic Cells and T Cells to Remodel the Tumor Microenvironment: Cellular and Spatial Effects of Cancer Immunotherapy\\

Cancer immunotherapy, including immune checkpoint inhibitors such as pembrolizumab or nivolumab, has revolutionized cancer treatment, yet immunologically cold tumors, such as pancreatic ductal adenocarcinoma (PDAC), remain a major challenge to treat effectively. Cold tumors are defined by a low T cell infiltration and a highly immunosuppressive tumor microenvironment (TME) where dendritic cells (DCs), key mediators between innate and adaptive immunity, often become dysfunctional. This PhD thesis explored strategies to restore both DC and T cell functionality within the TME, aiming to promote sustained anti-tumor immunity in cold tumors following immunotherapy. In the first part of this work, we characterized the tumor models MC38 huCEA, KPC huCEA and EMT6 to uncover immune cell patterns linked to response or non-response to FAP-CD40, a DC-activating therapy. These tumor models exhibit distinct immune landscapes, with MC38 huCEA having an inflamed TME, while KPC huCEA and EMT6 are classified as cold phenotypes. FAP-CD40 treatment showed strong efficacy in the inflamed MC38 huCEA model, moderate efficacy in the KPC model and no efficacy in the EMT6 model. In the responsive models, FAP-CD40 reduced intratumoral cDC1s – suggesting migration to lymph nodes – and expanded intratumoral stem-like CD8+ T cells, a subset with self-renewal capacity and the capacity to differentiate into effector T cells. In contrast, the non-responsive EMT6 tumors showed no such changes. Given the apparent essential roles of cDC1s and stem-like CD8+ T cells in driving anti-tumor immunity, in subsequent studies we focused on enhancing the efficacy of these immune players in the moderately responsive KPC model. We combined FAP-CD40 with PD1-IL2v, a novel targeted immunocytokine designed to selectively expand the stem-like CD8+ T cells and promote their differentiation. This combination demonstrated powerful synergistic anti-tumor efficacy, resulting in significant tumor regression and clearance. Importantly, this synergy was linked to the formation of unique intratumoral immune structures, which we termed “T cell-DC clusters” (TDCs). These TDCs, characterized by dense co-localization of CD4+ and CD8+ T cells – including stem-like T cells – with cDC1s (but lacking B cells), were strongly associated with therapeutic success. Our findings suggest that these TDCs function as localized immune hubs that support T cell activation and bypass the need for continuous lymph node priming. In summary, this thesis demonstrates a promising therapeutic strategy to convert immunologically cold tumors into immune-responsive ones by combining FAP-CD40 with PD1-IL2v. This approach not only enhanced DC functionality and T cell infiltration but also induced the formation of organized immune hubs directly within the TME. These findings provide a novel understanding of how spatially coordinated immune responses can be therapeutically induced in treatment-resistant pancreatic tumors. $\textit{Full-text embargoed until: 2026-11-02}$