Adenosine blockade combined with FOLFIRINOX chemotherapy enhances T follicular helper cell function in pancreatic adenocarcinoma draining lymph nodes from mice and humans

Over 90% of all pancreatic cancer cases are pancreatic ductal adenocarcinoma (PDAC), an aggressive disease with poor prognosis and limited treatment options. Chemotherapy and immunotherapy efficacy are both limited by the immunosuppressive PDAC tumor microenvironment (TME). Treatment-related tumor cell death leads to accumulation of extracellular adenosine, a potent immunosuppressive metabolite in the PDAC TME; accordingly, we investigated the effects of blocking adenosine during chemotherapy treatment. In an orthotopic murine model of PDAC, CD73 - the primary enzyme responsible for extracellular adenosine - inhibition in combination with FOLFIRINOX reduced tumor size by 57% (P=0.04) and significantly increased survival compared to treatment with FOLFIRINOX alone. FOLFIRINOX+CD73i significantly decreased suppressive SPP1+ tumor-associated macrophages, increased Type I and Type II interferon signaling of multiple cell types in the TME, and was CD4+ and CD8+ T cell dependent. To track antigen-specific CD4+ T cells, we engineered expression of an MHC-II restricted epitope in orthotopically implanted tumor cells and found that FOLFIRINOX+CD73i treatment increased follicular helper T (Tfh) cells in tumor-draining lymph nodes but that these cells were not enriched for tetramer-positive, tumor antigen-specific cells. In contrast, Treg cells in the TME were enriched for tetramer-positive cells suggesting that CD73i may drive antigen-reactive T cells toward a suppressor phenotype. Targeting activated T cells by adding anti-CCR8 to FOLFIRINOX+CD73i significantly decreased tumor masses compared to FOLFIRINOX+CD73i alone (P=0.02). To investigate the effect of CD73i on adaptive immune responses in PDAC patients, we performed Xenium image-based spatial transcriptomics on 53 tumor-draining lymph nodes from patients treated with neoadjuvant FOLFIRINOX+anti-PD-1+CD73i, FOLFIRINOX+anti-PD-1, FOLFIRINOX alone, or untreated. In line with our mouse model, we found a significant CD73i-dependent increase in intra-follicular antigen-experienced B cell density (P=0.037) with a concomitant increase in intra-follicular CD8+ T cell density (P<0.001), which could be explained by enhanced function of Tfh cells. Accordingly, we found enrichment in IL-21 expressing Tfh cells in tumors treated with FOLFIRINOX+anti-PD-1+CD73i compared to FOLFIRINOX+anti-PD-1. These results indicate that CD73i may drive distinct immune remodeling in PDAC, boosting Tfh cell function in draining lymph nodes but favoring antigen-specific Treg enrichment in the tumor microenvironment.