TGFß-specific T cells induced by a TGFß-derived immune modulatory vaccine both directly and indirectly modulate the phenotype of tumor-associated macrophages and fibroblasts.

The tumor microenvironment (TME) of pancreatic cancer is highly immunosuppressive. We recently developed a transforming growth factor (TGF)ß-based immune modulatory vaccine that controlled tumor growth in a murine model of pancreatic cancer by targeting immunosuppression and desmoplasia in the TME. We found that treatment with the TGFß vaccine not only reduced the percentage of M2-like tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) in the tumor but polarized CAFs away from the myofibroblast-like phenotype. However, whether the immune modulatory properties of the TGFß vaccine on TAM and CAF phenotypes are a direct consequence of the recognition and subsequent targeting of these subsets by TGFß-specific T cells or an indirect consequence of the overall modulation induced within the TME remains unknown. Recognition of M2 macrophages and fibroblast by TGFß-specific T cells was assessed by ELISpot and flow cytometry. The indirect and direct effects of the TGFß vaccine on these cell subsets were evaluated by culturing M2 macrophages or fibroblasts with tumor-conditioned media or with T cells isolated from the spleen of mice treated with the TGFß vaccine or a control vaccine, respectively. Changes in phenotype were assessed by flow cytometry and Bio-Plex multiplex system (Luminex). We found that TGFß-specific T cells induced by the TGFß vaccine can recognize M2 macrophages and fibroblasts. Furthermore, we demonstrated that the phenotype of M2 macrophages and CAFs can be directly modulated by TGFß-specific T cells induced by the TGFß vaccine, as well as indirectly modulated as a result of the immune-modulatory effects of the vaccine within the TME. TAMs tend to have tumor-promoting functions, harbor an immunosuppressive phenotype and are linked to decreased overall survival in pancreatic cancer when they harbor an M2-like phenotype. In addition, myofibroblast-like CAFs create a stiff extracellular matrix that restricts T cell infiltration, impeding the effectiveness of immune therapies in desmoplastic tumors, such as pancreatic ductal adenocarcinoma. Reducing immunosuppression and immune exclusion in pancreatic tumors by targeting TAMs and CAFs with the TGFß-based immune modulatory vaccine emerges as an innovative strategy for the generation of a more favorable environment for immune-based therapies, such as immune checkpoint inhibitors.

TGFß-derived immune modulatory vaccine: targeting the immunosuppressive and fibrotic tumor microenvironment in a murine model of pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is associated with very poor survival, making it the third and fourth leading cause of all cancer-related deaths in the USA and European Union, respectively. The tumor microenvironment (TME) in PDAC is highly immunosuppressive and desmoplastic, which could explain the limited therapeutic effect of immunotherapy in PDAC. One of the key molecules that contributes to immunosuppression and fibrosis is transforming growth factor-ß (TGFß). The aim of this study was to target the immunosuppressive and fibrotic TME in PDAC using a novel immune modulatory vaccine with TGFß-derived peptides in a murine model of pancreatic cancer. METHODS: C57BL/6 mice were subcutaneously inoculated with Pan02 PDAC cells. Mice were treated with TGFß1-derived peptides (major histocompatibility complex (MHC)-I and MHC-II-restricted) adjuvanted with Montanide ISA 51VG. The presence of treatment-induced TGFß-specific T cells was assessed by ELISpot (enzyme-linked immunospot). Changes in the immune infiltration and gene expression profile in tumor samples were characterized by flow cytometry, reverse transcription-quantitative PCR (RT-qPCR), and bulk RNA sequencing. RESULTS: Treatment with immunogenic TGFß-derived peptides was safe and controlled tumor growth in Pan02 tumor-bearing mice. Enlargement of tumor-draining lymph nodes in vaccinated mice positively correlated to the control of tumor growth. Analysis of immune infiltration and gene expression in Pan02 tumors revealed that TGFß-derived peptide vaccine increased the infiltration of CD8+ T cells and the intratumoral M1/M2 macrophage ratio, it increased the expression of genes involved in immune activation and immune response to tumors, and it reduced the expression of myofibroblast-like cancer-associated fibroblast (CAF)-related genes and genes encoding fibroblast-derived collagens. Finally, we confirmed that TGFß-derived peptide vaccine actively modulated the TME, as the ability of T cells to proliferate was restored when exposed to tumor-conditioned media from vaccinated mice compared with media from untreated mice. CONCLUSION: This study demonstrates the antitumor activity of TGFß-derived multipeptide vaccination in a murine tumor model of PDAC. The data suggest that the vaccine targets immunosuppression and fibrosis in the TME by polarizing the cellular composition towards a more pro-inflammatory phenotype. Our findings support the feasibility and potential of TGFß-derived peptide vaccination as a novel immunotherapeutic approach to target immunosuppression in the TME.