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The tumor microenvironment (TME) of pancreatic cancer is highly immunosuppressive. We recently developed a transforming growth factor (TGF)β-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 TGFβ 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 TGFβ vaccine on TAM and CAF phenotypes are a direct consequence of the recognition and subsequent targeting of these subsets by TGFβ-specific T cells or an indirect consequence of the overall modulation induced within the TME remains unknown. Recognition of M2 macrophages and fibroblast by TGFβ-specific T cells was assessed by ELISpot and flow cytometry. The indirect and direct effects of the TGFβ 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 TGFβ vaccine or a control vaccine, respectively. Changes in phenotype were assessed by flow cytometry and Bio-Plex multiplex system (Luminex). We found that TGFβ-specific T cells induced by the TGFβ vaccine can recognize M2 macrophages and fibroblasts. Furthermore, we demonstrated that the phenotype of M2 macrophages and CAFs can be directly modulated by TGFβ-specific T cells induced by the TGFβ 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 TGFβ-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., Competing Interests: Competing interests: MHA has developed an invention based on the use of transforming growth factor-β-derived peptides for vaccinations. A patent application directed to the invention is owned by the company IO Biotech ApS and lists MHA as the sole inventor. MHA is advisor and shareholder at IO Biotech. IL and EM are employees at IO Biotech. The additional authors declare no competing financial interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which subverts T-cell immunity at multiple levels, is itself subject to inherent T-cell reactivity. This intriguing deviation from central tolerance has been interpreted as counterbalancing IDO1-mediated immunosuppression. Based on this hypothesis, clinical studies employing an IDO1 peptide-based vaccine approach for cancer treatment have been initiated, but there remains a pressing need to further investigate the immunological ramifications of stimulating the anti-IDO1 T-cell response in this manner., Methods: CT26 colon carcinoma tumors were evaluated for expression of IDO1 protein by western blot analysis, immunofluorescence microscopy and flow cytometry. Mouse IDO1-derived peptides, predicted to bind either major histocompatibility complex (MHC) class I or II of the H2 d BALB/c strain, were emulsified in 50% Montanide for prophylactic or therapeutic vaccine treatment of CT26 tumor-bearing mice initiated either 7 days prior to or following tumor cell injection, respectively. In some therapeutic treatment experiments, administration of programmed cell death protein 1-binding antibody (anti-PD1 antibody) or epacadostat was concurrently initiated. Tumor size was determined by caliper measurements and comparative tumor growth suppression was assessed by longitudinal analyses of tumor growth data. For adoptive transfer, T cells from complete responder animals were isolated using paramagnetic beads and fluorescence-activated cell sorting., Results: This study identifies mouse MHC class I-directed and II-directed, IDO1-derived peptides capable of eliciting antitumor responses, despite finding IDO1 expressed exclusively in tumor-infiltrating immune cells. Treatment of established tumors with anti-PD1 antibody and class I-directed but not class II-directed IDO1 peptide vaccines produced an enhanced antitumor response. Likewise, class I-directed and II-directed IDO1 peptides elicited an enhanced combinatorial response, suggesting distinct mechanisms of action. Consistent with this interpretation, adoptive transfer of isolated CD8 + T cells from class I and CD4 + T cells from class II peptide-vaccinated responder mice delayed tumor growth. The class II-directed response was completely IDO1-dependent while the class I-directed response included an IDO1-independent component consistent with antigen spread., Conclusions: The in vivo antitumor effects demonstrated with IDO1-based vaccines via targeting of the tumor microenvironment highlight the utility of mouse models for further exploration and refinement of this novel vaccine-based approach to IDO1-directed cancer therapy and its potential to improve patient response rates to anti-PD1 therapy., Competing Interests: Competing interests: IO Biotech, which provided financial support for this project, is conducting IDO1 peptide vaccine-based clinical trials. KLK, IL and AWP are employed by IO Biotech, M-BZ is a Founder, Chief Executive Officer and a shareholder in IO Biotech, MHA is a Founder, Chief Scientific Officer and shareholder in IO Biotech and AJM is a Scientific Advisory Board member and shareholder in IO Biotech., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)