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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Res. Author manuscript; available in PMC 2010 September 15.
Published in final edited form as:
PMCID: PMC2754806
NIHMSID: NIHMS140253

IN SITU STIMULATION OF CD40 AND TOLL-LIKE RECEPTOR 3 TRANSFORMS OVARIAN CANCER-INFILTRATING DENDRITIC CELLS FROM IMMUNOSUPPRESSIVE TO IMMUNOSTIMULATORY CELLS

Abstract

Boosting therapeutically relevant immunity against lethal epithelial tumors may require targeting tumor-induced immunosuppression on an individualized basis. Here we demonstrate that, in the ovarian carcinoma microenvironment, CD11c+MHC-II+ dendritic cells (DCs) spontaneously engulf tumor materials, but rather than enhancing anti-tumor immunity, suppress T-cell function. In situ co-stimulation of CD40 and TLR3 on tumor-infiltrating DCs decreased their L-arginase activity, enhanced their production of Type-I interferon and IL-12(p70), augmented their capacity to process antigens, and upregulated co-stimulatory molecules in vivo in mice and in vitro in human dissociated tumors. Synergistic CD40/TLR activation also induced the migration of activated DCs to lymphatic locations and promoted their capacity to present antigens. Correspondingly, without exogenous antigen, combined CD40/TLR agonists boosted measurable T-cell-mediated anti-tumor immunity and induced the rejection of otherwise lethal intraperitoneal ovarian carcinomas. Our results highlight the potential of transforming tumor-infiltrating DCs (the most abundant leukocyte subset in the solid ovarian carcinoma microenvironment) from an immunosuppressive to an immunostimulatory cell type. Combined administration of synergistic CD40 and TLR3 agonists could enhance their individual therapeutic effects against ovarian and other lethal epithelial cancers.

Keywords: Immune evasion, Ovarian Cancer, Immunotherapy, anti-CD40, TLR agonist

INTRODUCTION

Dendritic cells (DCs) are best known for initiating T-cell-mediated immune responses. Consequently, multiple strategies aimed at using the immunostimulatory potential of adoptively administered DCs have been attempted to boost therapeutically relevant immune responses against established human cancers. Unfortunately, although measurable immunological enhancement has been routinely attained and new promising strategies are under investigation, the emerging view is that the DC-based vaccination approaches tested so far do not effectively induce cancer regression in most patients (12).

In the United States alone, epithelial ovarian cancer claimed the lives of more than 15,000 women in 2008 (3). Regrettably, chemotherapies implemented in the last 30 years have led to a 5-year survival rate of 30%, at best, for patients with metastatic ovarian carcinoma, the stage at which most cases are diagnosed (3). Although the lethality of the disease has traditionally reduced the number of potential advocates, the need for new complementary treatments is increasingly clear. Interestingly, ovarian cancer is an ideal target for novel immunotherapies for several reasons: Firstly, studies pioneered by Coukos and colleagues indicate that ovarian cancer naturally triggers anti-cancer immune responses (47). Secondly, although ovarian cancer is a devastating disease, metastases are frequently restricted to the peritoneal cavity where the tumor microenvironment is directly accessible, which prevents the need for systemic delivery of immunostimulatory treatments. Thirdly, CD11c+DEC205+MHC-IIlowCD11b DCs expressing low to undetectable levels of co-stimulatory CD80 represent the most frequent leukocytic subset in the microenvironment of mouse and human solid ovarian carcinomas (811), hence promoting their inherent immunostimulatory potential may prove the most effective therapy yet.

To boost endogenous immunity against different epithelial tumors, signaling through CD40 has also been attempted with promising results, although therapeutic effectiveness was limited by toxicity (1213). In independent trials, Toll like receptor (TLR) agonists have also been individually implemented as adjuvants (14). Furthermore, recent reports in non-tumor systems indicate that a combinatorial stimulation of TLRs and TRAF-signaling by CD40 cross-linking, generates a 10–20 fold increase in the number of activated CD8+ T-cells, compared to either agonist alone (15). In murine non-epithelial tumors, CD40/TLR7 agonists have been utilized as adjuvants in vaccination using exogenous tumor antigen, which resulted in stronger and less toxic anti-tumor memory T-cell responses compared to monotherapy (16).

We have previously demonstrated that CD11c+ DCs sorted from ovarian cancer-bearing mice are competent phagocytes but cannot efficiently present ovalbumin (OVA) to transgenic T-cells before receiving an aggressive stimulatory cocktail (9). In addition, the elimination of DCs from tumor locations delays ovarian cancer progression by boosting measurable T-cell-mediated anti-tumor immunity (17), and previous reports demonstrate that human ovarian cancer-associated DCs express functional levels of immunosuppressive PD-L1 (18). However, there is no direct evidence that CD11c+MHC-II+ DCs from ovarian carcinoma specimens suppress antigen-specific T-cell responses. We hypothesized that, if ovarian DCs acted as bona fide immunosuppressive cells in the ovarian carcinoma microenvironment where they massively accumulate (910), reversing their tolerogenic phenotype in vivo while promoting their capacity to present tumor antigens that they spontaneously engulf, could elicit therapeutically effective anti-tumor immunity. To test this hypothesis we administered a cocktail of CD40 and TLR3 agonists (16). Preceding synergistic cancer interventions were limited to TLR7 targeting, however plasmacytoid DC are the main subset of DCs which express TLR7 (and TLR3 at almost undetectable levels) in both mouse and human (1920). We show here that the activation of TLR3, expressed on ovarian tumor-associated CD11c+MHCII+ DCs, synergizes with CD40 agonists to induce DC maturation in situ, which reverses their immunosuppressive phenotype and induces T-cell responses that lead to the rejection of otherwise lethal ovarian carcinomas.

Materials and Methods

Animals, tissues and treatments

Mice were procured from the National Cancer Institute or Jackson Laboratories (Bar Harbor, ME). Experiments were approved by our Institutional Animal Care and Use Committee. Stage III–IV human ovarian carcinoma specimens were procured through Research Pathology Services at Dartmouth, under an approved protocol. Single cell suspensions were generated as previously described (21).

For treatments, wild-type C57BL/6 mice were intraperitoneally injected with 1.5 × 106 ID8-luciferase (17), ID8-GFP or ID8-Defb29/Vegf-A ovarian carcinoma cells. At days 7, 14, 21 and 28 post-tumor challenge received intraperitoneal injections of PBS, irrelevant Rat IgG (50 µg/mouse), anti-CD40 antibody (FGK4.5, BioExpress) (50 µg/mouse) or Polyinosine-polycytidylic acid (poly(I:C), Invivogen) (100 µg/mouse) in combination or alone, or one dose 21 days post-tumor challenge unless otherwise stated. Flagellin was purified as previously described (22).

For visualization of tumor burden, mice were injected with 0.2 ml of 15 mg/ml luciferin (Promega). After 10 min, animals were anaesthetized with isoflurane and imaged using the IVIS 200 system (Xenogen Corp.).

We obtained the CP-870,893 monoclonal antibody from Pfizer Inc. (Groton, CT).

In Vitro Suppression Assay

Ascites from mice bearing ID8-Defb29/Vegf-A for 31 days was sorted for CD45+CD11c+MHCII+ dendritic cells (tumor-derived DC;tDC) and used in the assay. Bone marrow-derived DCs (BMDC) or tDC were cultured with 50 µg/ml full length Ovalbumin (Sigma) at 106 cells/ml, for three hours. Negatively bead selected CD3+ OTI splenocytes (1×105) were then CFSE labeled and added to Ova pulsed bone marrow DCs (BMDC) or Ova pulsed tumor-derived DC (tDC; sorted from tumor ascites) in a 10:1 ratio. Either un-pulsed tDC or BSA (50 µg/ml) pulsed BMDC were introduced into ‘BMDC/Ova + CFSE OTI’ co-cultures at various ratios. (At day5, the total T-cell count was ~ 4x105). We then collected 30,000 events to detect CFSE dye dilution peaks.

Arginase Activity Assay

In vivo activated dendritic cells were sorted from either the peritoneal cavity or combined, inguinal, axillary and brachial lymph nodes of tumor bearing mice. Quantitative colorimetric arginase determination was performed using an Arginase Activity detection kit (BioAssay Systems). Briefly, 0.05 – 0.25 × 106 cells were washed and lysed for 10 min in 50 µL of 10 mM Tris-HCl (pH 7.4) containing 0.15 mM pepstatin A, 0.2 mM leupeptin, and 0.4% (v/v) Triton X-100. Lysates were then used to complete the assay according to manufacturer’s instructions.

ELISPOT

Total cells were either obtained from peritoneal washes or dissociated spleens of treated or untreated ID8-Defb29/Vegf-a bearing mice. Peritoneal cells were co-cultured for 48 hours, in coated and blocked ELISPOT plates, among BMDC in a 10:1 ratio, which were previously pulsed (overnight) with irradiated and UV-treated ID8-Defb29/Vegf-A cells (10 DC : 1 tumor cell). Splenocytes were first primed in 24-well plates in a 10:1 ratio with BMDC either pulsed with doubly treated tumor cells or with the peptide Mesothelin (GQKMNAQAI; New England Peptide), for 7 days. Cells were then collected and re-stimulated in 96-well ELISPOT plates with specified antigen or tumor pulsed BMDC (10:1 ratio) for 24hours. All cultures were maintained in complete RPMI containing 10% FBS. Analysis was then continued according to manufacture’s protocol (eBioscience).

Immunohistochemistry

Ovaries of mice were collected and embedded in Tissue-Tek, after which 8µm sections were made from frozen tissue blocks. Slides were then fixed with acetone and washed with PBS. Sections were then blocked using α-CD32 followed by staining with either biotinylated α-CD8 (53-6.7) or α-CD3 (145-2C11; eBioscience) and completion of immunohistochemical procedure according to manufactures instructions (Vector Labs). Slides were then viewed at ×200 and positive cells were quantified using an image analyzing software (NIS-Element Imaging).

Cytokine Detection

Either peritoneal lavages (10ml of PBS) or culture supernatants were used in ELISA assays for IL-2, IL-12p(70), IFN-α and IFN-β (eBioscience and PBL Biomedical Lab respectively) according to manufactures’ instructions. TNF-α was detected using a Mouse-5-Plex panel cytokine assay (Bio-Rad, Hercules, CA), following manufacturer’s instructions. For in vivo analysis; sorted peritoneal cells (106/ml) from treated or control animals were stimulated for 4 hrs with PMA/ionomycin (50ng/1µg/ml).

Flow Cytometry

Flow cytometry was performed on a FACSCanto (BD Biosciences, San Jose, CA). Sorting was performed on a FACSAria sorter (BD Biosciences).

Anti-mouse antibodies: CD45 (30-F11), CD69 (H1.2F3) and CD11c (HL3; all from BD Biosciences); MHC-II (NIMR-4, eBioscience). Anti-human antibodies: CD45 (HI30), DEC205 (MG38), CD11c (B-ly6), CD3 (UCHT1), CD11b (ICRF44), HLA-DR (L234) and CD14 (M5E2), all from Biolegend.

An allophycocyanin-labeled tetramer consisting of Kb folded with GQKMNAQAI peptide was provided by the NIH Tetramer Core Facility (Atlanta, GA).

Statistical Analyses

Differences between the means of experimental groups were analyzed using the Mann-Whitney test and survival was analyzed with the Log-rank test, both using the GraphPad Prism 4.0 software.

Division Indices, defined as average number of cell divisions that the responding cells underwent, were calculated using FlowJo software (Ashland, OR).

RESULTS

CD11c+MHC-II+ DCs from the ovarian carcinoma microenvironment suppress T-cell function but can be activated to become efficient APCs in vitro

To define the regulatory nature of ovarian cancer-associated DCs, we first confirmed that, CFSE-labeled, negatively selected, transgenic OT-I splenic T-cells proliferated very poorly in response to tumor-derived DCs pulsed with ovalbumin (OVA) (9), while bone marrow-derived DCs (BMDC) induced a robust expansion (Fig. 1A). To demonstrate the ability of tumor-derived DCs to suppress naive CD8 T-cell proliferation, we next analyzed the proliferation of OT-I lymphocytes incubated with the same OVA-pulsed BMDCs, in the presence of two different ratios of CD11c+MHC-II+ DCs sorted from tumor ascites. Notably, the addition of tumor DCs abrogated the expansion of transgenic T-cells (Fig. 1B). This was not caused by a potential interference with access of lymphocytes to pulsed DCs, because OVA-pulsed and washed BMDCs, mixed with identical ratios of irrelevant protein pulsed BMDCs, also induced strong proliferation of OT-I lymphocytes (Fig.1B and Suppl.Fig. 1A; P< 0.05 for the division index).

Figure 1
Tumor derived CD11c+MHC-II+ DCs suppresses T-cell responses but can be converted to efficient APCs

Confirming previous reports (1718, 23), ovarian cancer-associated CD11c+MHC-II+ DCs express significant levels of PDL-1 (Suppl.Fig.1B), and secreted immunosuppressive VEGF (17) (not shown). More importantly, as recently identified in breast and lung tumor models (2425), CD11c+DEC-205+MHC-II+ DCs sorted from ovarian cancer locations, but not DCs sorted from the non-tumor draining lymph nodes of the same animals, showed strong L-arginase activity (Fig. 1C; P=0.05). Underscoring the role of the tumor microenvironment in the regulatory differentiation of tumor DCs, incubation of BMDCs in tumor-conditioned media increased their production of functional L-arginase (Suppl.Fig. 1C).

Previous reports identified a synergistic effect for combined CD40 and TLR agonists, with combinatorial CD40/TLR3 agonists producing one of the best T-cell response in healthy mice (15). Because ovarian cancer-associated DCs express detectable levels of CD40 (Suppl.Fig. 1D) and TLR3 (Suppl.Fig. 1E), we next determined whether the synergistic stimulation of CD40 plus TLR3 could reverse the regulatory phenotype of tumor DCs. As shown in Fig. 1D, tumor-derived, OVA-pulsed CD11c+MHC-II+ DCs induced a significant proliferation of transgenic OT-I lymphocytes only after ex vivo activation with agonistic anti-CD40 antibody plus the TLR3 agonist poly(I:C). Notably, the combined intraperitoneal administration of CD40/TLR3 agonists to ovarian cancer-bearing mice, but not individual treatments, induced a significant decrease in the immunosuppressive L-arginase activity of tumor DCs (Fig. 1C; P=0.05).

Taken together, these data indicate that ovarian cancer-associated DCs suppress T-cell responses, but their immunostimulatory capacity can be promoted by the synergistic stimulation of CD40 and TLR3.

Synergistic stimulation of CD40 and TLR3 activates mouse and human ovarian cancer-infiltrating DC

To confirm activation of tumor-associated DCs by concurrent stimulation of CD40 and TLR3, we first treated ascites from ID8-Defb29/Vegf-a-tumor bearing mice with agonistic anti-CD40 plus poly(I:C), the individual treatments, or PBS. As shown in Fig.2A, combinatorial treatment induced a stronger up-regulation of CD80, CD86, CD70 and MHCII (P<0.05) in tumor CD11c+ DCs after 24-hours, compared to incubation with anti-CD40 or poly(I:C) alone. Correspondingly, combined anti-CD40 plus poly(I:C) treatment of CD11c+MHC-II+ DCs sorted from ovarian cancer locations induced a significant increase in the production of TNF-α (Fig. 2B). Most importantly, sorted peritoneal DCs from ovarian cancer-bearing mice treated with combined CD40/TLR3 agonists secreted significantly higher amounts of immunostimulatory IFN-β and IL-12(p70) in response to PMA/ionomicyn, compared to tumor DCs from mice receiving individual treatments (Fig. 2B; P=0.05).

Figure 2
Triggering of CD40 and TLR3 upregulates activation markers on both mouse and human ovarian cancer-infiltrating DC

To define the applicability of our findings, we stimulated single-cell suspensions from mechanically dissociated stage III–IV human ovarian carcinoma specimens (17) for 48 h with the fully human IgG2 mAb, CP 870, 893 (Pfizer) (12, 26). As shown in Fig. 2C, CD80 and MHC-II were upregulated in some specimens when poly(I:C) was simultaneously added. Therefore, although the synergistic stimulation of CD40 and TLR3 on tumor-associated DCs using currently available human antibodies may be weaker than that of our murine ovarian cancer model, co-administration of CD40 plus TLR3 agonists could also promote the maturation of human ovarian cancer-associated DCs in selected patients.

Concurrent stimulation of CD40 and TLR3 enhances the capacity of tumor-associated CD11c+ DCs to process antigens that they spontaneously engulf in the tumor microenvironment

Efficient antigen presentation requires competent antigen engulfment and processing. To confirm the capacity of tumor-infiltrating DCs to spontaneously take-up tumor materials in vivo, we first analyzed the ascites of C57BL/6 mice bearing intraperitoneal ID8-Defb29/Vegf-a engineered to express GFP (9). Up to 18% of total CD11c DCs at this temporal point (Day35) co-exhibited green fluorescence, suggesting recent engulfment of tumor materials (Fig. 3A). Then, to define the effect of co-stimulation of CD40/TLR3 on tumor-associated DCs, on their capacity to take-up and process antigens, we treated mice bearing highly aggressive intraperitoneal (i.p.) ID8-Defb29/Vegf-a tumors (9) with 3 weekly i.p. injections of agonistic αCD40 antibody plus poly(I:C). To monitor antigen processing, six days after the last treatment we administered ovalbumin (Ova) conjugated to a self-quenched fluorophore (DQ-Ova; Invitrogen), which emits bright green fluorescence only if proteolytic digestion occurs in the endosome (27). As expected, the proportion of DQ-Ova+CD11c+ DCs cells that took-up and efficiently processed antigen at tumor locations was significantly increased (≈10 fold) in mice receiving combinatorial treatment 24hrs before (Fig. 3B and Suppl.Fig. 2A; P<0.05). Supporting their in situ activation by CD40/TLR3 co-stimulation, CD11c+ DCs cells at tumor locations in treated mice also expressed higher levels of CD80, CD40 and MHCI (Fig. 3B and Suppl.Fig. 2B). Correspondingly, the percentages and total number of CD11c+ DCs processing GFP+ tumor materials and the upregulation of co-stimulatory CD80 in ID8-Defb29/Vegf-a-GFP bearing mice was also significantly higher in treated mice, compared to controls (Fig.3C and Suppl.Fig2C). Jointly, these results indicate that co-activation of CD40 and TLR3 dramatically enhances the capacity of tumor-infiltrating DCs to process the antigens that they spontaneously phagocytose at tumor locations, and induces the upregulation of co-stimulatory determinants.

Figure 3
Activation through CD40 and TLR3 augments CD11c+ DCs antigen processing capability

Co-stimulation of CD40 and TLR3 in vivo promotes the migration of activated DCs carrying antigens from tumor to lymphatic locations and their capacity to present antigens

Efficient antigen presentation in vivo also requires migration of mature DCs to lymphatic locations. As we’ve established that synergistic stimulation of CD40 and TLR3 is the most efficacious regimen for DC activation, compared to single treatments, we next sought to define its effect on the migration of activated DCs from tumor locations to draining lymph nodes. We first challenged mice with flank ID8-Defb29/Vegf-a tumors, as described (9) and when tumors reached a size of 200 mm2, mice were shaved and painted with 50 µl of 0.1% FITC dissolved in a 50:50 (v/v) acetone-dibutylphthalate (28), followed by an intra-tumoral treatment of CD40/TLR3 agonists, or PBS. As expected, the percentage of activated (CD80+MHCII+) CD11c+ DCs carrying FITC from the tumor location to its draining (inguinal) lymph node (LN) increased 2-fold in treated mice, compared to controls (Fig. 4A).

Figure 4
The migratory capacity of activated DCs carrying antigens from tumor to lymphatic locations are enhanced upon co-stimulation along with their ability to present antigen

There was also a substantial increase in the proportion of CD11c+FITC+CD80+MHCII+ DCs in the auxiliary-LN, where the inguinal-LN drains, in treated mice, compared to controls (Fig. 4B).

To confirm the capacity of CD40/TLR3-matured DCs to activate T-cells, we sorted CD45+CD11c+MHCII+ DCs from the peritoneal cavity of treated and control ID8-Defb29/Vegfa tumor-bearing mice and incubated them with CFSE-labeled allogeneic splenic CD3+ negatively selected from Balbc mice. DCs sorted from mice receiving PBS did not induce the expansion of allogeneic T-cells, in contrast, DCs derived from the ascites of αCD40/poly(I:C) treated mice, elicited significant proliferative responses after 5 days in culture (Fig. 4C). Finally, ELISPOT analysis revealed that the number of negatively immunopurified T-cell splenocytes from tumor-bearing mice, producing Granzyme-B in response to directly sorted (unpulsed) tumor-associated DCs, was significantly higher when tumor DCs were procured from mice synergistically treated in vivo, compared to mice receiving individual treatments (Fig.4D; P<0.05). Therefore, synergistic co-stimulation of CD40 and TLR3 enhances the migration of immunostimulatory DCs carrying local antigens from the immunosuppressive tumor microenvironment to T-cell rich lymphoid organs and also enhances antigen presentation and T-cell activity.

Administration of CD40/TLR3 agonists to ovarian cancer-bearing hosts boosts T-cell-mediated anti-tumor immunity

We hypothesized that, if synergistic co-stimulation of CD40 and TLR3 promotes the capacity of tolerogenic DCs from tumor locations to efficiently present the tumor antigens that they carry to lymph nodes, enhanced tumor-specific T-cell responses should become measurable. Supporting this proposition, mice bearing established ID8-Defb29/Vegf-a tumors treated with combinatorial CD40/TLR3 agonists contained three-folds more antigen-experienced (CD44+) CD8+ T-cells in peritoneal wash samples, compared to mice receiving irrelevant IgG or PBS (Fig.5A). Most importantly, the number of peritoneal T-cells producing IFN-γ in response to tumor antigens in ELISPOT analyses was significantly increased in ID8-Defb29/Vegfa ovarian cancer-bearing mice treated with combinatorial anti-CD40 agonistic antibody plus poly(I:C), compared to control mice receiving PBS (Fig.5B; P<0.05). Further confirming the synergistic effect, individual treatment with either anti-CD40 antibodies or poly(I:C) did not result in any measurable increase in the number of T-cells producing IFN-γ upon stimulation with tumor antigens (Fig.5B). Comparable results were found using splenic T-cell (Fig.5B; P<0.05). Finally, the percentage of tumor antigen-specific splenic CD8+ T-cells exhibiting a central memory (CD44+CD62L+) phenotype, specifically recognizing an H-2Db-restricted mesothelin epitope expressed by ID8 tumor cells (29) in tetramer analyses was significantly higher in mice treated with combinatorial CD40/TLR3 agonists, compared to mice receiving separate agonists or PBS (Fig.5C and Suppl.Fig.2D; P<0.05) (even though we did not detect differences in tetramer+CD8+ among groups). This result was corroborated by the enhanced secretion of IFN-γ from combinatorial treated splenocytes, in response to the tumor peptide, mesothelin (Fig.5C; P<0.05).

Figure 5
Anti-tumor T-cell immunity is enhanced upon treating with CD40/TLR3 agonists

Consistent with a dramatic enhancement of tumor-reactive T-cell-mediated responses elicited by in situ administration of CD40 plus TLR3 agonists, the infiltration of tumor islets in resected ID8-Defb29/Vegf-a tumor-bearing ovaries by CD8+CD3+ cytotoxic T-cells (the only known cell type in the ovarian cancer microenvironment that elicits immune pressure against tumor progression (7)) was significantly stronger in samples from mice treated with combinatorial CD40/TLR3 agonists (Fig.5D and Suppl.Fig.2E; P<0.05). Collectively, these data indicate that the immunostimulatory phenotype promoted by CD40/TLR agonists on otherwise immunosuppressive tumor-infiltrating DCs is associated with the dramatic expansion and activation of tumor-reactive T-cells.

Synergistic stimulation of CD40 and TLR3 induces the rejection of otherwise lethal intraperitoneal ovarian carcinomas

As we demonstrated that the synergistic use of CD40 and TLR3 agonists elicits enhanced anti-tumor immunogenic boosts, we next investigated its therapeutic potential. We first challenged mice with intraperitoneal ovarian carcinomas, developed with parental ID8 cells transduced with luciferase, for intravital monitoring of tumor burden (17). Mice growing established tumors received weekly combinatorial CD40/TLR3 agonists, rat IgG or PBS for four weeks, with no evidence of toxicity in any group. Notably, in the absence of direct targeting of tumor cells, administration of CD40/TLR3 agonists resulted in the elimination of any obvious disease in ≈50% of mice, which remained healthy >300 days after tumor injection, while all mice receiving control injections succumbed to the disease ≈190 days earlier (Fig.6A). Correspondingly, synergistic treatment significantly reduced tumor burden as determined by luminescence 68 days post tumor injection (Fig.6B and Suppl.Fig.2F).

Figure 6
Synergistic stimulation of CD40 and TLR3 confers therapeutic effects in addition to increased cytokine production at tumor sites

To confirm the therapeutic potential of combinatorial treatment against in an even more aggressive ovarian carcinoma model, we also treated mice growing established intraperitoneal ID8-Defb29/Vegf-a tumors which accelerates tumor progression by ~3 fold (9). In the absence of complementary therapies, synergistic stimulation of CD40 and TLR3 also induced a significant 15% increase in lifespan with only 4 injections (Fig.6C; P<0.001). Notably, 90% of mice treated with CD40/TLR3 agonists were alive at the time (51days) when all control mice succumbed to tumor malignancy. Additionally, when we administered a TLR3 agonist with αCD40 for treatments, we saw that it’s effect was inferior to the αCD40/poly(I:C) combination (Suppl.Fig.2G: P<0.01), although ovarian-cancer associated DCs express TLR5 (23). Interestingly, CD40/TLR3 agonists treatments induced an astonishing 450-fold increase in IFN-α levels in peritoneal wash supernatants, accompanied by a ~2.5-fold increase in IL-12(p70) and a significant increase of IL-2 (Fig.6D; P<0.05). Therefore, synergistically activating CD40 plus TLR3 in hosts bearing established ovarian cancer results in a significant therapeutic benefit even in the absence of additional surgical or chemotherapeutic interventions and, depending on the tumor, induces durable curative results.

DISCUSSION

Ovarian cancer is one of the most aggressive and frequent forms of epithelial cancer and claims >15,000 lives every year in the United States alone (3). The most abundant leukocyte subset in the microenvironment of human and mouse solid ovarian carcinoma specimens is CD11c+DEC205+MHC-II+CD11b DCs with pro-angiogenic and immunosuppressive activity (910, 17). Here we demonstrate that the synergistic stimulation of CD40 and TLR3 transforms tumor-associated mouse DCs in vivo from an immunosuppressive to an immunostimulatory cell type that efficiently processes spontaneously engulfed tumor antigens, upregulates co-stimulatory molecules and migrates to lymphatic locations to activate antigen-specific T-cells. Consequently, the administration of CD40/TLR3 agonists to established ovarian cancer-bearing hosts boosts T-cell-mediated anti-tumor immunity, resulting in the rejection of otherwise lethal intraperitoneal ovarian carcinomas.

Despite inducing measurable immune responses in most cancer patients, vaccination approaches using ex vivo conditioned DCs have so far induced limited therapeutic effects (12). The success of DC-based immunotherapy in stimulating anti-tumor cellular immunity is decidedly dependent on trafficking of mature DCs to T-cell rich lymphoid organs after tumor antigen processing. Ovarian cancer infiltrating DCs spontaneously engulf tumor cells, avoiding the need to prime them ex vivo or in vivo with exogenous tumor antigens. CD40/TLR triggering enhances both antigen processing and lymphatic migration, which results in increased numbers of tumor-specific T-cells with central memory (CD44+CD62L+) attributes in the spleen, in conjunction with IFN-γ secreting T effector cells in spleens and at tumor locations. Although the phenotypic transformation induced by combined CD40/TLR3 agonists on human ovarian cancer-infiltrating DCs was clearly weaker than that of our mouse model, we also observed that CD80 and MHC-II were upregulated in some specimens. Therefore, combining CD40 and TLR3 agonists, by promoting the immunostimulatory potential of immunosuppressive/pro-angiogenic tumor antigen-presenting DCs in situ in selected patients, could enhance the therapeutic effect of these reagents, currently being individually tested (12, 3031)

We have previously shown that the elimination of immunosuppressive/pro-angiogenic DCs from ovarian cancer locations results in measurable therapeutic effects (17). Rather than depleting them, this new approach transforms tumor-infiltrating DCs into “Trojan Horses”, resulting in stronger therapeutic benefits. As preliminary reports indicate that the persistence of anti-tumor T-cells adoptively transferred into ovarian cancer patients may be significantly reduced by tumor microenvironmental factors, it will be therefore interesting to determine whether eliminating this abundant immunosuppressive component from the ovarian cancer microenvironment while boosting endogenous anti-tumor immunity enhances the persistence and/or the effectiveness of T-cell adoptive therapies in a future clinical setting.

Supplementary Material

ACKNOWLEDGEMENTS

This research was supported by a 2006–2011 Liz-Tilberis Award and the NCI Grant #RO1CA124515. UKS was supported by NIH Training Grant T32AI007363. We thank the NIH Tetramer Core Facility for providing the tetramer, RJ Noelle for providing reagents and critical expertise, and Pfizer for providing the anti-CD40 Ab.

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