P25. Pancreatic Cancer Derived Cancer Associated Fibroblast Inhibition of Cytotoxic T-cells is PD-L1 Dependent

Introduction: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with limited treatment options. Better understanding of cancer-associated fibroblasts (CAFs) in the immunosuppressive tumor microenvironment (TME) may improve therapeutic targeting of PDAC. Here, we show that immunosuppression mediated by patient-derived CAFs is dependent on its expression of immune checkpoints PD-1 and PD-L1.


Methods: Under IRB approved protocol, human surgical PDAC specimens were used to examine tumor immune components and its stromal interactions. Tumor was sectioned for histology, flow cytometry, organoid creation, tumor infiltrating lymphocyte (TIL) expansion, and fibroblast culture. Tissue slides were stained with fluorescent antibodies against CAF markers alpha smooth muscle actin (a-SMA) and fibroblast activation protein (FAP). Single-cells from tumor digest were used for isolation of TILs and expanded with IL-2 and anti-CD3/CD28 antibodies. CD45, CD3, CD4, CD8, CD25, CD69, PD-1, and ICOS antibodies were used to phenotype T-cells. Sections were stained with antibodies against immune markers CD3, CD69, CD25, FOXP3, PD-1 and PD-L1. CAFs were cultured from tumor in 3D matrix for 4-5 passages with FGF, and analyzed by flow cytometry, western blot, and qPCR. TILs were co-cultured with CAFs/CAF culture supernatant +/- PD-L1 inhibitor Atezolizumab or PD-1 inhibitor Pembrolizumab. TILs co-cultured were analyzed by flow cytometry, and co-culture media was analyzed IFN-g ELISA.


Results: Flow cytometry showed that CAFs express PD-L1 and PD-1 on cell surface, and verified by western blot and qPCR. TILs co-cultured with CAFs or CAF supernatant showed >90% decreased IFN-g release vs control, however Atezolizumab and Pembrolizumab both significantly reduced release to 60% of control (P< 0.05). T-cells had increased surface PD-1 and reduced CD69, a marker of T-cell activation, however pre-treatment of CAFs with Atezolizumab significantly reduced reduction of CD69 on T-cells vs T-cells co-cultured with non-pretreated CAFs (P< 0.05).


Conclusions: Our results indicate that CAFs contribute to T-Cell suppression in PDAC by expression of PD-L1. Reversibility of this process suggests a novel mechanism to incorporate immunotherapies to improve the cytotoxic immune response in PDAC.