Vaccination with TGFβ-derived peptides activates and expands TGFβ-specific T cells in vivo

To identify immunogenic peptides within the murine TGFβ1 protein sequence, we used NetMHC V.4.0 and NetMHCII V.2.325 servers to predict MHC-I-restricted and MHC-II-restricted epitopes, respectively. We selected five peptides: one 15-mer peptide (mTGFβ−18–32) and four 8-9mer peptides (mTGFβ−4–11, mTGFβ−215–223, mTGFβ−282–289, and mTGFβ−334–342) predicted to be MHC-II (H2-Ab) and four MHC-I (H2-Kb)-restricted, respectively. To test the immunogenicity of the selected peptides, C57BL/6 mice were vaccinated with the TGFβ-derived peptides using Montanide ISA 51 VG as an adjuvant. The immune response in the spleen was evaluated 7 days post-vaccination by interferon (IFN)γ ELISpot. It was confirmed that all five peptides could induce an immune response upon vaccination (figure 1A,B). Next, we investigated the phenotype of TGFβ-specific T cells induced by the TGFβ-derived peptides in IFNγ ELISpot by sorting CD4⁺ and CD8⁺ T cells (online supplemental figure 1) from the splenocytes of vaccinated mice. We confirmed the in silico predictions, as the peptides predicted to bind MHC-I and MHC-II generated CD8⁺ and CD4⁺ peptide-specific T cells, respectively (figure 1C). Interestingly, mTGFβ−215–223, mTGFβ−282–289, and mTGFβ−334–342 peptides were also able to induce a CD4⁺ peptide-specific response.

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Figure 1

Vaccination with TGFβ-derived peptides activates and expands CD8⁺ and CD4⁺ TGFβ-specific T cells in vivo. (A) mTGFβ−18–32-specific IFNγ-secreting cells in the spleens of mTGFβ−18–32-vaccinated mice (n=4 mice; left) and representative examples of IFNγ ELISpot responses (right). (B) mTGFβ−4–11, 215–223, 282–289, and 334–342-specific IFNγ-secreting cells in the spleens of mTGFβ−4–11, 215–223, 282–289, and 334–342-vaccinated mice, respectively (n=4 mice per peptide; left) and representative examples of IFNγ ELISpot responses (right). For (A) and (B), mice were vaccinated once, and vaccine-induced responses were assessed 1 week after vaccination. Data are presented as mean±SEM. Dots indicate individual mice. (C) Peptide-specific IFNγ-secreting cells in CD4⁺ or CD8⁺ sorted T cells (>93% purity, online supplemental figure 1) from the spleens of mTGFβ−18–32, 4–11, 215–223, 282–289, or 334–342-vaccinated mice, assessed by IFNγ ELISpot. Four mice were vaccinated with all five TGFβ-derived peptides on days 0 and 7. On day 14, mice were sacrificed, spleens harvested and pulled, and CD4⁺ and CD8⁺ T cells sorted and set up in an IFNγ ELISpot in co-culture with bone marrow-derived dendritic cells (BMDC) as antigen presenting cells (APC) in a 1:2 ratio (T cell:APC). Bars represent mean±SEM. Dots represent technical replicates. ELISpot, enzyme-linked immunospot; IFN, interferon; MHC, major histocompatibility complex; TGFβ, transforming growth factor-β.

TGFβ-derived peptide vaccination induces antitumor immunity in a murine tumor model of pancreatic cancer

We evaluated the antitumor activity of TGFβ-derived peptide vaccination in Pan02, a model of PDAC characterized by high expression of TGFβ132 (online supplemental figure 2), high infiltration of immunosuppressive cells such as TAMs, limited tumorous T-cell infiltration,33 and minimal response to immune checkpoint blockade.34 We assessed the effect of a vaccine containing the TGFβ1-derived MHC-II-restricted peptide alone, all four MHC-I-restricted peptides, or a combination of all five peptides on Pan02-tumor bearing C57BL/6 mice. Mice were vaccinated when tumors became palpable (day 10 post-inoculation) and three additional times (days 17, 24, and 35). We observed that a significant delay in tumor growth was only achieved when MHC-I-restricted 8-9mers were combined with the MHC-II-restricted peptide in a multipeptide vaccine (hereafter referred to as ‘TGFβ vaccine’; figure 2A and B and online supplemental figure 3). Next, we examined the immune response generated against all peptides in each treatment group by IFNγ ELISpot. We verified that mice developed a strong and specific immune response towards the peptide(s) with which they were vaccinated (figure 2C).

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Figure 2

Vaccination with TGFβ-derived peptides delays tumor growth in the Pan02 model of pancreatic cancer. (A) Average Pan02 tumor growth for untreated mice and mice vaccinated with a TGFβ-derived MHC-II-restricted peptide (mTGFβ−18–32, referred to as "CD4 epitoe"), a pool of four 8-9mers predicted to bind MHC-I (mTGFβ−4–11, 215–223, 282–289, and 334–342, referred to as "CD8 epitopes"), or a combination of all five peptides (referred to as ‘TGFβ vaccine’) in a 4-dose regimen. Mice (n=5–8 mice per group) were vaccinated on days 10, 17, 24, and 35, as indicated by the arrows. Data are presented as mean±SEM. (B) Tumor volume at endpoint (day 38) for the tumor study shown in (A). n=5–8 mice per group. Dots represent individual mice. Data are presented as mean±SEM. (C) TGFβ-derived peptide-specific responses in the spleen on day 38 for each treatment group for the tumor study shown in (A) assayed by IFNγ ELISpot. n=5–8 mice per group. Data are presented as mean±SEM. (D) Average Pan02 tumor growth for mice that were either untreated or vaccinated with a TGFβ vaccine consisting of a pool of one TGFβ-derived MHC-II predicted peptide (mTGFβ−18–32) and four MHC-I-restricted peptides (mTGFβ−4–11, 215–223, 282–289, and 334–342) in a 2-dose regimen. Mice (n=7–8 per group) were vaccinated on days 10 and 17, as indicated by the arrows. Data are presented as mean±SEM. Antitumor effect was confirmed in six independent experiments. A representative example is shown. (E) Individual tumor growth for Pan02 tumor-bearing mice shown in (D). n=7–8 mice per group. Curves represent individual mice. Arrows indicate vaccination days. (F) Tumor volume and (G) tumor weight at endpoint (day 35) for the tumor study shown in (D). n=7–8 mice per group. Dots represent individual mice. Data are presented as mean±SEM. (H) Correlation between tumor weight and tumor-draining lymph node (LN) weight at the endpoint in untreated or TGFβ-vaccinated Pan02 tumor-bearing mice. n=10 mice per group. Dots represent individual mice. (I) Correlation between tumor-draining LN weight and the expression of Cd3e relative to the housekeeping gene Hprt1 shown in arbitrary units (a.u.) in the tumor-draining LN at the endpoint in TGFβ-vaccinated Pan02 tumor-bearing mice. n=10 mice per group. Dots represent individual mice. (J) TGFβ vaccine-specific responses in the tumor-draining LN at the endpoint in Pan02 tumor-bearing mice assayed by IFNγ ELISpot. n=6–8 mice per group. Dots represent individual mice. Data are presented as mean±SEM. *p<0.05 and **p<0.01 according to TumGrowth software for (A) and (D); unpaired two-tailed t test for (F),(G) and (J); and linear regression for (H) and (I). ELISpot, enzyme-linked immunospot; IFN, interferon; MHC, major histocompatibility complex; TGFβ, transforming growth factor-β.

Next, we confirmed that the TGFβ vaccine could significantly delay Pan02 tumor growth when limiting the treatment schedule to only two vaccinations, on days 10 and 17 post-inoculation (figure 2D–2E). A significant reduction in tumor volume (figure 2F) and tumor weight (figure 2G) at endpoint was observed when Pan02 tumor-bearing mice were treated with the TGFβ vaccine compared with the untreated group. Interestingly, a significant negative correlation between tumor weight and tumor-draining lymph node weight at endpoint was observed only in the group treated with the TGFβ vaccine (figure 2H). In this group, bigger lymph nodes correlated with higher expression of Cd3e (figure 2I). In addition, a strong vaccine-specific immune response was observed in the tumor-draining lymph nodes in the group treated with the TGFβ vaccine (figure 2J), suggesting that a stronger vaccine-induced immune response is associated with a better control of tumor growth.

We confirmed that the TGFβ vaccine-induced antitumor effect was not a consequence of general immune activation by the adjuvant (Montanide ISA 51 VG), as a significant delay in tumor growth was only observed in TGFβ vaccine-treated Pan02 tumor-bearing mice, whereas no effect on tumor growth was observed in a group of mice that only received Montanide (online supplemental figure 4A). In addition, we concluded that the antitumor effect observed for the multipeptide TGFβ vaccine was only achieved when TGFβ-derived peptides were combined with Montanide (online supplemental figure 4A), as a TGFβ vaccine-specific immune response was only developed when TGFβ-derived peptides were administered in the presence of an adjuvant (online supplemental figure 4B). The TGFβ vaccine was well tolerated, as this treatment did not affect the evolution of body weight with time, compared with untreated Pan02 tumor-bearing mice (online supplemental figure 4C).

TGFβ-derived peptide vaccination favors the presence of pro-inflammatory immune subsets in the TME and modulates CAF phenotype

To investigate the mechanism of action underlying the antitumor effect of the TGFβ vaccine, changes in the TME induced by the vaccine were characterized by multicolor flow cytometry. The TGFβ vaccine did not alter the fraction of cancer cells in the tumor (figure 3A and online supplemental figure 5A) or the overall infiltration of leukocytes (figure 3B and online supplemental figure 5B). Interestingly, a reduction in the percentage of endothelial cells upon vaccination was observed (figure 3C and online supplemental figure 5B). Regarding the T-cell compartment, although vaccination with TGFβ-derived peptides did not alter the percentage of total T-cell infiltration (figure 3D and online supplemental figure 5C), we observed a significant increase in tumor-infiltrating CD8⁺ T cells (figure 3E–G) with no change in the percentage of infiltrating CD4⁺ T cells (figure 3H). This resulted in a significant increase in the CD8⁺/CD4⁺ T cell ratio (figure 3I). No changes were detected in the percentage of Tregs among the CD3⁺ population (figure 3J and online supplemental figure 5D), which led to a significant increase in the CD8⁺/Treg ratio (figure 3K). When examining changes in the myeloid compartment, we observed that the infiltration of both polymorphonuclear and monocytic myeloid-derived suppressor cells (MDSC) remained unchanged after TGFβ vaccination (online supplemental figure 5E–5G). Interestingly, although TGFβ vaccination did not affect the percentage of tumor-infiltrating macrophages (figure 3L and online supplemental figure 5H), a drastic increase in the M1 population, followed by a marked decrease in M2 infiltration, was observed (figure 3M, N and P). This resulted in a significant increase in the M1/M2 ratio (figure 3O).

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Figure 3

Vaccination with TGFβ-derived peptides increases the tumoral-infiltration of CD8⁺ T cells and polarizes tumor-associated macrophages from an M2-like to an M1-like phenotype. Pan02 tumor-bearing mice (n=8 per group) were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested on day 33, pooled in pairs among treatment groups, and analyzed by flow cytometry. Bar plots show (A) cancer cells gated as CD45⁻ CD31⁻FAP⁻, (B) leukocytes gated as CD45⁺CD31⁻, (C) endothelial cells gated as CD45⁻ CD31⁺, (D) CD3⁺ T cells gated as CD45⁺CD3⁺, (E) CD8⁺ T cells gated as CD45⁺CD3⁺CD8⁺CD4⁻, (H) CD4⁺ T cells gated as CD45⁺CD3⁺ CD8⁻ CD4⁺, (I) CD8/CD4 ratio calculated by dividing the percentage of CD8⁺ T cells among CD3⁺ cells by the percentage of CD4⁺ T cells among CD3⁺ cells, (J) Tregs gated as CD45⁺CD3⁺ CD8⁻ CD4⁺ CD25⁺ FoxP3⁺, (K) CD8/Treg ratio calculated by dividing the percentage of CD8⁺ T cells among CD3⁺ cells by the percentage of Tregs among CD3⁺ cells, (L) macrophages gated as CD11b⁺ F4/80⁺, (M) M1 macrophages gated as CD11b⁺ F4/80⁺ mannose receptor (MR)⁻, (N) M2 macrophages gated as CD11b⁺ F4/80⁺ MR⁺ and (O) M1/M2 ratio calculated by dividing the percentage of M1 macrophages by the percentage of M2 macrophages in the tumor of untreated or mice treated with the TGFβ vaccine. All populations were gated on single live cells. Gating strategy can be found in online supplemental figures 8–10. Data are presented as mean±SEM. Dots represent pooled tumors (n=3–4 per group). (F) and (G) Representative dot plots of CD4⁺ and CD8⁺ cells in the CD3⁺ population of (F) untreated or (G) TGFβ vaccine-treated mice. (P) Representative histograms of MR on macrophages in untreated mice or mice treated with the TGFβ vaccine shown in (M) and (N). ns, not significant; *p<0.05 and **p<0.01 according to unpaired two-tailed t test. TGFβ, transforming growth factor-β; Tregs, regulatory T cells.

It is well known that TGFβ is highly expressed by CAFs.35 We confirmed that Tgfb1 was mainly expressed in the CD45⁻ compartment of Pan02 tumors from untreated mice (figure 4A), which is mainly composed of cancer cells and CAFs. As TGFβ plays a key role in the recruitment of stromal fibroblasts to the tumor, induces local CAF proliferation and promotes a fibroblast-to-myofibroblast transition,36 we investigated whether vaccination with TGFβ-derived peptides had an impact on the proportion and phenotype of CAFs in Pan02 tumors. We assessed the expression of five CAF biomarkers commonly identified in PDAC37: Fap, S1004a, Acta2, Pdgfra, and Pdgfrb (figure 4B–F) and found that Pan02 tumors from vaccinated mice had significantly lower expression of S1004a (figure 4C) and Acta2 (figure 4D). The same trend was observed for Pdgfrb (figure 4F). We next performed flow cytometric analysis of the CAF compartment and found that the percentage of CAFs (identified as CD90⁺ PDPN⁺, as described in a study by Grauel et al38) in Pan02 tumors was reduced upon treatment with the TGFβ vaccine (figure 4G). Grauel et al characterized two subsets of murine CAF according to their CD26 and Ly6C expression.38 When we examined these two CAF subsets in Pan02 tumors, we found that the TGFβ vaccine induced a trend towards an increase in the percentage of CD26^lo Ly6C^lo CAFs (figure 4H and online supplemental figure 5I), which was followed by a respective trend towards a decrease in the percentage of CD26^hi Ly6C^hi population (figure 4I and online supplemental figure 5I). As myofibroblasts are characterized by a high expression of Acta2,37 we assessed the expression of αSMA (protein encoded by the Acta2 gene) based on mean fluorescence intensity (MFI) in the defined CAF subsets. Based on MFI, we found that αSMA expression was not affected in CAFs (figure 4J) or CD26^lo Ly6C^lo CAFs (figure 4K) on vaccination. However, the TGFβ vaccine resulted in a significant reduction in αSMA expression in the CD26^hi Ly6C^hi CAF population (figure 4L).

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Figure 4

TGFβ vaccine reduces the percentage of cancer-associated fibroblasts (CAF) and the expression of Acta2 (αSMA), a classic myofibroblast marker, in Pan02 tumors. (A) Tgfb1 expression relative to the housekeeping gene Hprt1 in the CD45⁺ and CD45^- fractions of tumors from untreated Pan02-tumor bearing mice assessed by RT-qPCR at endpoint. Data are presented as mean±SEM. Dots represent individual mice (n=2 per group). (B)–(F) Expression of CAF-related genes: (B) Fap, (C) S1004a, (D) Acta2, (E) Pdgfra and (F) Pdgfrb in tumors from untreated or TGFβ vaccine-treated Pan02 tumor-bearing mice. Mice were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17 post-inoculation. Tumors were harvested at endpoint (day 29) and gene expression was assessed by RT-qPCR. Gene expression relative to the housekeeping gene Hprt1 is shown. Data are presented as mean±SEM. Dots represent individual mice (n=8–9 per group). (G)–(L) Flow cytometric analysis of the CAF population in tumors from untreated or TGFβ-vaccinated Pan02 tumor-bearing mice. Pan02-inoculated mice (n=8 per group) were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested at endpoint (day 29), pooled in pairs among treatment groups, and analyzed by flow cytometry. (G, left) Percentage of CAFs, gated as CD90⁺ PDPN⁺, cells across treatment groups. (G, middle) and (G, right) Representative contour plots of CD90⁺ PDPN⁺ cells in the CD45⁻ CD31⁻ population shown in (G) of (G, middle) untreated or (G, right) TGFβ vaccine-treated mice. (H) and (I) Percentage of (H) CD26^lo Ly6C^lo and (I) CD26^hi Ly6C^hi CAF subsets—as previously described38—across treatment groups. (J)–(L) Mean fluorescence intensity (MFI) of αSMA (x10³) in (J) CAFs, (K) CD26^lo Ly6C^lo CAFs and (L, left) CD26^hi Ly6C^hi CAFs. (L, right) Representative histograms of αSMA on CD26^hi Ly6C^hi CAFs in untreated mice or mice treated with the TGFβ vaccine shown in (L, left). All populations were gated on live cells. Gating strategy can be found in online supplemental figure 11. Data are presented as mean±SEM. Dots represent pooled tumors (n=4 per group). a.u., arbitrary units; ns, not significant; *p<0.05, **p<0.01 according to unpaired two-tailed t test. RT-qPCR, reverse transcription-quantitative PCR; TGFβ, transforming growth factor-β.

TGFβ vaccine promotes changes in gene expression in the tumor associated with an increased anti-tumor immune response and a reduced fibrotic environment

Next, we performed bulk RNA sequencing on tumors from Pan02 tumor-bearing mice that were either untreated or treated with the TGFβ vaccine. Differential gene expression analysis identified a total of 545 upregulated and 258 downregulated genes on TGFβ vaccination (figure 5A, online supplemental figure 6A, online supplemental table 1). Gene Ontology analysis for biological processed revealed that 74% of the enriched processes within the upregulated genes were related to immune function. (online supplemental figure 7 and online supplemental table 2). Both innate and adaptive immune-related processes were enriched. Interestingly, ‘CD8⁺ T-cell activation’, ‘Antigen presentation’, ‘T-cell-mediated cytotoxicity’, and ‘B cell receptor signaling’-related processes were enriched, which is indicative of the development of an antitumor immune response on vaccination (figure 5B). Specifically, a higher expression of Cd8a, Tnfa, Gzmb and Cd69 was observed in the vaccinated group (online supplemental figure 6B). Moreover, tumors from vaccinated mice had increased average expression of a panel of genes involved in antigen presentation, lymphocyte activation, cytokines and chemokines (figure 5C), which indicates that the TGFβ vaccine activates an immune response against the tumor. Tumors from vaccinated mice additionally had increased average expression of genes involved in inflammation, IFN signaling and Toll-like receptor signaling (figure 5C), demonstrating that the TGFβ vaccine can generate a pro-inflammatory TME. Lastly, the TGFβ vaccine resulted in an increased average expression of genes involved in phagocytosis, immune response to tumors and apoptosis (figure 5C), which suggests that the response induced by the vaccine is indeed antitumorigenic. Next, we quantified the infiltration level of different immune cells through RNAseq expression data deconvoluted with the ImmuCC algorithm. The results supported the TGFβ vaccine-induced increase in the CD8/CD4 and CD8/Treg ratios observed by flow cytometry (online supplemental figure 6C). In addition, a trend towards higher infiltration of natural killer cells and B cells on TGFβ vaccination was observed.

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Figure 5

TGFβ vaccine promotes changes in intratumoral gene expression associated with the generation of a pro-inflammatory environment, the development of an antitumor immune response and a shift in the phenotype of CAF from fibrotic to inflammatory. Pan02 tumor-bearing mice were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested (n=4 for untreated, n=3 for TGFβ vaccine) on day 30, RNA extracted, and bulk RNAseq performed. (A) Volcano plot showing differentially expressed genes (false discovery rate (FDR)<0.05 and absolute log2 fold-change>0.585) in Pan02 tumors from TGFβ-vaccinated mice compared with untreated tumors. n=545 upregulated and n=258 downregulated genes. (B) Gene ontology (GO) analysis for immune-related biological processes associated with significantly upregulated genes in Pan02 tumors from TGFβ-vaccinated mice compared with untreated tumors. Representative processes for each category are shown. Remaining processes can be found in online supplemental table 2. (C) Heatmaps illustrating the average gene expression level of a panel of genes related to antigen presentation, lymphocyte activation, cytokines and chemokines, inflammation, interferon signaling, TLR pathway, phagocytosis, immune response to tumors and apoptosis in Pan02 tumors from untreated (U1–U4) or TGFβ-vaccinated mice (T1–T3). Gene lists can be found in online supplemental table 3. All gene lists were obtained from nanoString panels, except for the cytokine and chemokine gene list, which was self-generated. Columns represent individual mice. RNAseq counts were VST-normalized and row mean centered (z-score). Rows represent the mean expression (mean z-score) across all genes in each gene list. (D–E) Box plots showing the expression level assessed by RNAseq and presented as VST-normalized counts of (D) genes encoding ligands, receptors, and molecules involved in TGFβ signaling, assembly, or activation, and (E) myCAF-related genes38 in Pan02 tumors from untreated or TGFβ-vaccinated mice. Dots represent individual mice. *p<0.05, **p<0.01 according to unpaired two-tailed t test. (F) Heatmap illustrating the gene expression level of a panel of 15 fibroblast-derived collagen genes in Pan02 tumors from untreated (U1–U4) or TGFβ-vaccinated mice (T1–T3). Gene lists were obtained as per a study by Nissen et al28 and can be found in online supplemental table 3. Rows represent individual genes and columns represent individual mice. RNAseq counts were VST-normalized, log2 transformed, and row mean centered (z-score). *differentially expressed genes identified by DESseq2 (FDR<0.05 and absolute log2 fold-change >0.585). (G) Heatmaps illustrating the average gene expression level of a panel of genes representative of a pro-fibrotic fibroblast phenotype and an inflammatory fibroblast phenotype in Pan02 tumors from untreated (U1–U4) or TGFβ-vaccinated mice (T1–T3). Gene lists were obtained as per a study by Ledoult et al27 and can be found in online supplemental table 3. Columns represent individual mice. RNAseq counts were VST-normalized and row mean centered (z-score). Rows represent the mean expression (mean z-score) across all genes in each gene list. CAF, cancer-associated fibroblast; MHC, major histocompatibility complex; myCAF, myofibroblastic CAF; RNAseq, RNA sequencing; TGFβ, transforming growth factor-β; TLR, Toll-like receptor.

We examined the expression of several TGFβ-related genes in the tumor and found that TGFβ ligands and receptors did not have distinctive alterations in the expression patterns upon TGFβ vaccination (figure 5D, top). When assessing the expression of genes coding for proteins involved in canonical TGFβ downstream signaling, we found that TGFβ vaccination significantly reduced the expression of Smad3, suggesting reduced TGFβ signaling in tumors from vaccinated mice (figure 5D, bottom). Interestingly, in the vaccinated group, we also observed a reduction in the expression of genes encoding latent transforming growth factor beta binding protein 1 (LTBP1; Ltbp1), a TGFβ-binding protein required for the structural assembly and secretion of TGFβ, and thrombospondin-1 (THBS1; Thbs1), a major regulator of TGFβ activation (figure 5D, bottom). In line with the significant reduction in endothelial cells in tumors from mice treated with the TGFβ vaccine (figure 3C), we observed a tendency towards reduced expression of Vegfa and Flt1, genes encoding vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor 1 (VEGFR1), respectively, in tumors from vaccinated mice (online supplemental figure 6D).

Using single-cell RNAseq, Grauel et al defined murine myofibroblasts (myCAFs) as a subset of Acta2+CAF subset characterized by a higher expression of genes associated with ECM remodeling, including Tagln, Lrrc15, and Mfap4.38 As vaccination with TGFβ-derived peptides resulted in a lower expression of Acta2 and αSMA expression based on MFI in CD26^hi Ly6C^hi CAFs in Pan02 tumors, we evaluated the effect of the vaccine on the expression of additional myCAFs-related genes reported by Grauel et al.38 RNAseq data showed a significant decrease in the intratumoral expression of Acta2 and Tagln, with a trend towards reduced expression of Lrrc15 on TGFβ vaccination (figure 5E) As myCAFs are characterized by a significant contribution to ECM deposition,10 we assessed whether the reduction in the expression of myCAFs-related genes had an impact on ECM deposition. We found that tumors from vaccinated mice expressed lower levels of fibroblast-derived collagens compared with untreated mice (figure 5F). Interestingly, Col6a1, Col6a2, Col6a3, and Col27a1 came up as differentially downregulated genes (online supplemental table 1). As TGFβ is a key a driver of fibrosis in many conditions, including PDAC,10 we assessed the fibrotic status of Pan02 tumors after treatment with the TGFβ vaccine by exploring the average expression of genes representative of a pro-fibrotic fibroblast phenotype and an inflammatory fibroblast phenotype, as described by Ledoult et al.27 We observed a marked reduction in the overall pro-fibrotic transcriptional program in Pan02 tumors treated with the TGFβ vaccine (figure 5G). In turn, a higher expression of inflammatory fibroblast-related genes upon treatment was found (figure 5G).

TGFβ vaccine reduces immunosuppression of T cells and macrophages in the TME

We assessed whether the generation of a pro-inflammatory environment by TGFβ vaccination would affect the intratumoral secretion profile of soluble immunosuppressive factors, and whether this could have an impact on T-cell function. Tumor-conditioned media (TCM) was generated from untreated or vaccinated mice by culturing Pan02 whole-tumor digests for 48 hours and harvesting the supernatant. We confirmed that TCM generated from mice treated with the TGFβ vaccine had lower concentration of TGFβ1, compared with TCM from untreated mice (figure 6A). We assessed whether this difference in levels of TGFβ1 in TCM would affect T-cell proliferation. While the proliferation of anti-CD3/CD28-stimulated T cells from an untreated tumor-free mouse was strongly impaired when cultured with TCM from untreated mice, the TCM from mice treated with the TGFβ vaccine had a limited impact on T-cell proliferation (figure 6B). We showed that Tgfb1 is mainly expressed by cancer cells and CAFs in Pan02 (figure 4A). However, treatment with the TGFβ vaccine drastically polarized macrophages from an M1-like to an M2-like phenotype (figure 3M–3P). We assessed whether the polarization of macrophages in the treated group could be explained due to an indirect effect of the TGFβ vaccine in this population. To do so, bone marrow-derived macrophages (BMDM) were polarized towards an M2-like phenotype with interleukin (IL)-4 and cultured with TCM generated from untreated or TGFβ vaccinated-mice. We observed that the fraction of M1-like macrophages was increased when M2-like BMDM were cultured with TCM from mice treated with the TGFβ vaccine, compared with TCM from untreated mice, leading to an increase in the M1/M2 ratio (figure 6C). This increase was followed by a reduction in the expression based on MFI of the M2-like markers arginase-1 (Arg1) and PD-L1 (figure 6D–6E).

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Figure 6

TGFβ vaccination results in reduced intratumoral secretion of TGFβ and reduces immunosuppression of T cells and macrophages in the tumor microenvironment. Pan02 tumor-bearing mice were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested (n=4 per group) on day 24 and pooled in pairs among treatment groups. Tumor-conditioned media (TCM) secreted by 0.1×10⁶ cells from the tumor digest during a 48 hours culture was harvested. (A) Quantification of TGFβ1 protein levels in TCM from untreated and TGFβ-vaccinated mice. (B) Effect of TCM from untreated or TGFβ-vaccinated mice on the proliferation of anti-CD3/CD28-stimualted naïve splenocytes from an untreated tumor-free mouse. Naïve splenocytes were CFSE-labeled and activated with Dynabeads Mouse T-Activator CD3/CD28 (1:1 ratio) for 48 hours in the presence or absence of TCM generated from tumors from untreated or TGFβ-vaccinated mice. Proliferation of live CD3⁺ cells was measured by flow cytometry. Gating strategy can be found in online supplemental figure 12. (B, left) Proliferation index in CD3⁺ cells across culture conditions. (B, right) Representative histograms of CFSE staining in live CD3⁺ cells (B). (C–E) Effect of TCM from untreated or TGFβ-vaccinated mice on the phenotype of bone-marrow derived macrophages (BMDM) from an untreated tumor-free mouse that were polarized with IL-4 to an M2-like phenotype. BMDM were cultured for 24 hours in the presence of TCM generated from tumors from untreated or TGFβ-vaccinated mice. The phenotype of BMDM was assessed by flow cytometry. (C, top) M1/M2 ratio in BMDM cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. Macrophages were gated as CD11b⁺ F4/80⁺, M1 and M2 macrophages were gated as CD11b⁺ F4/80⁺ mannose receptor (MR)⁻ and CD11b⁺ F4/80⁺ MR⁺, respectively. Gating strategy can be found in online supplemental figure 13. (C, bottom) Representative histograms of MR on macrophages across culture conditions shown in (C, top). (D, top) Mean fluorescence intensity (MFI) of Arg1 macrophages cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. (D, bottom) Representative histograms of Arg1 on macrophages across culture conditions shown in (D, top). (E, top) MFI of PD-L1 macrophages cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. (E, bottom) Representative histograms of PD-L1 on macrophages across culture conditions shown in (E, top). Bar plots are presented as mean±SD. Dots represent data derived from TCM generated from pooled tumors (n=2 per group). Arg1, arginase-1; IL, interleukin; PD-L1, programmed death-ligand 1; TGFβ, transforming growth factor-β.