IL-10 release upon PD-1 blockade sustains immunosuppression in ovarian cancer

Animals

Four- to 12-wk-old female C57BL/6J (B/6J) mice, obtained from an in-house breeding program or from The Jackson Laboratory (Bar Harbor, ME), were used for experimentation. Animal care and use was in accordance with the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic.

Cell lines

Leukocytes were cultured in modified RPMI 1640 media as previously described (4). ID8 tumor cells, obtained from Dr. K. Roby, University of Kansas in 2005, were derived from immortalized ovarian epithelial cells generated by repeated passage in culture and were grown in complete DMEM media (4). They were last authenticated as mouse origin by IDEXX BioResearch in 2014. ID8 cells used in the described experiments were used within 12 passages upon receipt.

Tumor implantation

ID8 tumor cells (5 × 10⁶ cells) were injected i.p. at a volume of 500 μl in PBS. Tumor and ascites were harvested from the mice when moribund, usually 50–100 days following challenge. For comparison of tumor burden, the weight of omentum was measured from tumor bearing mice as a surrogate. This was done to reduce variability associated with harvesting diffuse nodules scattered throughout the peritoneal cavity. We found in preliminary studies that the weight of the tumor bearing greater omentum is linearly proportional to the total tumor burden (R²=0.81, p<0.0001, n=56). In order to more accurately represent tumor burden, the weight of omentum of age-matched healthy mice were subtracted from the omentum of the tumor bearing mice.

In vivo PD-1 and IL-10 blockade

Mice were inoculated with ID8 cells and on day 25 received either 200 μg hamster IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) or 200 μg G4 clone PD-1–blocking monoclonal antibody i.p. as described (21, 22). Mice were treated until moribund up to 11 treatments (8 - 11 times up to 7 weeks). IL-10 neutralizing antibody (BioLegend, San Diego, CA) and IL-10R antagonist antibody (BD Pharmingen, San Diego, CA) were injected i.p alternating between the two antibodies at a concentration of 100 μg and 200 μg per mouse, respectively. The same amount of isotype matched antibodies (Rat IgG1κ or Rat IgG1χ) from the respective suppliers was injected in the control groups. Tumor and ascites were harvested as necessary when mice were moribund.

Leukocyte isolation and culture

Leukocytes from ascites of tumor-bearing mice were isolated by discontinuous Ficoll gradient as described previously (23). Tumor-infiltrating leukocytes (TILs) were harvested by mincing the tumor through a 70-μm nylon cell strainer and separation with a discontinuous Ficoll gradient. From single-cell suspensions, DCs were magnetically isolated based on the cells of interest using CD11c microbeads obtained from Miltenyi Biotec (Auburn, CA). Naive mouse leukocytes were obtained from BL/6J mice spleens by grinding the spleen through a 70-μm nylon cell strainer. The splenocytes were centrifuged at 3000 rpm for 10 min and resuspended in 4 ml (Ammonium-Chloride-Potassium) ACK buffer to lyse red blood cells. The remaining cells were then washed and resuspended in culture media. Bone marrow derived dendritic cells (BMDCs) were obtained from BL/6J by flushing femurs and tibias with media and centrifuging cells at 1500 rpm for 10 minutes, followed by ACK treatment. Cells were re-suspended with media containing 10 ng/ml mGM-CSF and 1 ng/ml mIL-4 and plated into 6 well plates. Culture media was changed with fresh media containing 10 ng/ml mGM-CSF and 1ng/ml mIL4 at 48 and 96 hours. The cells were matured with 40 ng/mL TNFα on day 5 of cell culture. After 2 additional days, the cells were washed with PBS, resuspended in fresh media and cytokines (VEGF-A, Genway Biotech, San Diego CA; IL-4, Peprotech, Rocky Hill, NJ); IL-10, Peprotech; IL-12, Peprotech) or control vehicle were added to respective wells.

Flow cytometry

Cell-surface molecule staining and flow cytometry were done essentially as previously described on whole populations and/or enriched populations (21, 22). For flow cytometric analysis, a similar number of events, usually 20,000 – 200,000 were collected for all groups. Antibodies against CD3, CD8, CD11c, F4/80, PD-1, CD45, PD-L1, PD-L2, CD40, CD27, CD69, CD80, and CD86 were purchased from eBioscience (San Diego, CA). Antibodies against CD4, CD25 were purchased from BD Pharmingen (San Diego, CA). Appropriate isotype-matched non-specific antibodies were used as controls.

STAT3 inhibition and siRNA knockdown

For STAT3 inhibition, STAT3 Inhibitor VI (InSolution™ Calbiochem, Billerica, MA) was used at 150 uM. IL-10 was added to the respective wells one hour following the addition of the STAT3 inhibitor. STAT3 siRNAs (siRNA I and siRNA II) and the control siRNA were purchased from Cell Signaling Technologies (Beverly, Massachusetts) and were used at a concentration of 150 nM. Mission siRNA transfection reagent (Sigma Aldrich, St. Louis, MO) was used as transfection agent. SiRNA-mediated reduction in STAT3 protein was confirmed using Western Blot analysis after 48 or 72 hrs. Forty-eight hours after addition of siRNA, cells were either left untreated or treated with IL-10 (10 ng/mL) for an additional 48 hours. Cells were then stained for flow cytometric analysis.

Western Blot

BMDCs in respective experiments were lysed in lysis buffer A (150 mM NaCl, 20 mM Tris Cl (pH7.4), 5 mM EDTA, 1 mM CaCl₂, 10 mM NaF, 1% Triton X 100, 5% glycerol, HALT protease/phosphatase inhibitor (Thermo Scientific). 10 μgs protein were loaded and separated by SDS-PAGE and analyzed by immunoblotting for PD-1 (Novus Biologicals, AF1021), STAT3 (79D7) and p-STAT3 (D3A7) (Cell Signaling Technology), and β-actin (LI-COR, Lincoln NE). Corresponding anti-goat, anti-mouse, and anti-rabbit secondary antibodies conjugated to IRDyes were purchased from LI-COR. Signal was detected using LI-COR Odyssey Imaging System.

Reverse-transcription-polymerase chain reaction

RNA was isolated from purified TIDCs using Qiagen RNeasy plus mini kit (QIAGEN, Valencia, CA). First-strand cDNA was synthesized using Superscript First-Strand Synthesis System III (Invitrogen, Carlsbad, CA). PD-1 specific quantitative PCR was performed using the PD-1 (NM_008798.2) primer/probe set (Mm01285676_m1) purchased from ThermoFisher. GAPDH was used as internal control using the primer/probe set (Mm99999915_g1) from ThermoFischer. IL-10 (NM_010548.2) specific PCR amplification was performed using the following primers: forward primer 5′-ATGCCTGGCTCAGCACTGCT -3′, reverse primer 5′-CATTCATGGCCTTGTAGACACCTTGG-3′. β-actin (NM_007393.5) was used as control. Following primers were used for β-actin specific PCR amplification: Murine forward primer 5′-GTCCCTCACCCTCCCAAAAG-3′, murine reverse primer 5′-GCTGCCTCAACACCTCAAC CC-3′. Bands were resolved by running PCR products on a 1% agarose gel.

Cytokine ELISA

TIDCs or BMDCs were treated with 10 μg/ml anti-PD1 antibody (G4 clone), 10 μg/ml isotype control IgG, or left untreated for 24 or 48 hrs, after which the supernatants were collected for IL-10 and soluble PD-L1 assessments. TIDCs, serum and ascites samples were also collected for IL-10 and PD-L1 assessments. To measure mouse IL-10, a Ready-Set-Go ELISA kit from eBioscience (San Diego, CA) was used according to manufacturer’s protocol. Soluble PD-L1 ELISA was performed on the cell culture supernatants according to the manufacturer’s protocol (My BioSource, San Diego, CA).

FRα antibody ELISA

ELISA plates were coated in bicarbonate coating buffer with recombinant mouse FRα (R&D Systems, Minneapolis, MN) at a concentration of 1 μg/mL. Wells that did not receive any serum were used to determine the background. Serial dilutions of purified mouse IgG were used as a standard curve to convert the O.D. reading to a concentration of antigen-specific IgG. After overnight incubation at 4C, the plates were washed with 0.05% Tween in PBS, blocked with 1% BSA in PBS for 1 hour at room temperature, washed again, and then serum samples were added. Plates were then incubated overnight at 4C followed by wash with PBS/Tween. Anti-mouse HRP secondary Ab was then added to each well and incubated for 1 hour at room temperature before washing and developing with TMB substrate. OD was read using an ELISA reader after reaction was stopped by reducing the pH of the reactions with acid.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 4.00 (GraphPad, San Diego, CA; http://www.graphpad.com). The Student t test, Mann–Whitney U test, or two-way ANOVA test was performed to determine statistically significant difference. A p value ≤0.05 was considered significant.

IL-10 increases the expression of PD-1 and its ligand PD-Ll on ovarian TIDCs

Our recent studies have shown that PD-1, which has been primarily studied in T cells, is also expressed on the TIDCs (4). In the present study, we wanted to determine what factors (e.g. cytokines), commonly found in tumor microenvironment, lead to the increased expression of PD-1 on the TIDCs. Since TIDCs already have upregulated PD-1, and in general are difficult to access, we developed an in vitro model using BMDCs which consistently have a much lower level expression of PD-1 relative to TIDCs (4). TNF-α-activated BMDCs were treated with a variety of well-described immunologic agonists (IL-10, 10 ng/mL; IL-12, 20 ng/ml; VEGF-A, 25 ng/ml; poly: IC, 50 ug/mL; CpG, 50 ug/ml; or IL-4, 5 ng/mL) for 48 hours after maturation, followed by flow cytometric analysis of PD-1 expression. As shown in Fig. 1A, of the agents tested, only IL-10 was able to induce significantly elevated expression of PD-1 on BMDCs. IL-10 dose assessments (Fig. 1B) showed dose dependency with maximal effective concentration of 5.1 ± 1.3 ng/ml (n=18). Fig. 1C shows a flow cytometry histogram of PD-1 expression on BMDCs upon treatment with 10 ng/mL Il-10. Time course experiments showed PD-1 was maximally upregulated at 48 hours following exposure to IL-10 (10 ng/ml) (Fig. 1D). To determine the validity of the BMDC, we also observed the effects of IL-10 on expression of PD-1 on the surface of murine ovarian TIDCs-derived from ascites. As shown in Figs. 1E–F, while TIDCs express high levels of PD-1, treatment with IL-10 for 48 hrs. led to a further increase in PD-1 expression by approximately double. Increased expression of PD-1 induced by IL-10 was due to augmented pdcd1 gene expression which resulted in increased total cellular levels of PD-1 protein (Fig. 1G).

We previously showed that PD-1 maintains an immune suppressive phenotype of TIDCs and that a primary mediator of T cell suppression is TIDC-expressed PD-L1 (4, 24). Thus, as a readout for immune suppressive potential, we also analyzed PD-L1 expression following IL-10 treatment of BMDCs. As shown in Figs. 2H–I, IL-10 also led to an increased expression of PD-L1 on the surface. We also observed that IL-10 treatment of BMDCs induced the release of soluble PD-L1 (sPD-L1) by these cells (Fig. 1J). In contrast, DCs express only low levels of PD-L2, which was not increased following treatment with IL-10 (Figs. 1K–L). The effects of IL-6 were also examined in comparison with IL-10 since both cytokines have common proximal signaling elements (e.g. JAK-STAT3) (25). IL-6 was able to induce PD-1 expression in BMDCs but was not nearly as effective as IL-10 (Fig. S1A). In fact, the effect of IL-6 was biphasic, peaking at 30 ng/ml and then ineffective at higher concentrations. In contrast, IL-6 appeared to be more efficient at driving PD-1 expression on CD4 T cells during T cells activation, whereas, IL-10 had no effect (Fig. S1B). Similar effects were observed with CD8 T cells, although much less pronounced (Fig. S1C). Thus, IL-10, which is typically at high levels in the ovarian cancer TME, is a key driver of PD-1 expression on TIDCs.

IL-10 mediated increase in PD-1 on the surface of DCs is STAT3 dependent

Various transcription factors have been implicated in either promoting or blocking PD-1 expression primarily in T cells (26, 27). We found that pSTAT3 and STAT3 levels were increased in the DCs isolated from ascites of ID8 ovarian tumor-bearing mice and correlated with increased PD-1 expression (Fig 2A). Since signaling emanating from IL-10 receptor involves the activation of STAT3 molecules via tyrosine phosphorylation, subsequent dimerization, and nuclear translocation resulting in binding to the DNA and gene transcription, we next asked if STAT3 activity has a role in IL-10-mediated up-regulation of PD-1 expression on DCs. BMDCs in this experiment were treated with STAT3 inhibitor (which prevents the dimerization and hence the nuclear translocation of STAT3) 1 hour prior to addition of IL-10. As shown in Figs. 2B–C, STAT3 inhibition completely reversed the IL-10-mediated increase in expression of PD-1 on the BMDCs. In a separate approach, we suppressed STAT3 in the BMDCs with siRNA (Fig. 2D) prior to the addition of IL-10, which resulted in a decrease in PD-1 expression (Figs. 2E–F). These results are consistent with the recent findings that implicate activated STAT3 as an important transcription factor in PD-1 expression (28).

PD-1 blockade results in compensatory release of IL-10 by TIDCs

PD-1 blockade has been shown to be therapeutic in mouse models as well as in humans for certain types of cancers; yet a majority of patients among various tumor types don’t benefit (29). Although the response of T and B cells to PD-1 therapy continues to be extensively explored, the effects of PD-1 blockade on DCs are not well established (21). We have shown that TIDCs respond to PD-1 blockade by increasing co-stimulatory molecule and MHC class I expression as well as increasing antigen presentation, the effects that were primarily dependent on release of inhibition on NFκB pathway (21). Here we show that disruption of PD-1: PD-L1 axis on TIDCs using PD-1 blocking or PD-L1 blocking antibody leads to increased release of the immune regulatory cytokine, IL-10 (Fig. 3A). We also show that PD-1 blockade on TIDCs leads to an increase of IL-10 mRNA suggesting that the increased production of IL-10 upon PD-1 blockade is not merely due to enhanced release of pre-existing intracellular stores of IL-10 (Fig. 3B). The effect of different concentrations of PD-1 blocking antibody on IL-10 release by TIDCs was also measured. As shown in Fig. 3C, progressive increases in the concentration of the blocking antibody beyond 10 μg/ml did not progressively increase the production of IL-10. In all the experiments onwards, 10 μg/mL antibody was used in the in vitro experiments. From time course experiments, a significant difference in IL-10 release was detected as early as 7 hrs. (Fig. 3D). In the ID8 tumor-bearing mice, a significant increase in the IL-10 levels were detected both in the blood serum and ascites of mice that were treated with PD-1 blocking antibody compared to the untreated ones (Fig. 3E).

The effect of PD-1 blockade on BMDCs was also evaluated. PD-1⁺ BMDCs (BMDCs post treatment with IL-10 as shown in Fig. 1C) were treated with PD-1 blocking Ab for 24 hrs. PD-1 blockade on these cells also led to significantly increased release of IL-10 (Fig. 3F). In order to check if IL-10, released upon PD-1 blockade, can in turn lead to increased expression of PD-1 on surface of BMDCs, we collected the cell culture supernatants from the BMDCs treated as in Fig. 3F. Cell culture supernatants were then either left untreated or pre-treated with IL-10 neutralizing Ab before adding to fresh BMDC cultures. As shown in Figs. 3G–H, IL-10 pre-neutralization prevented the increase in PD-1 expression on BMDCs treated with conditioned media from BMDCs treated with anti-PD1. This shows that PD-1 and IL-10 form a feedback loop that amplifies upon PD-1 blockade. These results identify a mechanism in DCs whereby the blockade of PD-1 on these cells leads to a compensatory release of IL-10, hence potentially maintaining and fostering the suppressive environment despite PD-1 blockade.

Combination treatment with PD-1 blocking Ab and IL-10 neutralization/IL-10R blockade reduces tumor burden and enhances survival in tumor bearing mice

Given the complex interdependent interaction between IL-10 and PD-1 that we observed in the above studies and their role in immune suppression in tumor microenvironment, we looked to see if combining these two therapies (PD-1 and IL-10/IL-10R blockade) has any effect in the generation of anti-tumor immunity. As shown in Fig. 4A, anti-PD-1 or anti-IL-10(R) as monotherapy had minimal effect on the overall survival or tumor burden; but the combination of these significantly delayed tumor growth and enhanced the survival of the tumor-bearing mice. Combination treatment also significantly reduced the tumor burden in the mice (Fig. 4B) compared to the control group.

Combination treatment enhances the infiltration of immune effectors in the ascites of tumor bearing mice

In order to determine if the combination treatment modulated the quantity and quality of infiltrating immune cells, we looked for T and B-cell infiltrates in the ascites of tumor-bearing mice. As shown in Fig. 5A, there was a significant increase in the total number of T cells in the ascites of tumor bearing mice that were treated with the PD-1 blocking antibody alone or the combination regimen of PD-1 blocking and IL-10(R) neutralizing antibodies. A slight increase in the fraction of CD8⁺ T cells and a slight decrease in the frequency of CD4⁺ T cells among the total CD3⁺ T cells compared to other treatment groups, were observed in the ascites of mice from the combination treatment group (Figs. 5B–C). Importantly and consistent with an inhibitory role of IL-10, we observed a robust increase in both the numbers of activated CD4⁺ and CD8⁺ T cells in the combination treatment group (Figs. 5D–E), whereas those levels were not different from controls in the monotherapy groups.

Similar to the changes in T cell numbers in the ascites, a significant increase in the frequency of B-cells (CD19⁺B220⁺) was observed in the ascites of tumor-bearing mice that were treated with PD-1 blocking antibodies alone or the combination regimen (Figs. 6A–B). Thus, we sought to determine if tumor antigen-specific antibody response was enhanced in the combination treatment group. Folate receptor alpha (FRα) is a well-known tumor antigen that has been shown to be overexpressed in various epithelial derived cancer cells, including ovarian cancer, and deemed capable of driving oncogenic transformation (30). In order to determine if an antigen specific antibody response was generated, the sera from different treatment groups were assayed for FRα-specific antibodies. As shown in Fig. 6C, there were significantly increased levels of circulating FRα specific antibodies in the serum of mice from combination treatment group. Also, increased levels of FRα specific antibodies were observed in ascites of mice from the combination treatment group compared to the other groups (Fig. 6D). These results demonstrate that the combination of PD-1 blockade and IL-10(R) neutralization modulates the adaptive immunity by enhancing the infiltration of activated and antigen specific immune effectors into the TME.

Combination treatment leads to a decrease in infiltration of MDSCs in the ascites

Immune suppressive cells present in the TME are adept at suppressing the effector responses against the tumor and hence promote tumor escape and progression. Myeloid derived suppressor cells (MDSCs) are a subset of such suppressive cells that have been shown to play a major role in immune suppression in the ovarian cancer microenvironment (31). In a murine model of breast cancer, we have shown that a combination regimen of peptide vaccine (MHC Class I binding immunogenic peptides from rat neu (neu), legumain and β-catenin targeting tumor cells, cancer stem cells, and tumor associated macrophages) and PD-1 blocking antibody leads to a decrease in the frequency of MDSCs in the tumor (22, 32). In this study, we found that although anti-PD-1 treatment alone did not change the frequency of MDSCs in the ascites, anti-IL-10 and the combination treatment significantly reduced the frequency of MDSCs in the ascites of tumor bearing mice (Fig. 7A and 7B).