Peter Chockley

Interleukin-6 modulates graft-versus-host responses after experimental allogeneic bone marrow transplantation

Purpose: The graft-versus-tumor (GVT) effect is a potent form of immunotherapy against many hematologic malignancies and some solid tumors. The beneficial GVT effect after allogeneic bone marrow transplantation (BMT) is tightly linked to its most significant complication, graft-versus-host disease (GVHD). The role of interleukin-6 (IL-6) after allogeneic BMT is not well understood. This study used a series of complementary knockout and antibody blockade strategies to analyze the impact of IL-6 in multiple clinically relevant murine models of GVHD and GVT. Experimental Design: We examined the effect of the source of IL-6 by analyzing the role IL-6 deficiency in donor T cells, donor bone marrow or in host tissues. We confirmed and extended the relevance of IL-6 deficiency on GVHD and GVT by treating BMT recipients with anti-mouse IL-6 receptor (IL-6R), MR16-1. Results: Deficiency of IL-6 in donor T cells led to prolongation of survival. Total inhibition of IL-6 with MR16-1 caused an even greater reduction in GVHD-induced mortality. The reduction in GVHD was independent of the direct effects on T effector cell expansion or donor regulatory T cells. GVT responses were preserved after treatment with MR16-1. Conclusion: MR16-1 treatment reduced GVHD and preserved sufficient GVT. Tocilizumab, a humanized anti–IL-6R monoclonal antibody (mAb), is approved in several countries including the United States and European Union for the treatment of rheumatoid arthritis and other inflammatory diseases. Blockade of IL-6 with anti–IL-6R mAb therapy may be testable in clinical trials as an adjunct to prevent GVHD in BMT patients without a significant loss of GVT. Clin Cancer Res; 17(1); 77–88. ©2010 AACR.

Natural killer cells eradicate galectin-1-deficient glioma in the absence of adaptive immunity

Natural killer (NK) cells safeguard against early tumor formation by destroying transformed target cells in a process referred to as NK immune surveillance. However, the immune escape mechanisms used by malignant brain tumors to subvert this innate type of immune surveillance remain unclear. Here we show that malignant glioma cells suppress NK immune surveillance by overexpressing the β-galactoside-binding lectin galectin-1. Conversely, galectin-1-deficient glioma cells could be eradicated by host NK cells before the initiation of an antitumor T-cell response. In vitro experiments demonstrated that galectin-1-deficient GL26-Cit glioma cells are ∼3-fold more sensitive to NK-mediated tumor lysis than galectin-1-expressing cells. Our findings suggest that galectin-1 suppression in human glioma could improve patient survival by restoring NK immune surveillance that can eradicate glioma cells. Cancer Res; 74(18); 5079-90. ©2014 AACR.

Ikaros-Notch axis in host hematopoietic cells regulates experimental graft-versus-host disease.

Host hematopoietically derived APCs play a vital role in the initiation of GVH responses. However, the APC autonomous molecular mechanisms that are critical for the induction of GVHD are not known. We report here that the Ikaros-Notch axis in host hematopoietically derived APCs regulates the severity of acute GVHD across multiple clinically relevant murine models of experimental bone marrow transplantation. In the present study, Ikaros deficiency (Ik(-/-)) limited to host hematopoietically derived APCs enhanced donor T-cell expansion and intensified acute GVHD, as determined by survival and other GVHD-specific parameters. The Ik(-/-) conventional CD8(+) and CD8(-)CD11c(+) dendritic cells (DCs), the most potent APCs, showed no increase in the expression of activation markers or in response to TLR stimulation compared with wild-type controls. However, Ik(-/-) DCs demonstrated an enhanced stimulation of allogeneic T cells. Deficiency of Ikaros in the conventional CD8(+) and CD8(-)CD11c(+) DCs was associated with an increase in Notch signaling, the blockade of which mitigated the enhanced in vitro and in vivo allostimulatory capacity. Therefore, the Ikaros-Notch axis is a novel pathway that modulates DC biology in general, and targeting this pathway in host hematopoietically derived APCs may reduce GVHD.

Natural killer cells require monocytic Gr-1+/CD11b+ myeloid cells to eradicate orthotopically engrafted glioma cells

ABSTRACT Malignant gliomas are resistant to natural killer (NK) cell immune surveillance. However, the mechanisms used by these cancers to suppress antitumor NK cell activity remain poorly understood. We have recently reported on a novel mechanism of innate immune evasion characterized by the overexpression of the carbohydrate-binding protein galectin-1 by both mouse and rat malignant glioma. Here, we investigate the cytokine profile of galectin-1-deficient GL26 cells and describe the process by which these tumors are targeted by the early innate immune system in RAG1−/− and C57BL/6J mice. Our data reveal that galectin-1 knockdown in GL26 cells heightens their inflammatory status leading to the rapid recruitment of Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells into the brain tumor microenvironment, culminating in tumor clearance. We show that immunodepletion of Gr-1+ myeloid cells in RAG1−/− mice permits the growth of galectin-1-deficient glioma despite the presence of NK cells, thus demonstrating an essential role for myeloid cells in the clearance of galectin-1-deficient glioma. Further characterization of tumor-infiltrating Gr-1+/CD11b+ cells reveals that these cells also express CCR2 and Ly-6C, markers consistent with inflammatory monocytes. Our results demonstrate that Gr-1+/CD11b+ myeloid cells, often referred to as myeloid-derived suppressor cells (MDSCs), are required for antitumor NK cell activity against galectin-1-deficient GL26 glioma. We conclude that glioma-derived galectin-1 represents an important factor in dictating the phenotypic behavior of monocytic Gr-1+/CD11b+ myeloid cells. Galectin-1 suppression may be a valuable treatment approach for clinical glioma by promoting their innate immune-mediated recognition and clearance through the concerted effort of innate myeloid and lymphoid cell lineages.

Immunological Consequences of Epithelial-Mesenchymal Transition in Tumor Progression.

Microenvironments that tumor cells encounter are different during the stages of cancer progression—primary tumor, metastasis, and at the metastatic site. This suggests potential differences in immune surveillance of primary tumor and metastasis. Epithelial–mesenchymal transition (EMT) is a key reversible process in which cancer cells transition into highly motile and invasive cells for dissemination. Only a tiny proportion successfully metastasize, supporting the notion of metastasis-specific immune surveillance. EMT involves extensive molecular reprogramming of cells conferring many clinically relevant features to cancer cells and affects tumor cell interactions within the tumor microenvironment. We review the impact of tumor immune infiltrates on tumor cell EMT and the consequences of EMT in shaping the immune microenvironment of tumors. The usefulness of EMT as a model to investigate metastasis-specific immune surveillance mechanisms are also explored. Finally, we discuss potential implications of EMT for tumor immunogenicity, as well as current immunotherapies and future strategies.

Epithelial-mesenchymal transition leads to NK cell–mediated metastasis-specific immunosurveillance in lung cancer

During epithelial-mesenchymal transition (EMT) epithelial cancer cells transdifferentiate into highly motile, invasive, mesenchymal-like cells, giving rise to disseminating tumor cells. Few of these disseminated cells successfully metastasize. Immune cells and inflammation in the tumor microenvironment were shown to drive EMT, but few studies investigated the consequences of EMT for tumor immunosurveillance. In addition to initiating metastasis, we demonstrate that EMT confers increased susceptibility to natural killer (NK) cells and contributes, in part, to the inefficiency of the metastatic process. Depletion of NK cells allowed spontaneous metastasis without affecting primary tumor growth. EMT-induced modulation of E-cadherin and cell adhesion molecule 1 (CADM1) mediated increased susceptibility to NK cytotoxicity. Higher CADM1 expression correlates with improved patient survival in 2 lung and 1 breast adenocarcinoma patient cohorts and decreased metastasis. Our observations reveal a novel NK-mediated, metastasis-specific immunosurveillance in lung cancer and present a window of opportunity for preventing metastasis by boosting NK cell activity.

IMPS-01Gr-1+/CD11b+MYELOID CELLS ARE REQUIRED FOR NATURAL KILLER CELL-MEDIATED ERADICATION OF GALECTIN-1-DEFICIENT GLIOMA

Glioblastoma (GBM) is highly infiltrated by immune cells of the myeloid lineage; however, the immunosuppressive microenvironment established by GBM favors anti-inflammatory activity in these cells and in turn promotes tumor immune escape. We have recently shown that shRNA-mediated knockdown of the β-galactoside-binding lectin galectin-1 (gal-1) in malignant glioma cells leads to glioma eradication within a week of intracranial engraftment (Baker et al, Cancer Research. 2014; 74:5079-90). NK cells are central to the rejection of gal-1-deficient glioma as immunodepletion of these cells with anti-asialo GM1 or NK1.1 antibodies permitted the growth of these tumors. We now report that immunodepletion of Gr-1+ myeloid cells in RAG1−/- mice also permits the growth of gal-1-deficient glioma, despite the presence of NK cells. This intriguing observation establishes that NK cells require collateral support by Gr-1+ myeloid cells to exert anti-glioma immune surveillance activity. We found that gal-1-deficient mouse GL26-Cit cells are pro-inflammatory as they upregulate expression of chemokines CXCL10/IP-10 and RANTES. Seven-fold more Gr-1+/CD11b+ myeloid cells infiltrated the gal-1-deficient GL26-Cit microenvironment compared to infiltration into control GL26-Cit tumors. Myeloid infiltration was followed by a 10-fold increase in the number of NK cells. Implantation of gal-1-deficient GL26-Cit cells into the brain of B6.Myd88−/- mice failed to recruit peripheral blood mononuclear cells (PBMCs) into the tumor microenvironment 72 hours post-engraftment, thus permitting gal-1-deficient glioma growth. This implied that TLR/Myd88−/- signaling is required to induce the entry of PBMCs into the gal-1-deficient microenvironment to cause tumor eradication. Experiments are now underway to determine the identity of the biomolecular factors produced by gal-1-deficient GL26-Cit glioma cells responsible for initiating Myd88 signaling and the receptors and cells in which Myd88 signaling is necessary for glioma rejection. These studies identify a novel innate immune network necessary and sufficient for early glioma rejection without requiring contributions from the adaptive immune system.

Abstract 3195: STAT3 inhibition using shRNA inhibits GBM proliferation, cell migration, anchorage-independent growth of mouse, rat, and human stem-like cells in vitro; and it induces long term survival and anti-GBM immunity in vivo

The purpose of this study was to elucidate the role of Signal Transducers and Activators of Transcription 3 (STAT3) signaling on the tumor microenvironment in the most commonly occurring and aggressive primary brain tumor, Glioblastoma Multiforme (GBM), including preclinical testing of inhibition of STAT3 expression in vivo in syngeneic and patient derived xenograft (PDX) models of GBM. GBM is genetically heterogeneous, but always overexpresses genes that are vital to cell cycle regulation, cell growth and proliferation, cell invasion, and angiogenesis. STAT proteins are transcription factors associated with gene regulation and expression signatures that are implicated in several survival pathways that enable human GBMs to grow in the brain parenchyma. In addition, STAT3 has been identified as a central mechanism in tumor-induced immunosuppression in GBM and other cancers. We studied the effects of STAT3 inhibition via shRNA down-regulation in vivo and in vitro using patient derived primary glioma cells (HF2303 & MGG8) in conjunction with other glioma lines from humans, mice, and rats (U251, GL26, and CNS-1, respectively). In this study, we found that GBM cells harboring down-regulated STAT3 signaling exhibit delayed proliferation, increased apoptosis, and anchorage independence. In vivo, STAT3 inhibition resulted in increased survival rates when tumor cells were treated with the STAT3 shRNA both pre- and post-GBM implantation (syngeneic GBM model). Our data demonstrate that STAT3 has a profound influence on the GBM microenvironment, which prevents the host from clearing the tumor. Down-regulating this signaling pathway using gene therapeutic strategies allows for infiltration of immune cells, decreased invasion, and a decrease in GBM cells’ proliferation that leads to the tumor elimination in ∼83% of the animals, this was associated with the development of an effective anti-tumor immunity that prevents tumor recurrence. Given the phenotype of STAT3 inhibition in in vitro assays and its success in in vivo GBM models, inhibition of STAT3 expression using shRNA and gene therapy technologies constitutes an attractive strategy for preclinical development as a potent therapeutic target for GBM. Citation Format: Nathan T. VanderVeen, Nicholas Raja, Elizabeth Yi, James Curtin, Peter Chockley, Hikmat Assi, Jonathan Savakus, Tom Mikkelsen, Samuel Rabkin, Pedro R. Lowenstein, Maria G. Castro. STAT3 inhibition using shRNA inhibits GBM proliferation, cell migration, anchorage-independent growth of mouse, rat, and human stem-like cells in vitro; and it induces long term survival and anti-GBM immunity in vivo. [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 3195. doi:10.1158/1538-7445.AM2015-3195

Abstract 3651: Natural killer cells eradicate galectin-1 deficient glioma in the absence of adaptive immunity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Natural killer (NK) cells safeguard against early tumor formation by seeking out and destroying transformed target cells in a process referred to as NK immunosurveillance. While it is clear that malignant brain tumors such as glioblastoma (GBM) evade NK-mediated tumor suppression, the precise mechanisms by which this occurs remain unknown. We now show that shRNA-mediated knockdown of the β-galactoside-binding lectin, galectin-1 (gal-1), in malignant glioma cells leads to the failure to form lethal intracranial tumors in RAG1-/- mice, a mouse strain devoid of adaptive immunity. However, gal-1 deficient glioma growth is fully restored on implantation into the brain of severely immunocompromised NOD-scid IL2Rg null mice, which lack both adaptive and innate immune function, thus implicating the innate immune response in the early rejection of gal-1 deficient glioma. Immunodepletion of NK cells in RAG1-/- or C57BL/6J mice using anti-asialo GM1 or anti-NK1.1 antibodies permit the growth of large gal-1 deficient gliomas, while macrophage depletion with clodronate liposomes only permits limited tumor growth. This combined result suggests that NK cells and macrophages may work together to achieve gal-1 deficient glioma rejection. Antigen-specific IFN-γ ELISpot assays using splenocytes from immunocompetent C57BL/6J mice indicate that gal-1 deficient glioma is cleared prior to the onset of an adaptive anti-tumor immune response. Flow cytometric analysis of brain tumor-infiltrating immune cells reveal that gal-1 deficient gliomas contain significantly more macrophages and granzyme B+ NK cells compared to gal-1 expressing gliomas. In-vitro experiments further show that gal-1 deficient glioma cells are inherently over 3-times more sensitive to NK-mediated tumor lysis, fail to suppress pro-inflammatory (M1) microglial activation, and secrete pro-inflammatory cytokines IL-1β, IL-12p70, and CXCL2. We conclude that glioma-derived gal-1 is a powerful inhibitor of NK-mediated cytotoxicity in-vivo, and predict that its suppression will be of therapeutic value in the treatment of human malignant brain tumors by dramatically heightening anti-tumor NK immunosurveillance. Citation Format: Gregory J. Baker, Viveka Nand Yadav, Peter Chockley, Robert Doherty, Michael Ritt, Sivaraj Sivaramakrishnan, Maria G. Castro, Pedro R. Lowenstein. Natural killer cells eradicate galectin-1 deficient glioma in the absence of adaptive immunity. [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 3651. doi:10.1158/1538-7445.AM2014-3651