Abstract 2572: Association of cancer-associated fibroblast composition with CD4 T cells in pancreatic ductal adenocarcinoma

Abstract Understanding interactions between cancer-associated fibroblasts (CAFs) and immune components in the tumor microenvironment (TME) is crucial for engineering antitumor immunity in pancreatic ductal adenocarcinoma (PDAC). While most studies have focused on CAF-mediated immunomodulation, the impacts of immune cells on CAF heterogeneity and function remain underexplored. In this study, we used flow cytometry and microscopy to profile three major CAF subsets—myofibroblasts (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (apCAFs)—in two orthotopic mouse models of PDAC presenting “immune-hot” (6421c2) and “immune-cold” (FC1245) TMEs. Consistent with subcutaneous models, orthotopic 6421c2 tumors exhibited smaller tumor size, elevated T cell infiltration, and reduced suppressive neutrophils compared to FC1245, highlighting distinct immune microenvironments. Although the overall frequency of PDPN+CAFs in the non-immune compartments was similar, CAF composition differed significantly between the two tumors. FC1245 was predominantly composed of myCAFs (80% of PDPN+CAFs), with low iCAF and apCAF proportions. In contrast, 6421c2 tumors displayed increased iCAFs (30%) and apCAFs (20%) alongside a reduced myCAF frequency (50%). Immunofluorescence and Trichrome staining revealed elevated αSMA+myCAFs and increased collagen deposition in FC1245, suggesting unique CAF composition and function in immunologically distinct TMEs. To examine whether intratumoral T cells modulate CAF heterogeneity in vivo, we examined CAF compositions in tumor-bearing mice administrated with CD8+ or CD4+ T cell depleting antibodies or in the Foxp3DTR mice given diphtheria toxin (DT) to deplete regulatory T cells (Tregs). Unlike CD8 or CD4 depletion, Treg depletion significantly improved primary tumor control in 6421c2, reduced metastasis, and prolonged survival in FC1245. CAF subset analysis also revealed Treg’s unique roles in regulating the myCAF and apCAF subsets in PDAC. Specifically, Treg depletion significantly reduced myCAFs, supporting prior findings that Tregs are essential for myCAF development during PDAC precursor lesion formation. Furthermore, Treg-mediated support of myCAF was CD4+ conventional T cell (Tconv)-dependent, as myCAF level was restored upon CD4 depletion in DT-treated mice. Conversely, apCAFs increased following Treg depletion but were completely abolished with CD4 depletion, suggesting that effector CD4 T cells are required for recruiting or maintaining MHC II-expressing CAFs within the TME. In summary, our study reveals distinct CAF compositions in immunologically diverse TMEs and uncovers the unique roles of Tregs and Tconv in regulating CAF subsets. These findings provide novel insights into CAF-CD4 T cell interactions in vivo and support further investigation of molecular mechanisms underlying stromal functions in PDAC. Citation Format: Jessica Hung, Michel DuPage. Association of cancer-associated fibroblast composition with CD4 T cells in pancreatic ductal adenocarcinoma [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 2572.