Published OnlineFirst July 13, 2018; DOI: 10.1158/1078-0432.CCR-17-3855 Translational Cancer Mechanisms and Therapy Clinical Cancer Research D-2-Hydroxyglutarate Is an Intercellular Mediator in IDH-Mutant Gliomas Inhibiting Complement and T Cells Lingjun Zhang1, Mia D. Sorensen2,3, Bjarne W. Kristensen2,3, Guido Reifenberger4, Thomas M. McIntyre5, and Feng Lin1 Abstract Purpose: Somatic mutations in the isocitrate dehydroge- Ex vivo experiments with D 2-HG identified immune inhib- nase (IDH)-1 and -2 genes are remarkably penetrant in diffuse itory mechanisms. gliomas. These highly effective gain-of-function mutations Results: IDH mutation associated with significantly reduced enable mutant IDH to efficiently metabolize isocitrate to D- complement activation and decreased numbers of tumor- þ þ þ 2-hydroxyglutarate (D 2-HG) that accumulates to high con- infiltrating CD4 and CD8 T cells with comparable FOXP3 / þ centrations within the tumor microenvironment. D 2-HG is an CD4 ratios. D 2-HG potently inhibited activation of com- intracellular effector that promotes tumor growth through plement by the classical and alternative pathways, attenuated widespread epigenetic changes in IDH-mutant tumor cells, complement-mediated glioma cell damage, decreased cellular but its potential role as an intercellular immune regulator C3b(iC3b) opsonization, and impaired complement-mediat- remains understudied. ed phagocytosis. Although D 2-HG did not affect dendritic cell þ Experimental Design: Complement activation and CD4 , differentiation or function, it significantly inhibited activated þ þ CD8 ,orFOXP3 T-cell infiltration into primary tumor T-cell migration, proliferation, and cytokine secretion. tissue were determined by immunohistochemistry using Conclusions: D 2-HG suppresses the host immune system, sections from 72 gliomas of World Health Organization potentially promoting immune escape of IDH-mutant (WHO) grade III and IV with or without IDH mutations. tumors. Clin Cancer Res; 24(21); 5381–91. Ó2018 AACR. Introduction within (10) and 3 mmol/L surrounding (11) gliomas carrying a mutant IDH-1 or IDH-2 gene. D 2-HG alters tumor cell metab- Site-specific mutations of isocitrate dehydrogenase (IDH)-1 or olism and epigenetic regulation (12–14), but the full significance -2 are present in 80% to 90% of patients with diffuse WHO grade of IDH mutations or more precisely the unique nature of II–III gliomas and a small subset of patients with WHO grade IV excessive D 2-HG accumulation is undefined. For instance, we glioblastomas (1–4). IDH mutations are also present but less now know that tumor IDH mutation tightly correlates to the penetrant in acute myeloid leukemia (5), angioimmunoblastic T- absence of microthrombi within the tumor vasculature of diffuse cell lymphoma (6), and chondrosarcomas (7). However, precisely gliomas, and that D 2-HG directly suppresses ex vivo activation how IDH mutations might confer an advantage to tumorigenesis and thrombosis of purified platelets (15). Potentially, then, is not well understood. tumor-derived D 2-HG functions as an intercellular mediator Missense mutations of R132 in IDH-1 or R172 in IDH-2 that that affects nonneoplastic cells of the tumor microenvironment. change this arginyl residue to any of several other residues confer þ þ Tumor-infiltrating CD4 helper and CD8 cytotoxic T cells are a remarkable gain of function to IDH catalytic activity enabling present in the glioma microenvironment (16), and mutant IDH mutant enzyme to stereospecifically reduce isocitrate to D-2- associates with fewer infiltrating immune cells, including macro- hydroxyglutarate (D 2-HG; refs. 2, 8, 9) rather than its normal phages, T cells, and B cells, in tumors (17–19), and IDH-mutant product a-ketoglutarate. D 2-HG accumulates to 30 mmol/L gliomas may escape from natural killer (NK) cell immune surveillance by downregulation of their NK group 2, member D (NKG2D) ligand expression (20). 1Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleve- Complement is a key component of the innate immune system land, Ohio. 2Department of Pathology, Odense University Hospital, Odense C, that defends against pathogen invasion and clears apoptotic cells Denmark. 3Department of Clinical Research, University of Southern Denmark, and immune complexes. When activated by either classical, 4 Odense C, Denmark. Department of Neuropathology, Heinrich Heine University, alternative, or lectin pathways, activated complement forms Dusseldorf,€ Germany. 5Department of Cellular and Molecular Medicine, Lerner membrane attack complex (MAC) pores that lyse targeted cells Research Institute, Cleveland Clinic, Cleveland, Ohio. (21). Complement activation also leads the deposition of C3b Note: Supplementary data for this article are available at Clinical Cancer (iC3b) fragments on target cells for "opsonization" that facilitates Research Online (http://clincancerres.aacrjournals.org/). phagocytosis through interactions with C3b(iC3b) receptors Corresponding Author: Feng Lin, Cleveland Clinic, Cleveland, OH 44095. Phone: (C3aR) expressed on phagocytes. Recent studies (22–24) also 2164456637; E-mail: [email protected] found that complement directly regulates T-cell function, in part doi: 10.1158/1078-0432.CCR-17-3855 through signaling of G-protein coupled C3aR and C5aR receptors Ó2018 American Association for Cancer Research. on antigen-presenting cells and T cells. www.aacrjournals.org 5381 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst July 13, 2018; DOI: 10.1158/1078-0432.CCR-17-3855 Zhang et al. Complement-mediated tumor cytotoxicity assay Translational Relevance Complement-mediated brain tumor cell damage assay was D-2-Hydroxyglutarate produced by gliomas expressing done based on the measurement of lactate dehydrogenase (LDH) mutant isocitrate dehydrogenase (IDH) is an intercellular leakage using a commercial kit (Sigma-Aldrich). modulator inhibiting innate and adaptive immune systems. These new insights could aid the development of better Complement C3b deposition assay immunotherapy for tumors with mutant IDH. EshA were incubated with 2% C5-depleted serum in gelatin þþ veronal buffer with calcium and magnesium (GVB )contain- ing defined concentrations of D 2-HG. For negative controls, 5 mmol/L EDTA was added to the buffer. After 10 minutes at shA Here, we determined whether the immunologic microenviron- 37 C, E were washed and stained with an Alexa Fluor 488- ment of adult diffuse gliomas is affected by IDH mutational conjugated anti-human C3 antibody (MP Biomedicals) for fl status. We find that IDH mutation associates with reduced com- additional 30 minutes on ice, followed by ow cytometry þ þ þ plement activation, decreased CD4 , FOXP3 , and CD8 T-cell analysis. infiltration in gliomas in situ, and that D 2-HG directly suppresses these essential elements of both innate and adaptive immunity. Complement opsonization-mediated phagocytosis assay The myeloid cell line U937 was differentiated into macro- Materials and Methods phages for the complement opsonization-mediated phagocytosis assay based on a published protocol (31, 32). Expanded Materials and Methods are presented in a supple- ment to this article. T-cell inhibition and migration assays Nylon wool-enriched T cells, or negative selection-purified þ þ Patient tissue CD4 and CD8 T cells from WT mice were activated by mono- Tissues were obtained from patients diagnosed with primary clonal antibodies against CD3 and CD28, then cultured in dif- high-grade astrocytoma between 1997 and 2017. All tumor ferent polarization conditions in the presence of different con- samples were classified or reclassified according to the WHO centrations of D 2-HG. The inhibitory effect of D 2-HG was Classification 2016 (25). Patients underwent initial surgery at assessed by measuring the proliferation of the activated T cells the Department of Neurosurgery, Odense University Hospital, using carboxyfluorescein succinimidyl ester (CFSE) dilution and Denmark, or at the Department of Neurosurgery, Heinrich bromodeoxyuridine (BrdU) incorporation. In addition, cytokines Heine University, Dusseldorf,€ Germany. None of the patients produced by the activated T cells were quantitatively assessed in had received treatment prior to surgery. Of the 72 patients the culture supernatants by ELISA, and the generation of Tregs þ þ þ included in the current study, 23 were WHO grade III anaplastic were assessed by analyzing CD4 CD25 FOXP3 cells using flow astrocytomas and IDH-mutant (mIDH), 16 were WHO grade cytometry. III anaplastic astrocytomas and IDH-wild-type (wtIDH), 14 Impact of D 2-HG on T-cell migration was assessed in a were WHO grade IV glioblastomas with mIDH, and 19 were conventional transwell migration assay. WHO grade IV glioblastomas with wtIDH. IDH status was determined by immunohistochemistry using an antibody Bone marrow–derived dendritic cell differentiation and against the most common IDH-1-R132H mutation (clone function assay H14, Dianova) using the BenchMark Ultra IHC/ISH staining Dendritic cells (DC) were generated from bone marrow using a system (Ventana Medical Systems, Inc.; ref. 26), and/or by next- published protocol (33), and their function was assessed using generation sequencing as previously described (27). Of the 37 antigen-specific T cells from ovalbumin peptide 323–339 detected IDH mutations, 31 were IDH-1-R132H, three were (OVA323-339)-specific TCR transgenic mice (OT II mice) and IDH-1-R132C, and one each corresponded