TGF{beta}-derived immune modulatory vaccine: targeting the immunosuppressive and fibrotic tumor microenvironment in a murine model of pancreatic cancer

Introduction

Pancreatic cancer is the seventh leading cause of cancer-related death globally, ranking third in the USA and fourth in the European Union.1 2 Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and accounts for 90% of the reported cases.1 Most patients have metastatic PDAC at the time of diagnosis, limiting surgery as a potential curative intervention and resulting in an overall 5-year survival rate of 5%.3 The poor survival rate can be attributed to late diagnosis due to the lack of disease-specific symptoms, early metastasis and the lack of effective treatment for non-resectable patients.4 Although the clinical benefit of nivolumab and ipilimumab in combination with radiotherapy in patients with refractory metastatic pancreatic cancer has been recently reported in a phase II trial,5 immunotherapy still shows limited efficacy in most patients with PDAC.6 This resistance can be attributed to the intrinsic immunosuppressive, non-immunogenic and desmoplastic nature of pancreatic tumors.6 PDAC is characterized by low T-cell infiltration and a high abundance of suppressive cells, including regulatory T cells (Treg), tumor-associated macrophages (TAMs), and cancer-associated fibroblasts (CAFs).7

Transforming growth factor β (TGFβ) is a major promoter of immunosuppression and is highly secreted in the tumor microenvironment (TME) by cancer cells, fibroblast, macrophages and Tregs.8 Its suppressive effects, including the inhibition of effector T cells, are well documented and have been confirmed in murine models of PDAC.9 In addition, TGFβ is a key player in the development of fibrosis and desmoplastic stroma in PDAC.10 For instance, it polarizes CAFs into myofibroblastic CAFs (myCAFs), a subset of fibroblasts characterized by a significant contribution to extracellular matrix (ECM) deposition,10 which has been linked to immune evasion and failure of cancer immunotherapy.11

Immune modulatory vaccines, which combat immunosuppression by targeting cells in the tumor that express suppressive molecules, offer an appealing and novel approach to cancer immunotherapy. In the last decade, we have described self-reactive, pro-inflammatory T cells, known as anti-Tregs, that specifically target immunosuppressive cells and hinder counter-regulatory feedback signals, particularly in patients with cancer.12 13 Anti-Tregs recognize major histocompatibility complex (MHA)-restricted epitopes derived from proteins expressed by regulatory immune cells, including indoleamine 2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1), arginase-1, arginase-2, and galectin-3.14–19 The clinical potential of immune modulatory cancer vaccines that activate anti-Tregs has been shown in murine models of cancer18–21 and in a recent clinical trial conducted at our center, where an impressive response rate was achieved in metastatic melanoma with an immune modulatory vaccine against IDO/PD-L1 in combination with nivolumab in a phase 1/2 trial.22

We recently described the presence of TGFβ-specific T cells in the blood of healthy donors and patients with cancer, and the ability of these cells to recognize and kill cancer cells in a TGFβ-dependent manner.23 24 In this study, we evaluated the efficacy of activating TGFβ-specific T cells by TGFβ-derived peptide vaccination to target immunosuppression and fibrosis in the TME in Pan02, a syngeneic murine model of PDAC. Our data show that TGFβ-derived multipeptide vaccination can control Pan02 tumor growth by polarizing the TME from a suppressive and fibrotic phenotype to a pro-inflammatory niche. We report that treatment with a TGFβ-derived peptide vaccine reduces the TGFβ-signature in the tumor and the infiltration of suppressive cells; increases the frequency of pro-inflammatory immune subsets, generates a pro-inflammatory environment that does not restrain T-cell proliferation, and reduces the expression of genes related to both, ECM-remodeling CAFs (myofibroblasts) and fibroblast-derived collagens. These findings support the therapeutic potential of TGFβ-derived peptide vaccine as a novel immunotherapeutic approach to target immunosuppression and fibrosis in PDAC.

MethodsMice

Animal experiments were performed at the animal facility of the Department of Oncology, Copenhagen University Hospital, Herlev, Denmark. Female C57BL/6 mice (8–18 weeks old) were bred in-house from a C57BL/6JBomTac background. Experimental procedures were conducted according to Federation of European Laboratory Animal Science Association (FELASA) guidelines and under licenses issued by the Danish Animal Experimentation Inspectorate.

Peptides

Murine TGFβ1-derived T cell epitopes were predicted using NetMHC V.4.0 and NetMHCII V.2.325 for MHC-I and MHC-II-restricted peptides, respectively. Five peptides were selected, one major histocompatibility complex (MHC)-II (H2-Ab)-predicted peptide: mTGFβ (mTGFβ)−18–32 (15mer, LLVLTPGRPAAGLST) and four MHC-I (H2-Kb)- predicted peptides: mTGFβ−4–11 (8mer, SGLRLLPL), mTGFβ−215–223 (9mer, QGFRFSAHC), mTGFβ−282–289 (8mer, TNYCFSST) and mTGFβ−334–342 (9mer, TQYSKVLAL). Peptides were purchased from Schäfer (purity of >90%). mTGFβ−18–32 peptide was reconstituted in 2 mM H2O. The remaining peptides were reconstituted in 20 mM dimethyl sulfoxide (DMSO).

Cell lines

Pan02 was retrieved from the cell line biobank at the National Center for Cancer Immune Therapy (Denmark) and cultured in RPMI-1640 GlutaMAX (Gibco), 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (P/S, Gibco). Cell lines were mycoplasma-free, assessed by PCR.

Mouse tumor models

Mice were injected subcutaneously (s.c.) on the right flank with 5×105 Pan02 cancer cells in 100 µL of RPMI-1640. When tumors became palpable, mice were divided into treatment groups by stratified randomization on tumor volume. Tumor length and width were measured three times a week with a digital caliper. Tumor volume was calculated as 0.5 × length × width2. The experimental endpoint was defined by the presence of early signs of tumorous ulceration. Survival could not be assessed due to tumorous ulceration.

Vaccination

Mice were vaccinated s.c. at the base of the tail with two emulsions, one containing 100 µg of the murine TGFβ1-derived MHC-II-restricted peptide (mTGFβ−18–32) and the other containing 50 µg of each MHC-I-restricted peptide (mTGFβ−4–11, mTGFβ−215–223, mTGFβ−282–289 and mTGFβ−334–342), unless otherwise stated in the figure legends. This treatment is referred to as ‘TGFβ vaccine’. Mice were vaccinated on randomization day and 7 days after, unless otherwise stated. The emulsions were generated by mixing the peptide solution with Montanide ISA 51 VG (Seppic) at a 1:1 ratio.

Organ collection and processing

Mice were sacrificed by cervical dislocation. Right inguinal lymph nodes, representing tumor-draining lymph nodes, were harvested and weighed. Spleens were collected. Spleens and lymph nodes were processed through a 70 µm cell strainer and red blood cells were lysed using RBC Lysis Buffer (QIAGEN). Tumors were collected, cut into smaller pieces and digested in RPMI-1640 supplemented with 1% P/S, 2.1 mg/mL collagenase type I (Worthington), 75 µg/mL DNase I (Worthington), and 5 mM CaCl2 for 30 min at 37°C and 300 rpm. Tumors were processed through a 70 µm cell strainer.

Cell sorting

Splenic CD4+ and CD8+ T cells were isolated by positive selection using mouse CD4 (L3T4) MicroBeads and mouse CD8a (Ly-2) MicroBeads (Miltenyi Biotec), according to manufacturer’s instruction. CD45+ and CD45− cells were isolated from Pan02 tumors using mouse CD45 MicroBeads (Miltenyi Biotec), following manufacturer’s instruction.

Generation of bone marrow-derived dendritic cells and macrophages

Femurs and tibias were harvested. Bone marrow cells were collected by flushing the bones with PBS. For the generation of bone marrow-derived dendritic cells (BMDC), 2×106 bone marrow-derived cells were cultured in 10 mL of RPMI-1640 with 10% FBS, 1% P/S and 20 ng/mL murine GM-CSF (PeproTech) in 10 cm Petri dishes (Sigma-Aldrich) and incubated at 37°C. On day 3, cells were supplemented with 10 mL of medium containing 20 ng/mL GM-CSF. On day 6, BMDC was harvested. For the generation of bone marrow-derived macrophages (BMDM), 4×106 bone marrow-derived cells were cultured in 10 mL of DMEM with 10% FBS, 1% P/S and 20 ng/mL murine M-CSF (PeproTech) in 10 cm Petri dishes (Sigma-Aldrich) and incubated at 37°C. On day 2, cells were supplemented with 4 mL of medium containing 20 ng/mL M-CSF. On day 4, media was changed and 10 mL of fresh media with 20 ng/mL murine M-CSF and 20 ng/mL human IL-4 (PrepoTech) was added per dish to polarize BMDM to an M2-like phenotype. On day 6, M2-like BMDM were harvested.

ELISpot

Enzyme-linked immunospot (ELISpot) was performed as described by Bendtsen et al.19 The 8×105 splenocytes, 4×105 cells derived from the lymph node or 2–3×105 CD4+ or CD8+-sorted T cells in 200 µL of RPMI-1640 supplemented with 10% FBS and 1% P/S were added per well in duplicates or triplicates. For experiments with sorted T cells, BMDC were added at a 1:2 ratio (BMDC:T cell). To assess the general response to the TGFβ vaccine, cells were stimulated with a peptide pool consisting of all five peptides included in the TGFβ vaccine to reach a working concentration of 5 µM/peptide. Specific responses are reported as the difference between average number of spots in peptide-stimulated wells and unstimulated wells.

Flow cytometry

The following antibodies/dies (purchased from BioLegend unless otherwise stated) were used for flow cytometry: CD45-PE-Cy7, CD31-FITC, FAP-biotin (R&D system), streptavidin-APC, CD90-BV605, PDPN-APC, Ly6C-AF700, CD26-PerCP-Cy5.5 (eBioscience), αSMA-Cy3 (Sigma), CD11b-PE-Cy7, CD11b-Pacific Blue, F4/80-APC, F4/80-FITC, MR-PE, MR-PE-Cy7, Ly6C-PerCP-Cy5.5, Ly6G-APC-Cy7, Arg1-PE (R&D system), PDL1-APC (BD Biosciences), CD45-FITC, CD3-AF700, CD4-BV421, CD8-BV605 (BD Biosciences), CD25-PE-Cy7, FoxP3-APC and carboxyfluorescein succinimidyl ester (CFSE, Sigma Aldrich). Viability was assessed with Zombie Aqua (BioLegend). Samples were Fc receptor-blocked using mouse FcR blocking reagent (1:10; Miltinyi Biotec). For intracellular staining, samples were fixated and permeabilized with eBioscience Fixation/Permeabilization Concentrate, Diluent and 10X Buffer (Invitrogen). Data were acquired on FACSCanto II (BD Biosciences) or ACEA NovoCyte Quanteon (Agilent) and analyzed with FlowJo V.10.6.1 (Tree Star). Gating strategies can be found in online supplemental figures 8–13.

Total RNA extraction

Tumors and lymph nodes (≤20 mg) were stored in RNAlater (Invitrogen) at −80°C. For RNA extraction, tumor fragments were transferred to RLT buffer (QIAGEN) and mechanically homogenized with a Tissue Lyser (QIAGEN). RNA was extracted with RNEasy Plus Mini Kit (QIAGEN), following manufacturer’s instructions. RNA concentration was measured with NanoDrop 2000 Spectrophotometer (Thermo Scientific).

RT-qPCR

Reverse transcription of 1 µg of total RNA was done with the iScript cDNA synthesis kit (Bio-Rad), following manufacturer’s instructions. Complementary DNA (cDNA) was diluted 1:3. Reverse transcription-quantitative PCR (RT-qPCR) was performed in technical triplicates on a thermocycler instrument (Roche LightCycler 480) using LightCycler 480 Probes Master (Roche Diagnostics) and the following TaqMan gene expressions assay probes (Life Technologies): Cd3e (Mm01179194_m1), Tgfb1 (Mm01178820_m1), Fap (Mm01329177_m1), S1004a (Mm00803371_m1), Acta2 (Mm01546133_m1), Pdgfra (Mm00440701_m1), Pdgfrb (Mm00435546_m1) and Hprt1 (Mm00446968_m1). Data were normalized to the expression level of Hprt1 (housekeeping gene) and analyzed with the 2−dCT method.

RNA sequencing

RNA sequencing (RNAseq) was performed as previously described26 on tumors from four untreated and three vaccinated mice. In short, 500 ng purified RNA (RNA Integrity Number (RIN) score >7) was enriched for polyadenylated messenger RNA followed by fragmentation, random-primed cDNA synthesis (NEBNext), PCR-mediated indexing (NEBNext), size selection and quantification (KAPA, Roche). The cDNA libraries were sequenced using Illumina NovaSeq 6000. Alignment to GRCm39 and quantification of reads was performed as previously described26 using STAR (V.2.7.8), featureCounts (V.1.6.4), and Ensembl gene transcripts (V.104). Differential gene expression was analyzed by the DESeq2 package (V.1.30, cut-off p adjusted value<0.05 and absolute log2 fold change >0.585). RNAseq data is available on GEO repository (GSE206764). Volcano plots were generated with EnhancedVolcano R package (V.1.8.0). The list of genes used for the generation of heatmaps can be found in online supplemental table 3. All gene lists were obtained from nanoString panel gene lists, except for the cytokine and chemokine gene list, which was self-generated; the pro-fibrotic fibroblast and inflammatory fibroblast gene lists, which were retrieved as per a study by Ledoult et al27 (gene lists named ‘TGFß1_upg’ and ‘TNFα_upg’); and the fibroblast-derived collagen gene list, which was retrieved as per a study by Nissen et al.28 Heatmaps were generated with pheatmap R package (.1.0.12). RNAseq counts were VST (variance stabilizing transformation)-normalized and row mean centered (z-score). Columns represent individual mice and rows represent either the z-score calculated for a specific gene or the mean expression (z-score) across all genes in each gene list, as indicated in the figure legends. Gene Ontology analysis for biological processes were performed using The Gene Ontology Resource software (http://geneontology.org/) using differently upregulated genes as an input. The most specific subclasses according to hierarchy were selected and classified as immune or non-immune processes. Immune-related processes were further classified into 10 different categories: T cell, antigen presentation, cytotoxicity, cytokine, leukocyte, B cell, chemotaxis, innate, neuroimmunity or pathogen immunity-related processes.

ImmuCC

The computational framework of the CIBERSORT analytical tool29 and the developed ImmuCC signature matrix (511 genes, non-tissue specific)30 suitable for the deconvolution of mouse bulk RNAseq data, were used to characterize and quantify 25 immune cell subtypes. For this study, two population schemes (compact and extended) were defined, resulting in the aggregation of some of the 25 immune subpopulations (online supplemental table 4). The signature matrix was available as an online supplemental table in30. The CIBERSORT software source code in R was obtained from the website: https://cibersort.stanford.edu/, after registration and request for access and download.

Cytokine measurements

Tumors were harvested and processed as previously described. A total of 0.1×106 cells were added per well to a 96-well plate and incubated for 48 hours at 37°C in RPMI-1640 with 10% FBS and 1% P/S. Cell culture supernatants, named tumor-conditioned media (TCM), were harvested. The concentration of TGFβ1 in TCM was quantified using Bio-Plex Pro TGF-β1 Set (Bio-Rad), following manufacturer’s instruction. Samples were acquired on Bio-Plex 200 system and analyzed with Bio-Plex Manager V.6.

Assays with TCM

The spleen of a tumor-free, untreated mouse was harvested and processed as previously described. 100 uL of a cell suspension containing 0.1×106 CFSE-labeled splenocytes were added per well to a 96-well plate. 100 uL of TCM were added per well. Proliferation was stimulated by adding Dynabeads Mouse T-Activator CD3/CD28 (Gibco, 1:1 bead-to-cell ratio). After a 48 hour-incubation, cells were harvested and stained with Zombie Aqua and CD3-AF700 for flow cytometric analysis. Proliferation index was calculated using FlowJo V.10.6.1 (Tree Star). 100 uL of a cell suspension containing 0.1×106 BMDM polarized towards an M2-like phenotype were added per well to a 96-well plate. 100 uL of TCM were added per well. After a 24 hour-incubation, cells were harvested and stained with F4/80-FITC, CD11b-Pacific Blue, MR-PE-Cy7, Arg1-PE and PD-L1-APC for cytometric analysis.

Data representation

Data was visualized using GraphPad Prism (V.8) or ggplot2 R package (V.3.3.5).

Statistical analysis

Analysis of tumor growth curves was performed using TumGrowth software31 (https://kroemerlab.shinyapps.io/TumGrowth) with default settings and with Bonferroni adjustment for correction for multiple comparison. Linear regression was performed with stats package (V.3.6.2) in R (V.4.0.3) and R Studio (V.1.2.5001). Statistical analyses for the comparison between treatments groups for tumor volume and tumor weight at endpoint, ELISpot responses, flow cytometry analyses, RT-qPCR analyses and RNAseq-derived normalized read counts were performed by an unpaired, two-tailed t test using the rstatix R package (V.0.7.0). Analyses were performed with R (V.4.0.3) and R Studio (V.1.2.5001) software. *p<0.05; **p<0.01; ***p<0.001.

ResultsVaccination 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.

Figure 1Figure 1Figure 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).

Figure 2Figure 2Figure 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).

Figure 3Figure 3Figure 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 CD26lo Ly6Clo CAFs (figure 4H and online supplemental figure 5I), which was followed by a respective trend towards a decrease in the percentage of CD26hi Ly6Chi 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 CD26lo Ly6Clo CAFs (figure 4K) on vaccination. However, the TGFβ vaccine resulted in a significant reduction in αSMA expression in the CD26hi Ly6Chi CAF population (figure 4L).

Figure 4Figure 4Figure 4

TGFβ vaccine reduces the percentage of cancer-associated fibroblasts (CAF) and the expression of Acta2 (

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