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.

CCL22-based peptide vaccines induce anti-cancer immunity by modulating tumor microenvironment.

CCL22 is a macrophage-derived immunosuppressive chemokine that recruits regulatory T cells through the CCL22:CCR4 axis. CCL22 was shown to play a key role in suppressing anti-cancer immune responses in different cancer types. Recently, we showed that CCL22-specific T cells generated from cancer patients could kill CCL22-expressing tumor cells and directly influence the levels of CCL22 in vitro. The present study aimed to provide a rationale for developing a CCL22-targeting immunotherapy. Vaccination with CCL22-derived peptides induced CCL22-specific T-cell responses in both BALB/c and C57BL/6 mice, assessed by interferon-γ secretion ex vivo. Anti-tumor efficacy of the peptides was evaluated in mouse models engrafted with syngeneic tumor models showing a reduced tumor growth and prolonged survival of the treated mice. Vaccination induced changes in the cellular composition of immune cells that infiltrated the tumor microenvironment assessed with multicolor flow cytometry. In particular, the infiltration of CD8+ cells and M1 macrophages increased, which increased the CD8/Treg and the M1/M2 macrophage ratio. This study provided preclinical evidence that targeting CCL22 with CCL22 peptide vaccines modulated the immune milieu in the tumor microenvironment. This modulation led to an augmentation of anti-tumor responses. This study provided a rationale for developing a novel immunotherapeutic modality in cancer.

Arginase-2-specific cytotoxic T cells specifically recognize functional regulatory T cells.

BACKGROUND: High expression of the metabolic enzyme arginase-2 (ARG2) by cancer cells, regulatory immune cells, or cells of the tumor stroma can reduce the availability of arginine (L-Arg) in the tumor microenvironment (TME). Depletion of L-Arg has detrimental consequences for T cells and leads to T-cell dysfunction and suppression of anticancer immune responses. Previous work from our group has demonstrated the presence of proinflammatory ARG2-specific CD4 T cells that inhibited tumor growth in murine models on activation with ARG2-derived peptides. In this study, we investigated the natural occurrence of ARG2-specific CD8 T cells in both healthy donors (HDs) and patients with cancer, along with their immunomodulatory capabilities in the context of the TME. MATERIALS AND METHODS: A library of 15 major histocompatibility complex (MHC) class I-restricted ARG2-derived peptides were screened in HD peripheral blood mononuclear cells using interferon gamma (IFN-γ) ELISPOT. ARG2-specific CD8 T-cell responses were identified using intracellular cytokine staining and ARG2-specific CD8 T-cell cultures were established by enrichment and rapid expansion following in vitro peptide stimulation. The reactivity of the cultures toward ARG2-expressing cells, including cancer cell lines and activated regulatory T cells (Tregs), was assessed using IFN-γ ELISPOT and a chromium release assay. The Treg signature was validated based on proliferation suppression assays, flow cytometry and quantitative reverse transcription PCR (RT-qPCR). In addition, vaccinations with ARG2-derived epitopes were performed in the murine Pan02 tumor model, and induction of ARG2-specific T-cell responses was evaluated with IFN-γ ELISPOT. RNAseq and subsequent GO-term and ImmuCC analysis was performed on the tumor tissue. RESULTS: We describe the existence of ARG2-specific CD8+ T cells and demonstrate these CD8+ T-cell responses in both HDs and patients with cancer. ARG2-specific T cells recognize and react to an ARG2-derived peptide presented in the context of HLA-B8 and exert their cytotoxic function against cancer cells with endogenous ARG2 expression. We demonstrate that ARG2-specific T cells can specifically recognize and react to activated Tregs with high ARG2 expression. Finally, we observe tumor growth suppression and antitumorigenic immunomodulation following ARG2 vaccination in an in vivo setting. CONCLUSION: These findings highlight the ability of ARG2-specific T cells to modulate the immunosuppressive TME and suggest that ARG2-based immunomodulatory vaccines may be an interesting option for cancer immunotherapy.

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.

Peptide vaccination activating Galectin-3-specific T cells offers a novel means to target Galectin-3-expressing cells in the tumor microenvironment.

Galectin-3 (Gal3) can be expressed by many cells in the tumor microenvironment (TME), including cancer cells, cancer-associated fibroblasts, tumor-associated macrophages, and regulatory T cells (Tregs). In addition to immunosuppression, Gal3 expression has been connected to malignant cell transformation, tumor progression, and metastasis. In the present study, we found spontaneous T-cell responses against Gal3-derived peptides in PBMCs from both healthy donors and cancer patients. We isolated and expanded these Gal3-specific T cells in vitro and showed that they could directly recognize target cells that expressed Gal3. Finally, therapeutic vaccination with a long Gal3-derived peptide epitope, which induced the expansion of Gal3-specific CD8+ T cells in vivo, showed a significant tumor-growth delay in mice inoculated with EO771.LMB metastatic mammary tumor cells. This was associated with a significantly lower percentage of both Tregs and tumor-infiltrating Gal3+ cells in the non-myeloid CD45+CD11b- compartment and with an alteration of the T-cell memory populations in the spleens of Gal3-vaccinated mice. These results suggest that by activating Gal3-specific T cells by an immune-modulatory vaccination, we can target Gal3-producing cells in the TME, and thereby induce a more immune permissive TME. This indicates that Gal3 could be a novel target for therapeutic cancer vaccines.

Arginase 1-Based Immune Modulatory Vaccines Induce Anticancer Immunity and Synergize with Anti-PD-1 Checkpoint Blockade.

Expression of the L-arginine catabolizing enzyme arginase 1 (ARG1) is a central immunosuppressive mechanism mediated by tumor-educated myeloid cells. Increased activity of ARG1 promotes the formation of an immunosuppressive microenvironment and leads to a more aggressive phenotype in many cancers. Intrinsic T-cell immunity against ARG1-derived epitopes in the peripheral blood of cancer patients and healthy subjects has previously been demonstrated. To evaluate the antitumor efficacy of ARG1-derived peptide vaccines as a monotherapy and as a combinational therapy with checkpoint blockade, different in vivo syngeneic mouse tumor models were utilized. To evaluate the antitumor effects, flow cytometry analysis and IHC were performed on tumors, and ELISPOT assays were performed to characterize immune responses. We show that ARG1-targeting therapeutic vaccines were able to activate endogenous antitumor immunity in several in vivo syngeneic mouse tumor models and to modulate the cell composition of the tumor microenvironment without causing any associated side effects or systemic toxicity. ARG1-targeting vaccines in combination with anti-PD-1 also resulted in increased T-cell infiltration, decreased ARG1 expression, reduced suppressive function of tumor-educated myeloid cells, and a shift in the M1/M2 ratio of tumor-infiltrating macrophages. These results indicated that the induced shift toward a more proinflammatory microenvironment by ARG1-targeting immunotherapy favors effective tumor control when combined with anti-PD-1 checkpoint blockade. Our data illustrate the ability of ARG1-based immune modulatory vaccination to elicit antigen-specific immunosurveillance and imply the feasibility of this novel immunotherapeutic approach for clinical translation.