Background Ovarian cancer (OC), a highly lethal cancer in women, has a 48% 5-year overall survival rate. Prior studies link the presence of IL-17 and Th17 T cells in the tumor microenvironment to improved survival in OC patients. To determine if Th17-inducing vaccines are therapeutically effective in OC, we created a murine model of Th17-inducing dendritic cell (DC) (Th17-DC) vaccination generated by stimulating IL-15 while blocking p38 MAPK in bone marrow-derived DCs, followed by antigen pulsing.
Methods ID8 tumor cells were injected intraperitoneally into mice. Mice were treated with Th17-DC or conventional DC (cDC) vaccine alone or with immune checkpoint blockade (ICB). Systemic immunity, tumor associated immunity, tumor size and survival were examined using a variety of experimental strategies.
Results Th17-DC vaccines increased Th17 T cells in the tumor microenvironment, reshaped the myeloid microenvironment, and improved mouse survival compared with cDC vaccines. ICB had limited efficacy in OC, but Th17-inducing DC vaccination sensitized it to anti-PD-1 ICB, resulting in durable progression-free survival by overcoming IL-10-mediated resistance. Th17-DC vaccine efficacy, alone or with ICB, was mediated by CD4 T cells, but not CD8 T cells.
Conclusions These findings emphasize using biologically relevant immune modifiers, like Th17-DC vaccines, in OC treatment to reshape the tumor microenvironment and enhance clinical responses to ICB therapy.
- CD4-Positive T-Lymphocytes
- Tumor Escape
- Immunotherapy, Active
Data availability statement
All data relevant to the study are included in the article or uploaded as online supplemental information.
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/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Increased levels of Th17 T cells and IL-17 cytokine are associated with improved survival in ovarian cancer. Dendritic cells (DCs) treated with IL-15 and p38 MAPK inhibitor stimulate IL-17 release from T cells in culture.
WHAT THIS STUDY ADDS
DC vaccines treated with IL-15 and p38 MAPK inhibitor are more effective than standard DC vaccines at generating antigen-specific Th17 T cells in vivo. Th17-inducing DC vaccines block ovarian cancer progression. Th17-inducing vaccines overcome IL-10-mediated resistance of ovarian cancer to anti-PD-1 immune checkpoint blockade therapy.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
The inclusion of Th17-DC vaccines during treatment with anti-PD-(L)1-based (eg, pembrolizumab, nivolumab) may lead to improved progression-free and overall survival in patients with advanced ovarian cancer.
Ovarian cancer (OC) causes ~14 000 deaths each year in the USA.1 While there have been advances in treatment, cure rates remain low.2 Outcomes in OC are heterogeneous and standard clinical/pathologic characteristics have limited prognostic power, suggesting that other host or environmental characteristics also contribute to survival.3 Many prior studies have shown the importance of the immune system in patient outcome.4 Despite extensive efforts, however, immunotherapies have been largely effective and none are approved for front-line therapy.
Several studies published over the last several years suggest that targeting Th17 T cell immunity may be an effective approach to immunotherapy that could result in more cures and longer survival in OC. For example, Krycek et al, found that increased IL-17 in ascites assoicates with increased overall survival in OC.5 Lan et al subsequently demonstrated association of Th17 T cells with improved recurrence-free survival (RFS).6 Higher levels of Th17 T cells are also associated with reduced regulatory T cells.5 7
In preclinical work, we have found that maturation of dendritic cells (DCs) with combiantion IL-15 costimulation and p38 MAPK inhibition, but not either agent alone, induces a DC phenotype that stimulates Th17 responses in vitro.8 9 The results of this study led us to hypothesize that this approach may be able to be used in vivo to drive antigen-specific T cell immunity, including Th17 T cells, that may be effective at preventing OC disease progression. We tested this hypothesis in a murine model of OC, evaluating the breadth and durability of the immune response, whether the Th17-inducing DC (Th17-DC) vaccine is therapeutically superior to conventional DCs (cDCs), and lastly if the PD-L1/PD-1 axis regulates its in vivo activity.
Materials and methods
Female C57BL/6J (B/6J) and STOCK IL17atm1.1(icre)Stck/J (Il17acre) mice aged 6–8 weeks old were from the Jackson Laboratory.
OC cell lines
ID8 tumor cells were obtained from Dr. K. Roby (University of Kansas, Lawrence, Kansas, USA). For biomarker purposes, the ID8 cell line was engineered to express SP17, a cancer-testis antigen overexpressed in OC (online supplemental materials and methods).10 For antigen pulsing of DC cultures, lysate from ID8-SP17 cells was prepared by resuspending in phospate buffered saline (PBS) and subjecting it to freeze/thaw cycles.
For assessment of p38 MAPK activity, DCs were harvested, sonicated in lysis buffer (Cell Signaling Technology) and proteins were denatured for 10 min at resolved on BOLT Bis-Tris gels (ThermoFisher), followed by transfer to PVDF membranes using iBlot and blocked for 1 hour at RT using Li-Cor Odyssey buffer. Blots were incubated overnight with a primary antibody against phospho-ATF-2(Thr71) (Cell Signaling Technology, # 9221) and anti-β-Actin (Li-Cor Bioscience, #92642212). Following washes, blots were incubated with the proper dye-labeled secondary antibodies for 1 hour in Odyssey buffer. Membranes were washed and imaged using the Li-Cor Odyssey DLx.
Generation of DC vaccines
Bone marrow cells were obtained from C57BL/6 J mice as previously described.11 Cells were resuspended in RPMI with 10% fetal bovine serum (FBS) media containing murine granulocyte macrophage colony stimulating factor (GM-CSF, 50 ng/mL, R&D systems, # 415-ML-050/CF), IL-4 (20 ng/mL, R&D systems, # 404-ML-100/CF) and with/without IL-15 (10 ng/mL, R&D systems and #447-ML-010/CF) and p38 MAPK inhibitor SB203580 (1.5 µM, Sigma Aldrich, # S8307). Cells matured in absence or presence of p38/SB203580 are referred here as cDC and TH17-DC, respectively. On days 2 and 6, a half volume of culture media was replenished with fresh cytokine-supplemented complete media. On day 7, DCs were pulsed with 1:10 dilution of ID8-SP17 tumor cell lysate, followed by stimulation with LPS (200 ng/mL) for 16 hours. The cells were removed by gentle scraping, washed twice with PBS, and resuspended in PBS for immunization of mice.
Peritoneal carcinomatosis, immunizations and antibody blockade
To establish peritoneal carcinomatosis, ID8-SP17 tumor cells (4×106 cells) were injected i.p. in PBS. Immunization with DC vaccines (cDC and TH17-DC) was done s.c./i.p. at a dose of 1×106 cells in 100 ml volume for varying weeks, which depended on the experiment (online supplemental figure S1). Priming was done with using s.c. injections every week and boosters were given i.p. every 2 weeks for a maximum of 10 weeks. Vaccinations were started 1 week after tumor inoculation. For T cell depletion studies, anti-CD4 antibody (GK1.5) or anti-CD8 antibody (53.6.7) was given at 0.5 mg/dose i.p. in parallel with the DC vaccines. Immune checkpoint blockade (ICB) was achieved by i.p. injection of 200 μg G4 clone PD-1 monoclonal antibody (Mayo Clinic Antibody Hybridoma Core) or 200 μg hamster IgG (Jackson ImmunoResearch) twice a week for 10 weeks.
Cells were collected, washed three times in PBS, and resuspended in FACS buffer (1X PBS+0.5% BSA+0.2 mM EDTA). The fixable viability stain (FVS520 or FVS570, BD Biosciences) was added for 20 min, cells washed with PBS, and then blocked for 10 min at 4°C. Fluorophore-conjugated cell surface antibodies (online supplemental materials and methods) were added to the samples and incubated for 30 min on ice. After washing, samples were run on an Attune NxT flow cytometer (ThermoFisher) and the data analysis was performed using Flow Jo software (FlowJo V.10.6.2). A similar number of events, usually 200,000–400,000, were collected.
Cytokine network analysis
For cytokine analysis, supernatants were harvested from DC cultures or 72 hours post co-culture of cDCs with in vivo primed T cells. T cells primed in vivo were obtained at day 42 from tumor-bearing mice immunized with either PBS, cDCs, or Th17-DCs. Spleens were collected in RPMI medium, ground into single cell suspensions via a 70 uM strainer, ACK (Ammonium-Chloride-Potassium) lysis performed for 1 min, washed and resuspended in PBS. CD4+T cells were collected with the CD4+isolation kit for AutoMacs (Miltenyi Biotech) and co-cultured with cDCs pulsed with antigen in 1:1 ratio in complete RPMI media for 24–72 hours depending on endpoint assay. Supernatants were centrifuged and profiled using the proteome profile mouse XL cytokine array kit (R&D Systems, # ARY028).
ELISA kits used to detect various cytokines (online supplemental materials and methods) were bought from ThermoFisher Scientific. Culture media samples were added neat while serum from blood or ascites were added at 1:10 dilution with an overnight incubation at 4°C. All wash steps were conducted with PBS containing 0.05% Tween 20 using an AquaMax 4000 plate washer. Absorbance was read at 450 nm on a plate reader and values were subsequently converted into a cytokine concentration based on the standard curve run for each kit.
Standard colorimetric ELISpot assays were performed to quantitate antigen-specific T cells with mouse IFN-γ (Mabtech, # 3321-2H), IL-4 (Mabtech, # 3311-2H), and IL-17 (R&D Systems # EL421) kits. Splenocytes were plated at 0.5×105 cells per well in triplicate in 96-well round bottom plates with/without tumor cell lysate and incubated at 37°C, 5% CO2 for 24 hours. Cells were transferred to ELISpot plates that were precoated with anti-IFN-γ, anti-IL-4, anti-IL-17 capture antibodies and that were blocked with complete medium. After cell addition, plates were incubated for 24 hours, washed, and incubated with biotinylated detection antibodies and streptavidin-alkaline phosphatase conjugates. After washing, BCIP/NBT chromogen was added and incubated in the dark, followed by a water rinse. All plates were dried and spots were read using an Advanced Imaging Devices (AID) ELISpot software and reader.
High avidity antibody ELISAs
Flat-bottom polystyrene 96-well plates were coated overnight at 4°C with PBS containing 10 µg/mL of ID8 tumor cell lysate. Mouse IgG (Sigma Aldrich) was used as a standard curve over a concentration range of 0.195–1000 ng/well. All wash steps were conducted with PBS containing 0.05% Tween 20. Wells were blocked with PBS+1% BSA and, after washing, mouse serum was added to the plate at a 1:25 dilution in triplicate and incubated at 37°C for 1 hour. The plates were washed, and the wells were treated with 6M urea or wash buffer for 30 min at 37°C. After washing, goat anti-mouse IgG HRP (Santa Cruz Biotechnology) was added at 1:2000 and incubated for 1 hour at 37°C. After a final wash, each well was incubated with 100 µL TMB substrate (BD Biosciences, # 555214). Color development was stopped with HCl. Absorbance was read at 450 nm on a plate reader. Wells that were not coated with the tumor cell lysate were used to subtract the background from the mouse serum samples. These values were subsequently converted into an antibody concentration using the IgG standard curve.
Immunohistochemistry of ascites, peritoneal cavity cells and tumor tissues
Ascites cells were purified from ascites fluid on a discontinuous Ficoll gradient. Peritoneal cavity cells derived from mice without ascites were obtained by flushing the peritoneal cavity of euthanized mice. Both ascites and peritoneal cells were washed in PBS, treated with ammonium-chloride-potassium treatment, and fixed in formalin (4% formaldehyde). Fixed cells were resuspended in Epredia Histogel (Fisher Scientific, # HG-4000-012) and air dried. The gel was transferred to a cassette and stained for various proteins (online supplemental materials and methods). The stained sections were scanned in an AT2 slide scanner (Leica Biosystems) and analyzed using eSlide Manager Software (Leica Biosystems, version # V.22.214.171.12408). For tumor tissues, mice were euthanized, and the peritoneum was opened to remove tumor tissues found lining the organs in the abdomen namely the intestines, spleen, and other regions, fixed and stained.
All statistical tests were two sided with a p<0.05 being considered as statistically significant. The statistical tests were calculated using GraphPad Prism V.8 and are noted in each figure.
IL-15 costimulation and p38 MAPK blockade selectively upregulates expression of MHC class II and IFN-γ while suppressing IL-10 and IL-12 production
SBA203580 (adezmapimod), a selective inhibitor, was used to block p38 MAPK.12 Initial studies verified effectiveness in DCs demonstrating abolition of phosphorylation of p38 MAPK target, activating transcription factor 2 (ATF-2) with little toxicity and improved recovery of DCs relative to conventional methods (figure 1A,B).13 The addition of p38 MAPK inhibitor and IL-15 selectively upregulated both the numbers of major histocompatibility complex (MHC) class II+ DCs as well as the magnitude of MHC class II expression (figure 1C,D, online supplemental figure S2). The impact on cell surface expression of T cell activators was specific with no significant increases in MHC I, CD80, CD86 and OX40L expression (figure 1C,D, online supplemental figure S2). Exposure to tumor antigen had no impact on expression of T cell activators (figure 1C,D, online supplemental figure S2). Similarly, IL-15 and p38 MAPK inhibitor demonstrated a selective cytokine production response. Both IL-10 (Treg inducer) and IL-12 (Th17 inhibitor) release were suppressed while IFN-γ release was elevated (figure 1E–G). Levels of inducers of Th17 T cell responses,14 IL-6, IL-1β and TGF-β were maintained at high levels and not impacted by treatment with IL-15 and p38 MAPK inhibitor (figure 1H–J). Additional qualitative proteomic analysis of myeloid-produced cytokines showed extensive similarity between cDC maturation and DCs matured in the presence p38-MAPK inhibition and IL-15 co-stimulation with notable exceptions (online supplemental figure S3 and table S1). Specifically, in the presence of p38-MAPK inhibitor and IL-15, B cell regulators, BAFF and IL-13 were both significantly elevated (figure 1K–L,). In contrast, the Treg chemoattractant, LIX (CXCL5) was suppressed (figure 1M).15 Blockade of p38 MAPK and co-stimulation with IL-15 modestly increased phagocytosis (online supplemental figure S4). Lastly, these differences were not due to an increased viability of DCs generated with IL-15 and p38-MAPK inhibitor (online supplemental figure S5). Overall, the combination of IL-15 and p38-MAPK inhibitor induces a phenotype of DCs that favors activation of Th17 T cells and B cells rather than Tregs.
IL-15 costimulation and p38-MAPK blockade specifically empowers DC vaccines to generate IL-17+ T cells and high affinity antibodies in vivo with negligible impact on generation of IFN-γ+or IL-4+ T cells.
To determine the ability of Th17-DC to generate Th17 T cells in vivo, DCs were either pulsed with tumor antigen or left unpulsed. ID8-SP17 tumor-bearing mice were vaccinated and IFN-γ+, IL-4+ and IL-17+ T cells were measured 2 weeks after completion of 4 weekly immunizations (online supplemental figure S1A). Both antigen-pulsed cDCs and Th17-DCs led to significant increases in the numbers of antigen-specific IFN-γ+ and IL-4+ T cells which was not evident when antigen was omitted (figure 2A,B). Consistent with expectations, only antigen-pulsed Th17-DCs reliably led to the generation of tumor antigen-specific IL-17+ T cell immunity (figure 2C). Vaccine-induced tumor antigen-specific antibodies were strongly induced in the serum, but the levels were not different between cDCs and Th17-DC vaccines (figure 2D). However, assay of antibodies under strong dissociation conditions (ie, 6M urea) revealed that Th17-DC vaccination induces a higher avidity antibody repertoire in many, but not all, of the mice (figure 2E). Overall, these results suggest that IL-15 co-stimulation and p38 MAPK blockade leads to highly selective modifications of DCs that specifically resulted in the activation of antigen-specific Th17 T cells and increased antibody avidity in some mice with negligible impact on Th1 and Th2 T cell frequencies.
IL-15 costimulation and p38-MAPK blockade specifically empowers DC vaccines to rapidly generate antigen-specific Th17 T cells
To determine if Th17-inducing DCs directly induce IL-17+ T cells, in vitro experiments were done using Th17-DC to stimulate splenocytes derived from naïve mice. As shown in figure 3A,B, Th17-DCs rapidly generated antigen-specific IL-17+ T cells within 72 hours from freshly prepared splenocytes. To ascertain that the antigen-specific IL-17+ T cells generated in vivo were CD4 T cells, splenocytes were harvested from immunized mice (online supplemental figure S1B) and CD4 T cells were purified and stimulated with antigen ex vivo. As demonstrated in figure 3C,D, CD4 T cells fractionated from splenocytes of immunized mice were highly enriched in antigen-specific Th17 T cells. Further analysis showed that Th17-DC vaccination specifically led to high levels of circulating IL-17 but not IL-10 in tumor-bearing animals (figure 3E), demonstrating that the vaccination likely did not result in Treg17 or rTh17 cells as has previously been reported.16 Lastly, to confirm that CD4 T cells were the source of IL-17 in vivo, mice were immunized as with antigen-pulsed Th17-DCs with or without CD4 or CD8 T cell depletion. As shown in figure 3F, depletion of CD4, but not CD8, T cells eliminated IL-17 release. Unexpectedly, we also found that elimination of CD8 T cells resulted in significantly elevated levels of IL-17, suggesting that treatment with CD8-depleting antibody modulates CD4 T cell levels or Th17 T cell induction activity in vivo. Alternatively, CD8 T cells may naturally dampen Th17 T cell responses, which was not addressed in this study. Nonetheless, these results demonstrate that IL-15 costimulation and p38 blockade in DCs results in rapid priming and expansion of CD4+ Th17 and not CD8+ Tc17 T cells that have been previously reported.17
Th17-DC vaccines induce a unique T cell-mediated cytokine network
To determine if Th17-DC-induced T cell immunity was associated with a unique cytokine/chemokine network relative to cDCs, proteomic analysis (online supplemental table S1) was performed on media derived from co-cultures of tumor-antigen-loaded cDCs (not Th17-DCs) incubated with purified CD4 T cells from mice immunized four times with either PBS, cDCs or Th17-DCs. As shown in figure 4, Th17-DC vaccine induced CD4 T cells capable of stimulating elevated levels of various myeloid cell modulating cytokines/chemokines such as CCL6, CX3CL1, CXCL16, GM-CSF, ICAM-1, IL-1a, LIX, osteoprotegerin and RBP-4, when compared with T cells from cDC vaccination (adjusted p<0.05). Thus, vaccination with Th17-DC vaccines leads to a distinct cytokine network following interaction of vaccine elicited T cells with antigen-presenting cells.
Th17-inducing DC vaccines eliminate circulating tumor cells in the peritoneal cavity
OC is unique among other cancers in that metastasis is mediated through exfoliation and intraperitoneal accumulation of tumor cells which can subsequently seed the omentum, the peritoneum and abdominal organs.18 To determine if Th17-DC vaccines can impact accumulation of tumor cells, peritoneal tumor burden was analyzed with Ki-67 and Sp17 IHC staining of peritoneal cavity-derived exfoliated cells following tumor challenge (online supplemental figure S1A). Figure 5A,B shows representative images and quantitative analyses of Ki-67 (inset graph) and Sp17 tumor marker (inset graph) staining in peritoneal cavity cells. The most significant observation was the suppression of formation of large tumor cell clusters in the mice immunized with antigen-pulsed vaccine. While the clusters were abundant in control animals, they were not often observed in samples from vaccinated mice. The numbers of positive Ki-67 or Sp17 staining (both in clusters and in single cells) were sharply decreased in animals immunized with either antigen-pulsed cDCs or antigen-pulsed Th17-DCs. Th17-DCs, however, were superior to cDC vaccines using both assessments. Thus, Th17-DC vaccines can reduce OC-associated carcinomatosis and are superior to cDCs.
Vaccination induces infiltration of T cells into solid tumor tissue and extends the lifespan of mice bearing OC
To assess the impact of Th17-inducing DC vaccines on survival, tumor-bearing mice were immunized as above and followed until moribund (online supplemental figure S1C). Tumor-bearing mice in the PBS-, unpulsed cDC-vaccinated and unpulsed Th17-DC-vaccinated groups succumbed to disease at days 60–80. However, mice immunized with either antigen-pulsed cDCs or antigen-pulsed Th17-DC displayed prolonged survival (p<0.0001) (figure 6A). Antigen-pulsed Th17-DCs were superior to antigen-pulsed cDC vaccines, with a median survival of 148 days as compared with 84 days (p<0.01). The survival advantage imparted by Th17-DC vaccination was reversed by depletion of CD4 T cells but not depletion of CD8 T cells (figure 6B). Ascites fluid derived from moribund animals showed accumulation of IL-17, suggesting that outgrowth was due to evasion strategies and not loss of immunity (figure 6C). Furthermore, tumor tissue in the peritoneal cavity at the time when Th17-DC vaccinated mice were moribund revealed strong infiltration of CD4 T cells but sparse CD8 T cell infiltration with either cDCs or Th17-DCs (figure 6D–G). Analysis of purified splenic CD4 T cells following vaccination with Th17-DCs and PBS demonstrated that a significant fraction produced Granzyme B (online supplemental figure S6). Additionally, tumors expressed MHC class II (online supplemental figure S7). Although levels of antigen-specific antibodies were higher in blood on death in the Th17-DC vaccinated mice, as compared with those vaccinated with cDCs, similar levels were observed in the ascites (figure 6H). The increased levels of antibody in the blood may have been due to the differential collection times. By the time animals were moribund, the avidities of the antibody repertoire were similar among those treated with cDCs and Th17-DCs (online supplemental figure S8). As shown in figure 6I (online supplemental figure S9), elimination of IL-17A had no impact on vaccine efficacy, suggesting that other cytokines or mediators released by Th17 T cells are more important. Overall, Th17-DC vaccination induces durable tumor-infiltrating immunity and improves survival over cDC vaccination in a CD4 T cell-dependent and IL-17-independent and CD8 T cell-independent manner. However, the mice succumb due to development of a yet unknown immune escape mechanism.
The PD-1/PD-L1 immunoregulatory axis limits the antitumor activity of Th17-DC vaccination
While Th17-DC vaccination can significantly improve survival, tumor-bearing mice succumbed to disease, likely due to immune editing. Prior work from our group and others suggests that the PD-1/PD-L1 axis is a potentially important immune regulatory pathway in OC, despite the observations that specifically targeting the pathway with ICB antibodies in the clinic has had limited results.11 19 20 It was questioned whether this axis regulated the activity of the vaccination by combining anti-PD-1 injections with Th17-DC vaccination, based on the following two observations. First, it was observed that ID8-SP17 cells express PD-L1 in vivo, including in both control animals and those treated with cDC and Th17-DC (figure 7A). The second observation was that CD4 T cells, but not CD8 T cells, derived from the peritoneal cavity of Th17-DC vaccinated mice demonstrated increased PD-1 expression following vaccination as compared with T cells derived from control animals (figure 7B). Figure 7C shows that anti-PD-1 therapy alone has little activity with only a marginal, although statistically significant (78 days in the PD-1 group vs 58 days in the PBS group, p=0.003), increase in survival relative to no treatment, which is consistent with our prior observations.11 20 In contrast, the combination of vaccine with anti-PD-1 doubled the median survival (p=0.02) (figure 7C). The synergistic activity of vaccination combination with anti-PD-1, like with vaccine monotherapy, was reversed by the elimination of CD4 T cells (online supplemental figure S10). One explanation for the augmented survival observed with the combination treatment is that it increased the number of antigen-specific effectors induced by vaccine. ELISpot analysis, however, revealed that the T cell immune responses were similar or slightly reduced (eg, Th1) by anti-PD-1 treatment (figure 7D). Similarly, the inclusion of anti-PD-1 did not alter levels or avidity of Th17-DC-induced antibodies (figure 7E, online supplemental figure S11). However, in vitro cytokine network analysis showed that the inclusion of anti-PD-1 along suppressed production of myeloid modulators CCL2, CCL3, CCL22, CXCL1 and Serpin E1 release suggesting that anti-PD-1 may impact the tumor-associated myeloid microenvironment (online supplemental figure S12).
Blockade of the PD-1/PD-L1 pathway selectively boosts myeloid immunity in the tumor microenvironment
Treatment of tumor-bearing mice with vaccine or combination therapy led to measurable shifts in cell number and immune cell infiltration into the peritoneal cavity at day 42 following four vaccinations, as shown in figure 7F–J. Vaccination was needed to induce infiltration into the peritoneal cavity as anti-PD-1 had negligible effect on the number of immune cells harvested from the peritoneum (figure 7F). In both control (PBS) and anti-PD-1-treated mice, lymphocytes were the dominant immune effector population (~50%–60%) followed by myeloid cells (DCs and macrophages, ~20%) and a small granulocyte fraction (~5%–10%) (figure 7G). Th17-DC vaccination markedly shifted the distribution, reducing the proportion of lymphocytes (~20%) and increasing monocyte and granulocyte fractions to ~50% and 20%, respectively. The inclusion of anti-PD-1 with vaccine further reduced the lymphocyte fraction to ~10% and markedly increased granulocytes to ~40%–45%.
Finer analysis of the lymphocyte fraction showed that the native (ie, control mice) lymphocyte response consisted of B cell infiltration (~60%), while CD4 (15%–20%) and CD8 (~10%) T cells constituted a small fraction. Treatment of mice with single agent anti-PD-1 slightly increased the fraction of B cells with minimal effect on the proportion of T cells (either total or activated) (figure 7H). Treatment of mice with Th17-DC vaccine significantly increased the proportion of total and activated CD4 T cells and reduced the proportion of B cells. It is notable that the inclusion of anti-PD-1 along with vaccine did not alter this distribution. Proportions of CD8 T and NK cells, which was a very minor fraction of the lymphocytes, were not affected by vaccination or anti-PD-1 treatment, although there was a low-level increase in numbers of activated CD8 T cells as assessed by CD69 expression. Thus, the major impact of vaccination overall, with or without anti-PD-1 therapy, was to augment myeloid immunity and shift lymphocyte immunity in favor of a CD4 T cell response and reduced B cell infiltration.
Given the observation that Th17-DC vaccination significantly induced myeloid cell infiltration, added analyses were conducted, evaluating the monocyte gate by flow cytometry. In the absence of treatment, monocytes, which represented about 20% of the peritoneal infiltrate (figure 7G), were composed of DCs (~25%–30%) and macrophages (~60%) with smaller components of myeloids derived suppressor cells (MDSCs, ~3%–4%) and CD19+ putative plasma or activated B cells (~15%) (figure 7I). Th17-DC vaccination alone or combined with anti-PD-1 significantly reduced the proportion of DCs (~5%), MDSCs (<1%) and CD19+plasma cells (~2%–3%) while the proportion of macrophages significantly increased to ~90%–100% of the total monocytes. While granulocytes were a minor fraction in control and anti-PD-1 monotherapy arms (<10%), vaccination alone or without anti-PD-1 results in a significant upregulation, particularly in the combination treatment group where granulocytes represented ~45% of the total immune cell infiltrate (figure 7G). Subsequent analysis of the granulocyte population revealed that the dominant cell type was eosinophils identified at Siglec-F+, CD193+ granulocyte, whereas Ly6G+ neutrophils and FceR1a+, CD200R3+ basophils were minor components (figure 7J). Overall, these results demonstrate that Th17-DC vaccines restructure the tumor immune microenvironment, favoring myeloid infiltration, which is further modified by the inclusion of anti-PD-1 during vaccination to boost eosinophil infiltration.
In prior studies, we found that IL-10 production is strongly induced in DCs within the microenvironment on administration of single agent anti-PD-1, thus sustaining local immune suppression of T cell responses.11 In that study, blockade of IL-10 signaling with anti-IL-10 and anti-IL-10 receptor antibodies along with anti-PD-1 led to improved survival, demonstrating that IL-10 release is a major mediator of adaptive resistance following anti-PD-1 treatment. Based on these findings and our observations that mice progressed despite therapy, we asked if IL-10 was present in the tumor microenvironment (ie, ascites) when animals were moribund. Consistent with the prior report, IL-10 was highly elevated in the ascites in mice that received only anti-PD-1 antibody but not when Th17-DC vaccine was given in combination (figure 7K). Thus, Th17-DC vaccination overcame IL-10-mediated resistance to anti-PD-1 therapy in the peritoneal cavity.
Although their involvement in OC requires further study, Th17 T cells have consistently been found to be associated with improved OC survival or beneficial pathology (eg, reduced Tregs).5 6 Thus, there is an increasing appreciation that therapeutic strategies that drive Th17 T cell immunity may benefit patients. We present evidence in this paper that specific induction of Th17 T cell immunity (along with Th1, Th2 and antibody) with Th17-inducing DC vaccination affords better protection against OC progression as compared with cDC approaches which drive a more restricted response lacking Th17 T cell immunity. We also present evidence that the PD-1/PD-L1 axis limits effectiveness of Th17-inducing vaccines and that blocking this pathway during vaccination improves survival, opening potential clinical pathways to combination therapy.
In parallel with this study, we also conducted and reported on a limited phase I trial testing immunogenicity of tumor antigen-loaded DC vaccines generated with p38 MAPK inhibitor and IL-15 costimulation in stage IIIC and IV OC patients in first remission following standard of care treatment.9 Although that study did not compare cDCs with Th17-DC, we found that vaccination was safe and all patients demonstrated coordinated multifunctional immunity including Th17 T cell immunity as well as Th1 and humoral immunity. While immunogenicity was the primary outcome objective, we observed scondarily that 39% (7/18) remained recurrence-free at the time of data censoring, with a median follow-up of 49.2 months, an RFS far superior to historical controls (<10%). The magnitude of the immune responses correlated significantly with improved RFS.
One key finding in this study was that CD4 T cells appeared to be the primary T cell mediator of antitumor immunity. It was expected that CD8 T cell immunity would be robustly activated based on prior studies that Th17 T cells stimulated cytotoxic T cell immunity.8 Despite that, we found that CD8 T cell depletion did not impact survival and little evidence of infiltration of CD8 T cells into the tumor following treatment. Similarly, in our parallel clinical trial we found that the vaccine did not induce CD8 T cell infiltration and that preexisting CD8 T cell infiltration was not associated with RFS. While CD4 T cells are often referred to as helper T cells, studies over the past two decades show that this subset of T cells has extensive plasticity and is central to immune responses for disease protection, either directly or by activating other innate and antigen-specific immune effectors. CD4 T cells are activated by either antigen-presenting cells or directly by MHC class II-expressing tumors. OC is unique among most other tumors in that it has high level MHC class II expression with up to 300,000 HLA-DR molecules per cell.21 The enhanced infiltration of CD4 T cells into the peritoneal cavity and tumor bed following vaccination suggests that vaccine-induced CD4 T cells might directly kill tumor cells. Indeed, prior work has shown that CD4 T cells can display direct tumor cell killing though ligation of death-inducing receptors (eg, through FAS or TRAIL death receptors) or the elaboration of toxic secretions (eg, granzyme and perforin) which can induce apoptosis and cell death.22–24 The finding that Th17-inducing vaccine induces granzyme-B secreting CD4 T cells supports a direct cytotoxic mechanism of Th17-inducing DC vaccines. CD4 T cells, however, are also known to produce cytokines which recruit and activate tumor macrophages to produce nitric oxide, super-oxide, and death ligands both of which can result in tumor killing, along with classical antibody dependent cellular phagocytosis.25 Subsets of CD4 T cells recruit and activate eosinophils that are also able to produce antitumor factors and kill via antibody dependent cellular cytotoxicity (ADCC).26 Mechanisms involving indirect myeloid cell killing are also supported by the present findings which show that Th17-inducing DC vaccine is associated with augmented antibody immunity. Interestingly, in our clinical trial we also found that Th17-DC vaccination in human generates cytotoxic antibodies capable of driving ADCC.9 These increased antibody responses could be due to the increased levels of BAFF and IL-13 produced specifically by the Th17-DC, both of which are known to increase expression of activation-induced deaminase which drives high affinity antibody responses.27 28 Given its dependence of CD4 T cells, Th17-DC vaccine may have important utility for the ~50% of OC patients that demonstrate loss of class I or TAP29 30 or who have age-related declines in CD8 T cell function.31–33
One key finding in this study was that the immunity driven by Th17 vaccine is regulated the PD-1/PD-L1 pathway and the inclusion of anti-PD-1 ICB led to long-term survival, even out to a year in some mice. Based on our prior work with peptide vaccines, we would have expected that blockade of PD-1 would have resulted in an increased priming to vaccine and increased activation of tumor associated T cells; however, this appeared not the be the case with Th17-DC vaccine.34 Rather, the major impact of PD-1 blockade was a strong induction of eosinophil infiltration. With combined treatment, granulocytes, of which eosinophils were dominant, represented about 40%–50% of the total peritoneal infiltrate, whereas they were a minor component of the infiltrate in mice treated with vaccine only. Eosinophils can eliminate tumor cells using both indirect and direct mechanisms.35 Indirect mechanisms include generation of inflammatory mediators which may further activate other immune cells in the tumor. Direct mechanisms include release of toxic granules, reactive oxygen intermediates and nitric oxide. While, the antitumor activity of eosinophils in the context of Th17-inducing vaccine and its regulation by the PD-1/PD-L1 axis require further study, emerging data suggest involvement of eosinophils in the efficacy of ICB therapy in patients with cancer.36
We had previously found in the same murine model that the effectiveness of ICB monotherapy is limited due to high level induction of myeloid-derived IL-10, a suppressor of T cell immunity.11 In this study, we show that vaccination with Th17-DC vaccines extensively modified the tumor microenvironment to eliminate myeloid-mediated IL-10 adaptive resistance. Similar findings have been observed in other vaccine model systems. For example, Baharom et al found that vaccination is effective in preventing the outgrowth of MC38 colon cancers by reducing tumor-induced Chil3+ + which have an anti-inflammatory gene expression signature associated with wound healing.37 Based on our findings, we have recently developed a phase II clinical trial evaluating the combination of vaccine with pembrolizumab in OC patients with early biochemical relapse following standard of care treatment, which has just started accruing (NCT05920798).
One unexpected finding from our study is that knockout of IL-17A did not reverse the effect of Th17-inducing vaccines. IL-17A has long been linked to increased pathogenesis in many cancers and its association with improved survival in OC has been puzzling.38 The finding of lack of activity of IL-17A suggests that either Th17 T cells have no role in in OC or that they may contribute to OC disease protection in other ways aside from IL-17A production. With respect to the former, additional studies in other models could be informative, for example, murine models that conditionally knockout Rorc, the gene encoding the RORgt transcription. If Th17 T cells are found to be involved in the antitumor immunity, one could speculate that it is independent of IL-17A. While IL-17A is one of the most abundant products of Th17 T cells, other cytokines and effector molecules are also highly expressed which may participate in preventing OC progression, including IL-17F, IL-21, IL-22, GM-CSF and granzymes. Indeed, it has become clear recently that IL-17A is dispensable for the pathogenic activity of Th17 in autoimmune disease and may be counterproductive in immunity mediated by Th17. For example, Chong et al recently reported that IL-17A deficiency increases the pathogenicity of Th17 T cells in experimental uveitis. In that system, the investigators observed that IL-17A bound to Th17 T cell IL-17 receptors stimulating IL-24 production which limited the release of critical effector cytokines, IL-17F and GM-CSF.39 In another study, Shibui et al found that IL-17 was dispensable for the B cell activating properties of Th17 T cells.40 These findings challenge the notion that IL-17A is the primary mediator of Th17 T cell activity in OC.
The results of the prior studies and our clinical trial which show that only 40%–50% may benefit from therapy provides for a compelling reason for continuing investigations of Th17-based therapies for OC to identify of biomarkers and improve the understanding of the mechanisms of action of. Analysis of pretreatment specimens from our initial phase I trial showed little difference in the tumor specimens between those who responded and those that recurred.9 In contrast, the ability to generate high levels of systemic immunity (T cell and antibody) were notable defining features. Heterogeneity in the human response to vaccine can be due to several factors, with DCs possible being a source of variation that can be targeted. Our proteomic profiling of surface markers and cytokine release showed that the addition of IL-15 and p38 MAPK inhibitor was selective in modulating protein expression. Out of the 55 proteins that we explored and found to be expressed by the DCs, only seven were significantly modulated by the inclusion of IL-15 and p38 MAPK. MHC class II was significantly upregulated, likely because of strong induction of IFN-γ production during maturation.41 IL-10 was released at (~5–10 fold) lower levels as compared with cDCs. Th17 T cells are known to express the IL-10R and are sensitive to IL-10 preventing their ability to clonally expand during antigenic challenge.42 Similarly, IL-12 was suppressed fivefold. Although IL-12 has been shown to be important in the development of Th1 T cell immunity, our results show little difference between cDC and Th17-DC in induction of Th1 T cell immunity in vivo.43 Rather, the reduced IL-12 may have been a crucial factor in the lack of generation or contribution of CD8 cytotoxic T cells following Th17-DC vaccination.44 In contrast to IL-10, IL-12, and IFN-γ, Th17-DC culture conditions did not impact DC production of Th17 T cell inducing cytokines IL-6, IL-1β, or TGF-β, exemplifying the tight independent control of cytokine production by DCs which enables their known plasticity in regulating adaptive immunity.45 Specific cytokine profiles, which could be readily acquired in the laboratory setting, may be useful in ensuring product quality across DC manufacturers or may be useful in selecting patients more likely to respond to therapy.
Limitations of our study include the use of a single mouse model and a single tumor injection site (ie, intraperitoneal). Testing of this approach in other OC models, such as the Friend Leukemia virus B (FVB) mouse-based spontaneously transformed ovarian surface epithelial (STOSE) cell line model, may provide additional insights into the utility of this immunotherapy strategy.46 Lastly, injection of tumor into other sites such as the ovary or the skin could potentially be useful when considering those rarer cases of patients with advanced OC that have intact ovaries or metastases to other sites such as the skin.
In conclusion, there remains an unmet need for developing novel treatments for OC. The current study provides further insights into the biology of Th17 vaccination and demonstration of its potential as a maintenance therapy or as a co-treatment to improve responsiveness to ICB in patients with OC, a disease where Th17 immunity positively impacts patient survival.
Data availability statement
All data relevant to the study are included in the article or uploaded as online supplemental information.
Patient consent for publication
Animal care and use was in accordance with the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic (Mayo Clinic IACUC # A36215-15-R21).
The authors are grateful for the technical support of the Mayo Clinic Florida Cytometry and Cell Imaging Laboratory. The authors are also grateful for the technical assistance of Emilie Perkerson.
Contributors Conceptualization: KLK, LY, PL, MC and MB. Methodology: LY, PL, BS, JJ and JD. Investigation: KLK, BS, LY, JJ, HS, KZ, DB, SK, KJ, JD, ND and BN: Statistical analysis: KLK and LY. Funding acquisition: KLK, LY, MC and MB. Supervision: KLK. Writing-original draft: KLK, LY, BN and BS. Writing-review and editing: KLK, LY, PL, BN, MC and MB. KLK is responsible for the overall content as guarantor.
Funding This work was funded by NIH/NCI grant P50CA136393 (KLK, MC and MB), R01CA276313 (KLK and MB) and the Marsha Rivkin Foundation (KLK).
Competing interests MC is an inventor on a patent filed by the University of Arkansas, entitled ‘Inhibition of dendritic cell-driven regulatory T cell activation and potentiation of tumor antigen-specific T cell responses by interleukin-15 and MAP kinase inhibitor.’ KLK and MB are inventors on a patent filed by the Mayo Clinic, entitled ‘Dendritic Cell Based Vaccines Combined with Pembrolizumab for the Treatment of Advanced Ovarian Cancer.’
Provenance and peer review Not commissioned; externally peer reviewed.
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