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Basic tumor immunology
TGFβ-specific T cells induced by a TGFβ-derived immune modulatory vaccine both directly and indirectly modulate the phenotype of tumor-associated macrophages and fibroblasts
  1. Maria Perez-Penco1,
  2. Lucia Lara de la Torre1,
  3. Inés Lecoq1,
  4. Evelina Martinenaite1 and
  5. Mads Hald Andersen1,2
  1. 1Department of Oncology, Copenhagen University Hospital, National Center for Cancer Immune Therapy (CCIT- DK), Herlev, Denmark
  2. 2Department of Immunology, University of Copenhagen, Kobenhavn, Denmark
  1. Correspondence to Dr Mads Hald Andersen; Mads.Hald.Andersen{at}regionh.dk

Abstract

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.

  • Transforming Growth Factors
  • Tumor Microenvironment
  • Immunomodulation
  • Macrophage
  • Vaccine

Data availability statement

Data are available upon reasonable request.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/jitc-2023-008405

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    Introduction

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly immunosuppressive tumor microenvironment (TME), which severely impacts the efficacy of immunotherapy.1 Immune modulatory vaccines (IMV) have emerged as a novel strategy to target immunosuppression.2 This therapy aims at activating anti-regulatory T cells that recognize cells that express immunosuppressive molecules, which act as TME antigens,3 such as indoleamine-pyrrole 2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1) and transforming growth factor-β (TGFβ).3 4 Recently, a combined IDO and PD-L1-based IMV showed a remarkable objective response rate of 80% as a first-line treatment in combination with anti-programmed cell death protein 1 (PD-1) in patients with metastatic melanoma.5 The complete response rate was recently updated to 50%, demonstrating long-term responses to IMV.6

    TGFβ is a potent immunosuppressive molecule and it is highly expressed in pancreatic cancer.7 We developed an IMV with TGFβ1-derived peptides formulated in Montanide ISA 51 (referred to as “TGFβ vaccine”) that can control tumor growth in Pan02, a murine model of PDAC.8 9 Treatment with the TGFβ vaccine promoted an antitumor immune response and generated a pro-inflammatory TME.8 Interestingly, vaccine-induced changes in the TME were not restricted to the lymphoid compartment. While the main population of tumor-associated macrophages (TAMs) in tumors from untreated mice exhibited a suppressive M2-like phenotype, vaccination with TGFβ-derived peptides induced a switch toward a dominant pro-inflammatory M1-like phenotype. Furthermore, the immune modulatory impact of the vaccine was also observed in cancer-associated fibroblasts (CAFs). Our study revealed that the TGFβ vaccine not only decreased the percentage of CAFs in the tumor, but also induced a shift in the phenotype of CAFs away from the myofibroblast (myCAF)-like subtype, a subset of fibroblasts characterized for its substantial contribution to the deposition of a collagen-rich extracellular matrix (ECM).10 In the current study, we examined whether the immune modulatory properties of the TGFβ vaccine on TAM and CAF phenotype are a result of the direct recognition and targeting of these subsets by TGFβ-specific T cells or an indirect consequence of the pro-inflammatory effects induced by vaccine-specific T cells in the TME.

    Materials and methods

    Comprehensive materials and methods are provided in online supplemental file 1.

    Supplemental material

    Results

    TGFβ-specific T cells present in the spleen of mice treated with a TGFβ-derived immune modulatory vaccine can recognize fibroblasts and M2 macrophages

    To investigate whether TGFβ-specific T cells could recognize fibroblasts and M2 macrophages in vitro, T cells were isolated from the spleen of mice treated with the TGFβ vaccine or a control vaccine, and co-cultured with fibroblasts or M2 macrophages. Immortalized murine embryonic fibroblasts were used as a source of fibroblasts, since their expression of Tgfb1 was comparable to that of CAFs sorted from KPC tumors (online supplemental figure 1). Macrophages were differentiated from bone marrow progenitors with macrophage colony-stimulating factor (M-CSF) and polarized towards an M2 phenotype with interleukin (IL-4) (figure 1A). We employed the terms “M2” and “M1” for in vitro IL-4-driven and interferon (INF)-γ-driven macrophage phenotypes, respectively; the term “M2-like” for macrophage phenotypes associated with tumor-promotion and immunosuppression and “M1-like” to refer to phenotypes linked to antitumor responses and cytotoxicity, as outlined by Mantovani et al.11

    Supplemental material

    Figure 1
    Figure 1

    Fibroblasts and M2 macrophages can be recognized by TGFβ-specific T cells present in the spleen of mice treated with the TGFβ-derived immune modulatory vaccine. (A) Diagrammatic representation of the experimental set-up. Macrophages were differentiated from bone marrow progenitors with macrophage colony-stimulating factor (M-CSF) and polarized towards an M2 phenotype with IL-4. Immortalized murine embryonic fibroblasts (MEF) were used as a source of fibroblasts. Cancer-associated fibroblasts (CAF) were isolated from subcutaneous KPC tumors and expanded in vitro. To assess recognition of M2 macrophages and fibroblasts by TGFβ-specific T cells induced by the TGFβ vaccine, T cells were isolated from the spleen of mice treated with a TGFβ immune modulatory vaccine (“TGFβ vaccine”) or a control vaccine, and co-cultured with M2 macrophages or fibroblasts. Recognition was assessed by INF-γ ELISpot and flow cytometry. To evaluate the direct effect of TGFβ-specific T cells induced by the immune modulatory vaccines on M2 macrophages, fibroblasts and CAF, T cells isolated from the spleen of mice treated with the TGFβ vaccine or a control vaccine were co-cultured with M2 macrophages, fibroblasts or CAF. Changes in the phenotype of the target cells were assessed by flow cytometry and multiplex ELISA. To investigate the indirect effects of the TGFβ-specific T cells on M2 macrophages, fibroblasts and CAF, these cells were cultured with tumor-conditioned media (TCM) generated from single-cell suspensions from Pan02 tumors from mice that were treated with the TGFβ vaccine or a control vaccine. Changes in the phenotype of the target cells were assessed by flow cytometry. Created with BioRender.com. (B–E) Normalized IFN-γ-secreting cells in T cells from mice that received the TGFβ vaccine or a control vaccine that were either (B) unstimulated, (C) stimulated with TGFβ-derived peptides, (D) co-cultured with fibroblasts in a 1:20 fibroblast:T cell ratio for 24 hours or (E) co-cultured with M2 macrophages in a 1:5 macrophage:T cell ratio for 48 hours (n=3 per group) assayed by IFN-γ ELISpot. Normalization was performed by dividing the number of IFN-γ-secreting cells for all samples for a given condition by the average number of IFNγ-secreting cells of the control group for that condition. (F–G) CD137 geometric mean fluorescence intensity (MFI) in T cells from mice that received the TGFβ vaccine or a control vaccine (F) alone or (G) in co-culture with M2 macrophages in a 1:5 macrophage:T cell ratio for 48 hours (n=3 per group). IFN-γ, interferon-γ; IL, interleukin; TGFβ, transforming growth factor-β.

    We confirmed that the TGFβ vaccine expanded TGFβ-specific T cells in vivo, as stimulation of T cells from treated mice with a pool of TGFβ-derived peptides resulted in the release of IFN-γ, assessed by ELISpot (figure 1B,C). We found that fibroblasts can be recognized by TGFβ-specific T cells, as a higher IFN-γ secretion was detected when T cells from mice that received the TGFβ vaccine were cultured with fibroblast for 24 hours, compared with the co-culture with T cells from mice treated with a control vaccine (figure 1D). However, the same pattern was not observed in a co-culture with M2 macrophages (figure 1E). Instead, we evaluated T cell activation after a co-culture with M2 macrophages by quantifying the expression of CD137, an activation marker that accurately identifies activated and reactive T cells.12 We detected a subtle yet significant increase in the mean fluorescence intensity of CD137 when T cells from mice treated with the TGFβ vaccine were cultured with macrophages for 48 hours, compared with the co-culture with control T cells (figure 1F,G).

    The TGFβ-derived immune modulatory vaccine can directly and indirectly modulate the phenotype of M2 macrophages

    We have previously reported that treatment with the TGFβ vaccine generates a pro-inflammatory TME.8 We demonstrated that culturing M2 macrophages with tumor-conditioned media (TCM) generated from single-cell suspensions from Pan02 tumors from mice that were treated with the TGFβ vaccine polarized macrophages towards an M1-like phenotype to a higher extent than TCM from mice that received a control vaccine.8 This suggested that TGFβ-specific T cells can indirectly modulate macrophage phenotype due to their pro-inflammatory effects within the TME. Here, we primarily aimed to explore whether the modulation properties of the TGFβ vaccine on the myeloid subset could also be a result of a direct targeting of macrophages by vaccine-induced T cells, and to assess how that would compare to the already described indirect modulation of the vaccine. To do so, M2 macrophages were co-cultured with splenic T cells from mice treated with the TGFβ vaccine or a control vaccine for 48 hours. We evaluated changes in the expression of M2-like markers by flow cytometry (figure 1A). We focused on mannose receptor (CD206), based on our observation that treatment with TGFβ vaccine reduced the percentage of CD206+ macrophages in Pan02 tumors.8 Additionally, we assessed arginase-1 (ARG1) and PD-L1, as these proteins are encoded by genes that are highly expressed in M2-like or alternatively activated macrophages and TAMs.11 Major histocompatibility complex (MHC)-II and CD86 were selected as M1-like markers.13

    As expected, exposure of M2 macrophages for 24 hours to TCM from mice that received the IMV notably reduced the expression of CD206, ARG1 and PD-L1 (figure 2A), and resulted in a slight reduction in MHC-II with no change in CD86 expression based on flow cytometry (online supplemental figure 2), compared to exposure to TCM from control mice. We performed a cytokine-focused Bio-Plex multiplex system to analyze the composition of TCM to identify possible drivers for the indirect modulation of M2 macrophage phenotype by the TGFβ vaccine. We found higher levels of IL-6, IL-2 and monocyte chemoattractant protein-1 (MCP-1) / chemokine ligand 2 (CCL2) in TCM from mice treated with the TGFβ vaccine, compared with the control group (online supplemental figure 3). IL-10 and IFN-γ were detected at very low levels and in a similar concentration in both treatment groups (online supplemental figure 3). When comparing these results to a co-culture of M2 macrophages with splenic T cells, we found that although no significant changes were observed in CD206 levels, M2 macrophages that were cultured with T cells from mice treated with the TGFβ vaccine had significantly lower levels of ARG1 and PD-L1, compared with a co-culture with T cells from control mice (figure 2B). Culture of T cells from mice that received the TGFβ vaccine with M2 macrophages showed no discernible impact on the expression of M1-like markers (online supplemental figure 1). Vascular endothelial growth factor (VEGF) can be secreted by M2-like TAMs with a pro-angiogenic phenotype.13 We measured VEGF levels in the supernatants of the co-cultures and found that VEGF secretion by M2 macrophages was decreased after a co-culture with T cells from mice treated with the IMV, compared with the control group (figure 2C). Culturing M2 macrophages with TCM or T cells from mice that received the TGFβ vaccine did not affect the recovered M2 macrophage cell counts and had a very limited impact on M2 macrophage viability and apoptosis, compared with the control conditions (online supplemental figure 4). Taken together, these results suggest that the direct recognition of M2 macrophages by TGFβ-specific T cells can lead to a reduction in their suppressive phenotype.

    Supplemental material

    Supplemental material

    Supplemental material

    Figure 2
    Figure 2

    The phenotype of macrophages can be subject to both direct and indirect modulation by a TGFβ-derived immune-modulatory vaccine. (A–B) Geometric mean fluorescence intensity (MFI) of M2-like markers (CD206, ARG1 and PD-L1) in macrophages that were (A) cultured for 24 hours with tumor-conditioned media derived from Pan02 tumors from mice that were treated with a TGFβ immune-modulatory vaccine (“TGFβ vaccine”) or a control vaccine or (B) co-cultured with T cells isolated from mice treated with the TGFβ vaccine or a control vaccine in a 1:5 macrophage:T cell ratio for 48 hours (n=3 per group) (A, top) and (B, top). Heatmaps displaying the normalized MFI values for M2-like markers across the different conditions. Normalization was performed by dividing the MFI values of all samples for a given marker by the average MFI of the control group for that specific marker (A, bottom) and (B, bottom). Raw MFI values and representative histograms of M2-like markers across the different conditions. (C) VEGF concentration in the supernatants of co-cultures of M2 macrophages with T cells isolated from mice treated with the TGFβ vaccine or a control vaccine in a 1:5 macrophage:T cell ratio for 48 hours (n=3 per group). ARG1, arginase-1; PD-L1, programmed death-ligand 1; TGFβ, transforming growth factorβ; VEGF, vascular endothelial growth factor.

    The phenotype of CAFs can be directly and indirectly modulated by the TGFβ-derived immune modulatory vaccine

    Next, we investigated whether the changes in the phenotype of CAFs induced by the TGFβ vaccine in vivo8 were mediated directly or indirectly by vaccine-induced TGFβ-specific T cells. To do so, fibroblasts were cultured with TCM generated from Pan02 tumors from mice treated with the TGFβ vaccine or a control vaccine for 24 hours, or co-cultured with T cells that were isolated from the spleen of mice that received the TGFβ vaccine or a control vaccine for 48 hours (figure 1A). By flow cytometry, we evaluated changes in the expression of the following fibroblast markers: α-smooth muscle actin (αSMA) and transgelin (TAGLN), as these proteins are encoded by genes whose expression is increased in myCAFs, compared with other fibroblast subsets.14 Notably, Acta2 (the gene that encodes for αSMA) and Tagln were identified as downregulated genes when performing bulk RNA sequencing (RNA-seq) analysis of Pan02 tumors from mice that received the TGFβ vaccine compared with untreated mice.8 We detected a significant reduction in the expression of αSMA and TAGLN based on flow cytometry in fibroblasts when cultured with TCM derived from mice treated with the TGFβ vaccine, compared with TCM from mice that received a control vaccine (figure 3A). Importantly, we found that the expression of the myCAF markers αSMA and TAGLN in fibroblasts was significantly reduced when fibroblasts were co-cultured with T cells from mice treated with the TGFβ vaccine, compared with T cells from control mice (figure 3B). Culturing of fibroblasts with either TCM or T cells from mice that received the TGFβ vaccine showed negligible effects on fibroblast recovery, viability and apoptosis, compared with control conditions (online supplemental figure 4).

    Figure 3
    Figure 3

    A TGFβ-derived immune-modulatory vaccine possesses the capacity to directly and indirectly shape fibroblast phenotype. (A–B) Geometric mean fluorescence intensity (MFI) of myCAF markers (αSMA and TAGLN) in fibroblasts that were (A) cultured for 24 hours with tumor-conditioned media derived from Pan02 tumors from mice that were treated with a TGFβ immune-modulatory vaccine (“TGFβ vaccine”) or a control vaccine or (B) co-cultured with T cells isolated from mice treated with the TGFβ vaccine or a control vaccine in a 1:20 fibroblast:T cell ratio for 48 hours (n=3 per group) (A, top) and (B, top). Heatmaps displaying the normalized MFI values for myCAF markers across the different conditions. Normalization was performed by dividing the MFI values of all samples for a given marker by the average MFI of the control group for that specific marker (A, bottom) and (B, bottom). Raw MFI values and representative histograms of myCAF markers across the different conditions. myCAF, myofibroblast cancer-associated fibroblasts; TAGLN, transgelin; TGFβ, transforming growth factorβ; αSMA, α-smooth muscle actin.

    To further validate these results, we sorted CAFs from KPC tumors based on the expression of the general fibroblast marker CD90.2 to use them as target cells. We found that culture of CAFs with TCM or T cells from mice that received the TGFβ vaccine resulted in a tendency towards a reduction in the expression of αSMA and a significant reduction in TAGLN levels in CAFs, compared with the culture with TCM or T cells from control mice (figure 4A,B). These results confirm the ability of TGFβ-specific T cells to both directly and indirectly modulate the phenotype of CAFs in the TME.

    Figure 4
    Figure 4

    CAF phenotype can be modulated both directly and indirectly by TGFβ-specific T cells induced by a TGFβ-derived immune-modulatory vaccine. (A–B) Geometric mean fluorescence intensity (MFI) of myCAF markers (αSMA and TAGLN) in CAFs that were (A) cultured for 48 hours with tumor-conditioned media derived from Pan02 tumors from mice that were treated with a TGFβ immune-modulatory vaccine (“TGFβ vaccine”) or a control vaccine or (B) co-cultured with T cells isolated from mice treated with the TGFβ vaccine or a control vaccine in a 1:12 CAF:T cell ratio for 48 hours (n=3 per group). CAFs were sorted from subcutaneous KPC tumors harvested from untreated mice (A, top) and (B, top). Heatmaps displaying the normalized MFI values for myCAF markers across the different conditions. Normalization was performed by dividing the MFI values of all samples for a given marker by the average MFI of the control group for that specific marker (A, bottom) and (B, bottom). Raw MFI values and representative histograms of myCAF markers across the different conditions. CAFs, cancer-associated fibroblasts; myCAF, myofibroblast CAF; αSMA, α-smooth muscle actin; TAGLN, transgelin; TGFβ, transforming growth factorβ.

    Discussion

    Overall, our results demonstrate that the TGFβ vaccine exerts both direct and indirect immune modulatory effects on important immune regulatory subsets in the TME: macrophages and fibroblasts. We observed practically negligible direct and indirect effects on the recovery, viability, and apoptosis of M2 macrophages and fibroblasts. Hence, the previously described alterations in TAMs and CAFs in pancreatic cancer induced by the TGFβ vaccine in vivo8 are likely mainly attributed to the direct and indirect modulation of their phenotype, rather than to the direct killing of these cell subsets. The indirect modulatory effects appear to be stronger than the direct effects for both cell subsets. However, a direct comparison of the phenotype modulation intensity is not possible, as it depends on experimental variables, such as the volume of TCM or the number of T cells used for each assay. When assessing the cytokine composition of TCM, we found higher levels of the pro-inflammatory cytokines IL-6 and IL-2, as well as increased concentration of CCL2 in TCM from mice treated with the TGFβ vaccine. This could suggest that the indirect effects might be, at least partially, mediated by one or more of these cytokines. Surprisingly, IFN-γ levels were comparable in both treatment groups. When evaluating whether TGFβ-specific T cells expanded by the IMV could directly modulate macrophage and fibroblast phenotype, neither TGFβ-specific T cells were enriched from the sorted T cell population, nor a TGFβ-specific T cell culture was generated. Instead, bulk T cells that were directly sorted from the spleen of mice treated with the TGFβ vaccine were used. Our results illustrate the remarkable ability of TGFβ-specific T cells to directly modulate the immune regulatory phenotype of macrophages and CAFs, considering that TGFβ-specific T cells constitute only a minor fraction of the overall splenic T cell population in vaccinated mice.8 We suggest that higher numbers of TGFβ-specific T cells might elicit an even more pronounced effect on these target cells.

    In our previous study, we reported that the TGFβ vaccine generated a pro-inflammatory environment and reduced the immunosuppression in the TME. We exemplified this by showing the ability of TCM from mice treated with the TGFβ vaccine to immune-modulate the phenotype of bone marrow-derived macrophages.8 Here, we confirmed these findings, which suggest that changes induced in the TME by TGFβ-specific T cells expanded by the TGFβ vaccine are enough to make macrophages transition from a suppressive to a pro-inflammatory state. When exploring whether vaccine-specific T cells exert a direct effect on macrophages that could be additive to the already described indirect effects, we found that TGFβ-specific T possesses the ability to directly recognize and target M2 macrophages, thereby reducing their immunosuppressive phenotype. This result is consistent with previous findings from our group that demonstrate that TGFβ-specific T cells from patients with pancreatic cancer can recognize and kill autologous regulatory myeloid cells in a TGFβ-dependent manner.15 TGFβ-targeting therapies have been shown to be effective in reducing the infiltration of macrophages in the tumor. Gunderson et al illustrated this by treating MC38-tumor-bearing mice with a small molecule inhibitor of the TGFβ type I receptor (galunisertib).16 Lan et al studied how the administration of bintrafusp alfa (BA), a bifunctional fusion protein composed of the extracellular domain of the TGFβ type II receptor to function as a trap for TGFβ and of an antibody blocking PD-L1, altered macrophages in the TME of 4T1-tumor bearing mice.17 They found that treatment with BA affected the transcriptomic profiles of macrophages, which lead to a decrease in the number of M2-like macrophages, identified as CD68+ ARG1+ macrophages.17 These results are consistent with our data, which show that targeting TGFβ-expressing cells with the TGFβ vaccine can both directly and indirectly decrease the levels of ARG1 in M2 macrophages in vitro.

    Here, we demonstrated that the effects of the TGFβ vaccine within the TME are sufficient to indirectly attenuate the matrix-producing phenotype of fibroblasts. Furthermore, we provide evidence that TGFβ-specific T cells exhibit direct recognition and targeting of fibroblasts, leading to a consequential decrease in the expression of myCAF-related markers. Notably, this phenomenon extends to CAFs directly isolated from pancreatic tumors, enhancing the clinical relevance of our findings. Although the mechanism of action of the TGFβ-derived immune modulatory vaccine does not rely on TGFβ-blockade but on the targeting and modulation of TGFβ-expressing cells, the results we report here align with the mounting evidence that supports that TGFβ-targeting therapies can reduce the abundance of myCAFs within the TME, which is in accordance with the established role of TGFβ in the myofibroblast subset. For instance, Lan et al reported a reduction in the number of αSMA+ cells in EMT-6 tumors following treatment with BA.18 Using an anti-TGFβ antibody (NIS793), Qiang et al demonstrated that TGFβ blockade can also decrease the frequency of αSMA+ cells in the orthotopic KPC model of pancreatic cancer.19 Finally, a comprehensive in-depth analysis by Grauel et al of how an anti-TGFβ blocking antibody impairs CAF populations in vivo elucidated that TGFβ blockade selectively targeted and ablated myCAFs in 4T1 and MC38 murine tumor models.14

    Although macrophage function is essential for maintaining homeostasis in various tissues and organs,20 mounting evidence supports the notion that this myeloid subset may exhibit several tumor-promoting functions, including promotion of vascularization, epithelial to mesenchymal transition, ECM remodeling and resistance to chemotherapy.21 Furthermore, within the TME, TAMs tend to adopt a suppressive phenotype, contributing to immunosuppression and immune escape.22 Notably, in pancreatic cancer, a higher infiltration of M2-like TAMs is associated with decreased overall survival.23 Myofibroblasts play a significant role in the generation of a stiff ECM, which hampers T cell infiltration in solid tumors,24 consequently impeding the efficacy of immune therapies such as immune checkpoint inhibitors (ICI) in highly desmoplastic cancers like PDAC.25 Using RNA-seq, Storrs et al recently reported that a higher abundance of communities comprised of aggressive basal-like malignant cells, tumor-promoting SPP1+ macrophages and myCAFs in human PDAC tumors correlated with especially poor prognosis.26 Here, we provide evidence that a TGFβ-based IMV can reduce the tumor-promoting phenotype of macrophages and CAFs in the TME. For all the reasons above stated and given that the TGFβ vaccine was proven to be well tolerated and safe in mice,8 targeting TAMs and CAFs with this immune-modulatory vaccine emerges as a novel approach to reduce immunosuppression and immune exclusion in PDAC, thereby generating a more susceptible environment for immune-based therapies, such as ICI. Interestingly, we reported that high numbers of TGFβ-specific T cells in the blood were associated with improved survival in patients with pancreatic cancer who underwent treatment with radiotherapy and ICI.15 In a phase-I clinical trial conducted at our institution, we are currently exploring whether the expansion of TGFβ-specific T cells with a TGFβ-based immune modulatory vaccine in combination with radiotherapy, nivolumab and ipilimumab would have an additive clinical benefit for patients diagnosed with pancreatic cancer (EudraCT no. 2022-002734-13).

    Data availability statement

    Data are available upon reasonable request.

    Ethics statements

    Patient consent for publication

    Ethics approval

    Animal experiments were conducted at the animal facility of the Department of Oncology, Copenhagen University Hospital, Herlev, Denmark, following Federation of European Laboratory Animal Science Association (FELASA) guidelines and under a license issued by the Danish Animal Experimentation Inspectorate (2021-15-0201-01001).

    Acknowledgments

    We would like to thank Anette Højgaard Andersen, Merete Jonassen and animal caretakers, Anne Boye and Ditte Stina Jensen, for excellent technical support.

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    Supplementary materials

    Footnotes

    • Twitter @marper2323

    • Contributors MHA and MP-P conceived the project design and study conceptualization. MP-P, LLdlT, IL and EM performed the experiments. MP-P and LLdlT analyzed and interpreted the data. MP-P and MHA wrote the manuscript. MHA supervised the project. All authors read and approved the final manuscript.

    • Funding This study was supported by Danish Health Authority grant ‘Empowering Cancer Immunotherapy in Denmark’ (grant number 4-1612-236/8), the Danish Cancer Society (R326-A18938), Independent Research Fund Denmark (grant number 0134-00072B), The Research Council at Herlev and Gentofte Hospital (grant number: N/A), and the following two Danish funds: Fonden til fremme af klinisk cancerforskning (grant number RL WSDOCS. FID1280670) and Tømrermester Jørgen Holm og hustru Elisa f. Hansens Mindelegat (grant number 20039).

    • 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.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.