Adaptive Antitumor Immunity Research Articles

Characterization of MHC Class I and β-2-Microglobulin Expression in Pediatric Solid Malignancies to Guide Selection of Immune-Based Therapeutic Trials.

Over 10,000 US children are diagnosed with cancer yearly. Though outcomes have improved by optimizing conventional therapies, recent immunotherapeutic successes in adult cancers are emerging. Cytotoxic T lymphocytes (CTLs) are the primary executioners of adaptive antitumor immunity and require antigenic presentation in the context of major histocompatibility complex (MHC) class I and the associated β-2-microglobulin (B2M). Loss of MHC I expression is a common immune escape mechanism in adult malignancies, but pediatric cancers have not been thoroughly characterized. The essential nature of MHC I expression in CTL-mediated cell death may dictate the success of immunotherapies, which rely on eliciting an adaptive response. We queried pediatric tumor microarray databases for MHC I and B2M gene expression. We detected MHC I in pediatric tumor cell lines by flow cytometry and characterized MHC I and B2M expression in patient samples by immunohistochemistry. To determine whether therapeutic approaches might enhance MHC I expression in selected models in vitro, we tested effects of exposure to IFN-γ and histone deacetylase inhibitors. Pediatric tumors overall, as well as samples within select individual tumor subtypes, exhibit wide ranges of MHC I and B2M gene and protein expression. For most cell lines tested, MHC I was inducible in vitro. MHC I and B2M expression vary among pediatric tumor types and should be evaluated as potential biomarkers, which might identify patients most likely to benefit from MHC I dependent immunotherapies. Modulation of MHC I expression may be a promising mechanism for enhancing MHC I dependent immunotherapeutic efficacy.

P74: Targeting tumor cells with Hsp70 chaperone

The Hsp70 chaperone is one of the major components of tumor microenvironment displaying multiple not yet established functions. It was shown to stimulate innate and adaptive anti-tumor immunity in a variety of cancer models. These effects were due to active release of endogenous Hsp70 to an extracellular matrix and therefore the extracellular chaperone can be a trigger of multiple events in the whole tumor. Factors inducing Hsp70 release are heat stress, inhibitors of important signaling proteins, anticancer drugs and X-ray treatment. Recently we have shown that delivery of Hsp70 can be efficient pusher of its intracellular analogue to extracellular milieu. In this study we show that intracellular cycling of exo- and endogenous Hsp70s increases the sensitivity of cancer cells to cytotoxic lymphocytes. Moreover, the results of in vivo studies employing B16 mouse melanoma and C6 rat glioblastoma as targets proved the therapeutic relevance of exogenous Hsp70 in intra-tumoral application. To uncover the mechanisms of the anticancer effect we used inhibitors and markers of intra- and extracellular protein transport. The data of these studies showed multiplicity of pathways using which exo-Hsp70 reaches cytosol and more usable ones was endocytosis. To be exported intracellular Hsp70 employs vesicular structures as well as intra-membrane lipid structures. Analyzing Hsp70 molecule we found the domain with potential vector activity, i.g. cell penetrating function. This peptide was synthesized and shown to cross a cellular membrane with efficacy exceeding that the whole Hsp70 molecule. Furthermore, the new peptide was used as a carrier for the delivery inside living cells antibody to Hsp70. The resulting construct was found to reduce the resistance of tumor cells to pro-apoptotic effect of staurosporin. Taking together these data, we can suggest that Hsp70 and its fragments can be effective players in communication between cells assembling functionally active tumor. The work was supported by Grant of Russian Scientific Foundation (N 14-50-00068 ).

Immune checkpoint inhibitors with radiotherapy and locoregional treatment: synergism and potential clinical implications.

Antigens recognized by T cells in tumors include differentiation antigens, overexpressed antigens, cancer-testis, and mutated tumor neoantigens. Ionizing radiation causes damage to multiple biomolecules by direct energy deposition or by generation of free radicals, leading to cell death when the damage cannot be repaired. Tumor cell death induced by radiation will generate specific molecular signals that are sensed by antigen-presenting cells and stimulate their maturation and ability to cross-present tumor-derived antigens to T cells. Immunogenic cell death will complement the activity of immune checkpoint inhibitors. We will provide the emerging information coming from preclinical and clinical testing about the combinations of immunotherapies and radiotherapy. Radiation induces chemokines that attract effector T cells to the tumor and vascular adhesion molecules that facilitate T-cell infiltration. This process, which has been named 'immunogenic modulation', plays a role not only in regression of the irradiated tumor but also in amplifying and strengthening adaptive antitumor immunity. The ongoing process of killing of tumor cells by cytotoxic T lymphocytes sustains release of more tumor antigens and possibly promotes antigenic spread, that is, activation of a broader T-cell repertoire. Results of several ongoing clinical trials are testing the combination of radiotherapy with immune checkpoint inhibitor treatment. Data support a model whereby 'waves' of tumor cell killing by T cells primed by the initial radiation-elicited antigen release boost the immune response. This process can eventually achieve systemic tumor control. Radiation therapy is confirmed to be a sensitizer of tumors to immune checkpoint inhibitors in clinical trials, and its application will be easy to implement and widespread. Conversely, many issues need to be addressed before radiotherapy can become such a valid immunogenic tool. An area of increasing importance will be the development of suitable biomarkers that will be able to reliably assess 'immunogenic tumor cell death', immune effector stimulation, and adaptive immunity. Such an immune profile of biomarkers will aid in searching for an optimal combination of radiotherapy and immunomodulation and allows patient selection and response prediction.

Abstract A49: Systemic oncolytic reovirus for the treatment of primary and secondary brain tumors

Abstract Reovirus is a systemically delivered oncolytic agent with evidence of activity in both pre-clinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, as well as activating anti-tumor innate and adaptive immunity. Having previously shown that intravenously delivered reovirus selectively accesses colorectal cancer metastatic to the liver in patients, in this study we explored the potential of systemic reovirus for the treatment of both primary and secondary brain tumors. We first showed that intravenous reovirus can be detected in tumors implanted into the brains of immunocompetent mice. We then tested our current most potent reovirus-based therapy (systemic reovirus plus granulocyte-macrophage colony stimulating factor [GMCSF] in reovirus pre-immune mice), and showed effective therapy in immunocompetent murine models of primary glioma, and of both directly reovirus sensitive, and insensitive, models of melanoma brain metastases. We also found that addition of reovirus/GMCSF to clinical ‘standard of care’ (radiotherapy and temozolamide chemotherapy) significantly enhances survival. In parallel to these pre-clinical experiments, we have initiated an open-label, non-randomized study of intravenous reovirus administered to patients prior to planned surgery for recurrent high grade glioma or metastatic brain tumors, to test whether the data showing access of the agent to tumors in the brains in mice, also applies to humans. In total, 10 patients will be treated with a single infusion of 1x1010 TCID50 of reovirus, of which 9 have completed the study to date. The primary objective is the presence of reovirus in the resected tumors as assessed by immunohistochemistry, RNA in-situ hybridization and retrieval of infectious virions. Early analysis of the first 3 patients, comprising one glioblastoma multiforme, one grade 3 oligodendroglioma and one colorectal brain metastasis, has shown that all 3 resected tumors contained reovirus RNA and protein. There was also evidence for productive reovirus infection in 2 of the tumors. Within all patients to date, the only grade 3-4 adverse reaction has been neutropaenia in 1 patient. Further tissue analysis is ongoing, as is testing of blood samples from these patients to further charactize how reovirus is carried in the blood and protected from neutralizing antibodies. This clinical study shows, for the first time that an oncolytic virus, reovirus, infects and replicates in brain tumors following intravenous administration. Together with pre-clinical data showing the efficacy of systemic reovirus in combination with GMCSF/radiation/temozolamide, these findings support the future development of trials and combination studies using reovirus in patients with high grade gliomas and brain metastases. Citation Format: Adel Jebar, Liz Ilett, Tim Kottke, Emma West, Karen Scott, Simon Thomson, Matt Coffey, Gerard Nuovo, Susan Short, Richard Vile, Alan Melcher. Systemic oncolytic reovirus for the treatment of primary and secondary brain tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A49.

DC vaccine generated by ALA-PDT-induced immunogenic apoptotic cells for skin squamous cell carcinoma

ABSTRACTDendritic cell (DC) vaccines were generated by apoptotic squamous cell carcinoma (SCC) cells induced by 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT). ALA-PDT-DC vaccine inhibited the growth of SCC in mice, indicating that immunogenic apoptotic cells can activate an effective antitumor adaptive immunity and lead to a DC vaccine-based cancer immunotherapy.

Molecular Pathways: Activating T Cells after Cancer Cell Phagocytosis from Blockade of CD47 "Don't Eat Me" Signals.

Recent advances with immunotherapy agents for the treatment of cancer have provided remarkable, and in some cases, curative results. Our laboratory has identified CD47 as an important "don't eat me" signal expressed on malignant cells. Blockade of the CD47:SIRP-α axis between tumor cells and innate immune cells (monocytes, macrophages, and dendritic cells) increases tumor cell phagocytosis in both solid tumors (including, but not limited to, bladder, breast, colon, lung, and pancreatic) and hematologic malignancies. These phagocytic innate cells are also professional antigen-presenting cells (APC), providing a link from innate to adaptive antitumor immunity. Preliminary studies have demonstrated that APCs present antigens from phagocytosed tumor cells, causing T-cell activation. Therefore, agents that block the CD47:SIRP-α engagement are attractive therapeutic targets as a monotherapy or in combination with additional immune-modulating agents for activating antitumor T cells in vivo.

Abstract 1278: Accumulation of tolerogenic human 6-sulfo LacNAc+ dendritic cells in renal cell carcinoma is associated with poor prognosis

Abstract 6-sulfo LacNAc+ (slan) DCs, representing a myeloid human blood DC subset, produce various proinflammatory cytokines, display cytotoxic activity and efficiently stimulate natural killer (NK) cells and T lymphocytes. Recent studies demonstrated that slanDCs accumulate in metastatic tumor-draining lymph nodes from cancer patients, indicating that slanDCs may contribute to antitumor immunity. We constructed tissue microarrays from primary clear-cell renal cell carcinomas (ccRCC) and paired non-neoplastic renal tissues from 265 patients who underwent radical/partial nephrectomy in the Department of Urology, University Clinic, TU Dresden. The slanDCs were identified by immunohistochemistry using a DD2 antibody, developed in the Institute of Immunology, TU Dresden. Freshly prepared tumor-infiltrating cells from 10 specimens were used for phenotypic characterization by flow cytometry. The effects of slanDCs and T-lymphocytes isolated from freshly prepared peripheral blood mononuclear cells on ACHN, CAKI-1 and on two native ccRCC cell lines were assessed by flow-cytomtery, proliferation assays. The frequency of slanDCs in ccRCC and paired non-neoplastic renal tissues were correlated with the clinico-pathological characteristics of patients. SlanDCs were detectable in the majority of primary tumors (93.2%), metastatic lymph nodes (87.5%) and distant metastases (85.1%). A significantly (p<0.001) higher number of slanDCs were detected in primary ccRCC tissues (mean: 3.74 slanDC/mm2, range: 0-19.82 slanDC/mm2) compared to tumor-free tissues (mean: 1.19 slanDC/mm2, range: 0-9.9 slanDC/mm2). A higher density of slanDCs in ccRCC tissues was significantly correlated with a higher grading and stage of the primary tumor (p = 0.001 and p = 0.006 respectively), as well as with lymph node- or distant metastases (p = 0.039 and p<0.001, respectively). Remarkably, a higher density of slanDCs was significantly correlated with a reduced progression-free, tumor-specific or overall survival of ccRCC patients. Freshly prepared ccRCC-infiltrating slanDCs expressed HLA-DR and low densities of CD86, but CD40, CD80, CD83 and CCR7 could not be detected. Whereas the proinflammatory cytokines TNF-α and IL-12 were not found, a proportion of slanDCs showed expression of the anti-inflammatory cytokine IL-10. Further studies revealed that primary ccRCC cells efficiently impair the capacity of slanDCs to stimulate CD4+ and CD8+ T-cell proliferation and Th1 programming. In addition, ccRCC cells significantly inhibited slanDC-mediated NK cell activation. These findings indicate that higher slanDC numbers in ccRCC tissues are associated with poor prognosis. The induction of a tolerogenic phenotype in slanDCs leading to an insufficient activation of innate and adaptive antitumor immunity may represent a novel immune escape mechanism of ccRCC. Citation Format: Marieta I. Toma, Rebekka Wehner, Anja Kloß, Kati Erdmann, Susanne Fuessel, Barbara Seliger, Dorothee Brech, Elfriede Noessner, Knut Schaekel, Manfred Wirth, Gustavo Baretton, Marc Schmitz. Accumulation of tolerogenic human 6-sulfo LacNAc+ dendritic cells in renal cell carcinoma is associated with poor prognosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1278. doi:10.1158/1538-7445.AM2015-1278

Abstract 252: Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of immune checkpoint blockades in K1735 and B16 mouse melanoma models

Abstract Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane and becomes exposed abundantly on tumor endothelial cells, tumor-secreted microvesicles, and tumor cells in response to chemo- and radiotherapy. Extensive studies have shown that PS is a global immune checkpoint and major contributor to tumor immunosuppression, which promotes the expansion of immunosuppressive cells, such MDSC and M2 macrophages, inhibits DCs maturation, while stimulates them to secrete immunosuppressive mediators. We have shown that PS targeting can override PS-mediated immunosuppression, reactivate innate tumor immunity, and evoke adaptive antitumor immunity. In the present study, we assessed the antitumor effect of the combination of PS blockade and anti-CTLA-4 or anti-PD-1 antibodies in B16 and K1735 melanoma models. Both combinations showed significantly superior tumor growth inhibition over single treatment, with many mice achieving complete tumor regression. Flow cytometry analysis showed that the combination treatment had significantly greater total and functional tumor-infiltrating CD8+ T, more IL-2- and IFNγ-producing splenic T cells, and lower number of splenic MDSCs than did single treatment. In addition, the ratio of M2 to M1 in the tumor was significantly lower in the combination treatment than that in single treatment. Finally, no toxicity was observed in any of the treatment groups following multiple treatment doses. These data suggest that combination of PS blockade with immune checkpoint blockade promotes strong, localized, enhanced therapeutic efficacy without the side-effects of systemic immune activation and represents a promising combinatorial strategy for cancer immunotherapy. A chimeric PS-targeting antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in multiple late-stage clinical trials. These data support our Phase I IST evaluation of our PS-targeting antibody bavituximab in combination with ipilimumab in advanced melanoma patients. Citation Format: Bruce Freimark, Jian Gong, Dan Ye, Rolf Brekken, Shen Yin, Jeff Hutchins, Van Nguyen, Chris Hughes, Xianming Huang. Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of immune checkpoint blockades in K1735 and B16 mouse melanoma models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2015-252

Abstract 393: Nicotinamide phosphoribosyl transferase (NAMPT) inhibitors: novel modulators of antitumor immunity

Abstract Nicotinamide phosphoribosyltransferase (NAMPT), an essential enzyme involved in NAD metabolism, is a pleiotropic player in cell signalling and is implicated in a number of diseases, including cancer, inflammatory and metabolic disorders. NAMPT inhibitors (e.g. FK866 and CHS828) have been shown to be potentially effective in cancer therapy and have entered clinical trials in oncology. NAMPT could represent a putative link between metabolism, inflammation and cancer. This study investigated the role of NAMPT in cancer-related inflammation. A mouse model of transplantable MNMCA1 fibrosarcoma was utilized. WT Mice or NAMPTflox CrelysM+/- mice were injected with 10^4 MN-MNCA1 or G217R MNMCA1 (NAMPT inhibitors resistant cell line) cells and subsequently treated or not with 10 mg/kg of NAMPT inhibitors. Next, tumor growth, metastasis formation, tumor angiogenesis and the inflammatory infiltrate were estimated. In addition, monocytic (M-MDSCs) and granulocytic (G-MDSCs) myeloid derived-suppressor cells (MDSCs), isolated from the spleen of tumour bearing mice (untreated and treated with NAMPT inhibitors), or differentiated in vitro from bone marrow precursors, were analysed for their suppressive activity, nitric oxide (NO) production, gene profile and expression of enzymes involved in cancer-associated immunosuppression (e.g. iNOS, ARG1, NOX2). In the fibrosarcoma mouse model, NAMPT inhibitors inhibited tumor growth, tumor vessel formation and delay metastasis formation. Phenotypic analysis of the immune infiltrate revealed that the number of macrophages and dendritic cells was not affected by NAMPT inhibition. However MDSCs displayed a marked decreased in blood, spleen and primary tumour, which was associated with an increase number of both CD4+ and CD8+ T lymphocytes. On the contrary an increase number of MDSCs have been found in the bone marrow. A detail analysis of CXCR4 expression revealed that NAMPT inhibitors affect MDSCs migration in vivo. Moreover, in NAMPTflox CrelysM+/- mice fibrosarcoma growth is reduced. Expression analysis revealed that NAMPT was up-regulated in MDSCs in response to M1-stimulation, suggesting a role of this enzyme during inflammatory process. Moreover, NAMPT inhibitors blocked the NO-mediated T cell suppressive activity induced by IFNγ in M-MDSC. Preliminary evidences indicate a role of both AMPK and SIRT 1/3 in the inhibition of M-MDSC suppressive activity operated by NAMPT inhibitors. Furthermore, both M-MDSCs isolated from spleen of NAMPT inhibitors treated mice and MDSCs isolated from NAMPTflox CrelysM+/- mice display a reduced T-cell suppression activity. Here, we demonstrate for the first time a role of NAMPT metabolism in the functional differentiation of suppressive M-MDSCs associated with cancer development and suggest that pharmacological inhibition of NAMPT may prevent cancer-associated immunosuppression, restoring adaptive antitumor immunity. Citation Format: Cristina Travelli, Sara Morlacchi, Ubaldina Galli, Gian Cesare Tron, Armando A Genazzani, Antonio Sica. Nicotinamide phosphoribosyl transferase (NAMPT) inhibitors: novel modulators of antitumor immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 393. doi:10.1158/1538-7445.AM2015-393

Molecular pathways: cbl proteins in tumorigenesis and antitumor immunity-opportunities for cancer treatment.

The Cbl proteins are a family of ubiquitin ligases (E3s) that regulate signaling through many tyrosine kinase-dependent pathways. A predominant function is to negatively regulate receptor tyrosine kinase (RTK) signaling by ubiquitination of active RTKs, targeting them for trafficking to the lysosome for degradation. Also, Cbl-mediated ubiquitination can regulate signaling protein function by altered cellular localization of proteins without degradation. In addition to their role as E3s, Cbl proteins play a positive role in signaling by acting as adaptor proteins that can recruit signaling molecules to the active RTKs. Cbl-b, a second family member, negatively regulates the costimulatory pathway of CD8 T cells and also negatively regulates natural killer cell function. The different functions of Cbl proteins and their roles both in the development of cancer and the regulation of immune responses provide multiple therapeutic opportunities. Mutations in Cbl that inactivate the negative E3 function while maintaining the positive adaptor function have been described in approximately 5% of myeloid neoplasms. An improved understanding of how the signaling pathways [e.g., Fms-like tyrosine kinase 3 (Flt3), PI3K, and signal transducer and activator of transcription (Stat)] are dysregulated by these mutations in Cbl has helped to identify potential targets for therapy of myeloid neoplasms. Conversely, the loss of Cbl-b leads to increased adaptive and innate antitumor immunity, suggesting that inhibiting Cbl-b may be a means to increase antitumor immunity across a wide variety of tumors. Thus, targeting the pathways regulated by Cbl proteins may provide attractive opportunities for treating cancer.

Engineering monocyte-derived dendritic cells to secrete interferon-α enhances their ability to promote adaptive and innate anti-tumor immune effector functions.

Dendritic cell (DC) vaccination has demonstrated potential in clinical trials as a new effective cancer treatment, but objective and durable clinical responses are confined to a minority of patients. Interferon (IFN)-α, a type-I IFN, can bolster anti-tumor immunity by restoring or increasing the function of DCs, T cells and natural killer (NK) cells. Moreover, type-I IFN signaling on DCs was found to be essential in mice for tumor rejection by the innate and adaptive immune system. Targeted delivery of IFN-α by DCs to immune cells could boost the generation of anti-tumor immunity, while avoiding the side effects frequently associated with systemic administration. Naturally circulating plasmacytoid DCs, major producers of type-I IFN, were already shown capable of inducing tumor antigen-specific T cell responses in cancer patients without severe toxicity, but their limited number complicates their use in cancer vaccination. In the present work, we hypothesized that engineering easily generated human monocyte-derived mature DCs to secrete IFN-α using mRNA electroporation enhances their ability to promote adaptive and innate anti-tumor immunity. Our results show that IFN-α mRNA electroporation of DCs significantly increases the stimulation of tumor antigen-specific cytotoxic T cell as well as anti-tumor NK cell effector functions in vitro through high levels of IFN-α secretion. Altogether, our findings mark IFN-α mRNA-electroporated DCs as potent inducers of both adaptive and innate anti-tumor immunity and pave the way for clinical trial evaluation in cancer patients.

Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation

Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo.

Accumulation of tolerogenic human 6-sulfo LacNAc dendritic cells in renal cell carcinoma is associated with poor prognosis

Dendritic cells (DCs) essentially contribute to the induction and regulation of innate and adaptive immunity. Based on these important properties, DCs may profoundly influence tumor progression in patients. However, little is known about the role of distinct human DC subsets in primary tumors and their impact on clinical outcome. In the present study, we investigated the characteristics of human 6-sulfo LacNAc (slan) DCs in clear cell renal cell carcinoma (ccRCC). slanDCs have been shown to display various tumor-directed properties and to accumulate in tumor-draining lymph nodes from patients. When evaluating 263 ccRCC and 227 tumor-free tissue samples, we found increased frequencies of slanDCs in ccRCC tissues compared to tumor-free tissues. slanDCs were also detectable in the majority of 24 metastatic lymph nodes and 67 distant metastases from ccRCC patients. Remarkably, a higher density of slanDCs was significantly associated with a reduced progression-free, tumor-specific or overall survival of ccRCC patients. Tumor-infiltrating slanDCs displayed an immature phenotype expressing interleukin-10. ccRCC cells efficiently impaired slanDC-induced T-cell proliferation and programming as well as natural killer (NK) cell activation. In conclusion, these findings indicate that higher slanDC numbers in ccRCC tissues are associated with poor prognosis. The induction of a tolerogenic phenotype in slanDCs leading to an insufficient activation of innate and adaptive antitumor immunity may represent a novel immune escape mechanism of ccRCC. These observations may have implications for the design of therapeutic strategies that harness tumor-directed functional properties of DCs against ccRCC.

OX40 ligand expressed in glioblastoma modulates adaptive immunity depending on the microenvironment: a clue for successful immunotherapy.

BackgroundGlioblastoma is the most malignant human brain tumor and has a dismal prognosis; however, some patients show long-term survival. The interaction between the costimulatory molecule OX40 and its ligand OX40L generates key signals for T-cell activation. The augmentation of this interaction enhances antitumor immunity. In this present study, we explored whether OX40 signaling is responsible for antitumor adaptive immunity against glioblastoma and also established therapeutic antiglioma vaccination therapy.MethodsTumor specimens were obtained from patients with primary glioblastoma (n = 110) and grade III glioma (n = 34). Quantitative polymerase chain reaction (PCR), flow cytometry, and immunohistochemistry were used to analyze OX40L expression in human glioblastoma specimens. Functional consequences of OX40 signaling were studied using glioblastoma cell lines, mouse models of glioma, and T cells isolated from human subjects and mice. Cytokine production assay with mouse regulatory T cells was conducted under hypoxic conditions (1.5% O2).ResultsOX40L mRNA was expressed in glioblastoma specimens and higher levels were associated with prolonged progression-free survival of patients with glioblastoma, who had undergone gross total resection. In this regard, OX40L protein was expressed in A172 human glioblastoma cells and its expression was induced under hypoxia, which mimics the microenvironment of glioblastoma. Notably, human CD4 T cells were activated when cocultured in anti-CD3-coated plates with A172 cells expressing OX40L, as judged by the increased production of interferon-γ. To confirm the survival advantage of OX40L expression, we then used mouse glioma models. Mice bearing glioma cells forced to express OX40L did not die during the observed period after intracranial transplantation, whereas all mice bearing glioma cells lacking OX40L died. Such a survival benefit of OX40L was not detected in nude mice with an impaired immune system. Moreover, compared with systemic intraperitoneal injection, the subcutaneous injection of the OX40 agonist antibody together with glioma cell lysates elicited stronger antitumor immunity and prolonged the survival of mice bearing glioma or glioma-initiating cell-like cells. Finally, OX40 triggering activated regulatory T cells cultured under hypoxia led to the induction of the immunosuppressive cytokine IL10.ConclusionGlioblastoma directs immunostimulation or immunosuppression through OX40 signaling, depending on its microenvironment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0307-3) contains supplementary material, which is available to authorized users.

Immune Suppression during Oncolytic Virotherapy for High-Grade Glioma; Yes or No?

Oncolytic viruses have been seriously considered for glioma therapy over the last 20 years. The oncolytic activity of several oncolytic strains has been demonstrated against human glioma cell lines and in in vivo xenotransplant models. So far, four of these stains have additionally completed the first phase I/II trials in relapsed glioma patients. Though safety and feasibility have been demonstrated, therapeutic efficacy in these initial trials, when described, was only minor. The role of the immune system in oncolytic virotherapy for glioma remained much less studied until recent years. When investigated, the immune system, adept at controlling viral infections, is often hypothesized to be a strong hurdle to successful oncolytic virotherapy. Several preclinical studies have therefore aimed to improve oncolytic virotherapy efficacy by combining it with immune suppression or evasion strategies. More recently however, a new paradigm has developed in the oncolytic virotherapy field stating that oncolytic virus-mediated tumor cell death can be accompanied by elicitation of potent activation of innate and adaptive anti-tumor immunity that greatly improves the efficacy of certain oncolytic strains. Therefore, it seems the three-way interaction between oncolytic virus, tumor and immune system is critical to the outcome of antitumor therapy. In this review we discuss the studies which have investigated how the immune system and oncolytic viruses interact in models of glioma. The novel insights generated here hold important implications for future research and should be incorporated into the design of novel clinical trials.

CN-08 * THERAPEUTIC CONCENTRATION OF TEMOZOLOMIDE DO NOT IMPAIR THE FUNCTION OF HUMAN DENDRITIC CELL SUBSETS

In humans, CD11c+ myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) represent two major subsets of DCs. They play distinct roles in innate and adaptive anti-tumor immunity by their expression of specialized cytokines and molecules. In addition, monocytes-derived DCs (moDCs) as in vitro-made myeloid DCs are commonly used as DC-vaccine therapy for several malignancies. However, the efficacy of DC-vaccine monotherapy has been reported to be limited, and therefore, combination use of appropriate chemotherapy would be required for the treatment strategy of immunotherapy. Because anti-tumor reagents generally have an activity to kill hematopoietic cells or inhibit lymphocyte functions, congeniality of anti-tumor reagents and DCs is important for the effective combination therapy. Temozolomide (TMZ) is an oral alkylating agent with established anti-tumor activity in patients with glioblastoma. In this study, we focused on the TMZ function and explored its influence on human DC subsets, including blood CD11c+ mDCs, pDCs, and moDCs. We found that over 300 mM of TMZ killed the DC subsets but that there were no killing effect at in vivo clinical concentration of less than 100 mM TMZ. Up to 100 mM TMZ did not affect IFN-a production from pDCs and IL-12 production from blood mDCs and moDCs and their expression of surface B7 and TNF superfamilies in response to various TLR ligands. In addition, TMZ did not inhibit moDC-mediated T cell responses. Thus, TMZ can elicit anti-tumor activity without affecting DC-mediated immune responses. Our present findings suggest that TMZ is a potential anti-tumor reagent suitable for the combination use with DC-vaccine therapy.

Tumors STING Adaptive Antitumor Immunity

Immunotherapy is revolutionizing the treatment of cancer patients, but the molecular basis for tumor immunogenicity is unclear. In this issue of Immunity, Deng etal. (2014) and Woo etal. (2014) provide evidence suggesting that dendritic cells detect DNA from tumor cells via the STING-mediated, cytosolic DNA sensing pathway.

Abstract 3636: Anti-CD20 therapy prevents protumor regulatory T-cell expansion and triggers a memory Th1 response in tumor-bearing mice

Abstract The long-lasting clinical response by lymphoma patients to anti-CD20 treatment has been attributed to a ‘vaccinal’-type effect through the induction of an anti-tumor adaptive immunity. Using a model where C57Bl/6 mice were injected intravenously with EL4 tumor cells expressing human CD20, we previously showed that CD4 cells are central to the host protection induced by anti-CD20 therapy. Here, we have first shown that the transfer of highly purified CD4+ T cells induces a long-term protection, demonstrating that this T-cell subset plays indeed a key role in the induction of an adaptive memory response. In addition, CD4+ effector memory T cells were detected in surviving animals. We have then analyzed the changes occurring in various T cell subsets in tumor-bearing animals receiving anti-CD20 treatment. On the one hand, untreated animals exhibited a large expansion of CD4+FoxP3+ regulatory T cells (Tregs), with reduced survival. Depletion of these cells using anti-CD25 injection led to long-term survival, indicating that a Treg-dependent immuno-suppressive environment is established after tumor injection in this model. On the other hand, anti-CD20 therapy, that induces a long-lasting protection, reversed the initial expansion of Tregs one week after the end of anti-CD20 treatment, and was accompanied by a marked increase in the number of Th1 cells. Concomitantly, an increase in the serum levels of interleukin-12 was observed, as well as an increased in the number of activated myeloid dendritic cells producing this cytokine. Moreover, a strong decrease in the survival was observed when Natural Killer cells were depleted using anti-asialo GM1 antibodies or following neutralization of interferon-γ. Thus, the host protection elicited by anti-CD20 treatment involves a network of cells from both innate and adaptive immunity that both are needed for long-term protection. In conclusion, our work demonstrates that anti-CD20 therapy promotes a strong anti-tumor adaptive immunity, opposes Treg expansion, and that a pro-tumor immune contexture shaped by the presence of growing tumor cells can be reversed by antibody treatment. Citation Format: Claire Deligne, Sophie Siberil, Jean-Luc Teillaud. Anti-CD20 therapy prevents protumor regulatory T-cell expansion and triggers a memory Th1 response in tumor-bearing mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3636. doi:10.1158/1538-7445.AM2014-3636

Newcastle disease virotherapy induces long‐term survival and tumor‐specific immune memory in orthotopic glioma through the induction of immunogenic cell death

The oncolytic features of several naturally oncolytic viruses have been shown on Glioblastoma Multiforme cell lines and in xenotransplant models. However, orthotopic glioma studies in immunocompetent animals are lacking. Here we investigated Newcastle disease virus (NDV) in the orthotopic, syngeneic murine GL261 model. Seven days after tumor induction, mice received NDV intratumorally. Treatment significantly prolonged median survival and 50% of animals showed long-term survival. We demonstrated immunogenic cell death (ICD) induction in GL261 cells after NDV infection, comprising calreticulin surface exposure, release of HMGB1 and increased PMEL17 cancer antigen expression. Uniquely, we found absence of secreted ATP. NDV-induced ICD occurred independently of caspase signaling and was blocked by Necrostatin-1, suggesting the contribution of necroptosis. Autophagy induction following NDV infection of GL261 cells was demonstrated as well. In vivo, elevated infiltration of IFN-γ(+) T cells was observed in NDV-treated tumors, along with reduced accumulation of myeloid derived suppressor cells. The importance of a functional adaptive immune system in this paradigm was demonstrated in immunodeficient Rag2(-/-) mice and in CD8(+) T cell depleted animals, where NDV slightly prolonged survival, but failed to induce long-term cure. Secondary tumor induction with GL261 cells or LLC cells in mice surviving long-term after NDV treatment, demonstrated the induction of a long-term, tumor-specific immunological memory response by ND virotherapy. For the first time, we describe the therapeutic activity of NDV against GL261 tumors, evidenced in an orthotopic mouse model. The therapeutic effect relies on the induction of ICD in the tumor cells, which primes adaptive antitumor immunity.

Anti-CD20 therapy induces a memory Th1 response through the IFN-γ/IL-12 axis and prevents protumor regulatory T-cell expansion in mice.

The long-lasting clinical response by lymphoma patients to anti-CD20 therapy has been attributed to the induction of an anti-tumor adaptive immunity. We previously demonstrated that a CD4-dependent mechanism is responsible for the long-term protection of CD20(+) tumor-bearing mice by anti-CD20 treatment. Here, we compare tumor immunity in tumor-bearing animals that did or did not receive anti-CD20 treatment. Splenic CD4(+)FoxP3(+) regulatory T cells (Tregs) expanded substantially in untreated mice that exhibited then a reduced survival, whereas Tregs depletion led to long-term survival of the animals, suggesting the establishment of a Treg-dependent immunosuppressive environment after tumor injection. Strikingly, anti-CD20 therapy reversed the initial expansion of Tregs, and was accompanied by a marked increase in the number of Th1 cells, with no detectable change in Th2 and Th17 cell numbers. Interleukin-12 serum level was also increased by the anti-CD20 treatment, and activated myeloid dendritic cells producing interleukin-12 could be detected in lymph nodes of treated animals, while interferon-γ blockade strongly reduced survival. Also, CD4(+) effector memory T cells were evidenced in surviving animals, and the transfer of CD4(+) T cells induced long-term protection. Thus, anti-CD20 therapy promotes strong anti-tumor adaptive immunity, opposes Treg expansion and inhibits tumor cells from maintaining an immunosuppressive environment.