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. 2018 Nov 1;24(21):5381-5391.
doi: 10.1158/1078-0432.CCR-17-3855. Epub 2018 Jul 13.

V体育2025版 - D-2-Hydroxyglutarate Is an Intercellular Mediator in IDH-Mutant Gliomas Inhibiting Complement and T Cells

Lingjun Zhang  1 , Mia D Sorensen  2   3 , Bjarne W Kristensen  2   3 , Guido Reifenberger  4 , Thomas M McIntyre  5 , Feng Lin  6
Affiliations

"VSports最新版本" D-2-Hydroxyglutarate Is an Intercellular Mediator in IDH-Mutant Gliomas Inhibiting Complement and T Cells

V体育ios版 - Lingjun Zhang et al. Clin Cancer Res. .

"V体育ios版" Abstract

Purpose: Somatic mutations in the isocitrate dehydrogenase (IDH)-1 and -2 genes are remarkably penetrant in diffuse gliomas. These highly effective gain-of-function mutations enable mutant IDH to efficiently metabolize isocitrate to D-2-hydroxyglutarate (D 2-HG) that accumulates to high concentrations within the tumor microenvironment. D 2-HG is an intracellular effector that promotes tumor growth through widespread epigenetic changes in IDH-mutant tumor cells, but its potential role as an intercellular immune regulator remains understudied. Experimental Design: Complement activation and CD4+, CD8+, or FOXP3+ T-cell infiltration into primary tumor tissue were determined by immunohistochemistry using sections from 72 gliomas of World Health Organization (WHO) grade III and IV with or without IDH mutations. Ex vivo experiments with D 2-HG identified immune inhibitory mechanisms VSports手机版. Results: IDH mutation associated with significantly reduced complement activation and decreased numbers of tumor-infiltrating CD4+ and CD8+ T cells with comparable FOXP3+/CD4+ ratios. D 2-HG potently inhibited activation of complement by the classical and alternative pathways, attenuated complement-mediated glioma cell damage, decreased cellular C3b(iC3b) opsonization, and impaired complement-mediated phagocytosis. Although D 2-HG did not affect dendritic cell differentiation or function, it significantly inhibited activated T-cell migration, proliferation, and cytokine secretion. Conclusions: D 2-HG suppresses the host immune system, potentially promoting immune escape of IDH-mutant tumors. Clin Cancer Res; 24(21); 5381-91. ©2018 AACR. .

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The authors declare no potential conflicts of interest

Disclosure of Potential Conflicts of Interest: None

Figures

Figure 1.
Figure 1.. IDH mutations associate with diminished complement activation in astrocytic brain tumors
Immunohistochemical staining for C3b(iC3b) deposition in tumor tissue sections from patients with anaplastic astrocytoma (AA) WHO grade III or glioblastoma (GBM) WHO grade IV. A, representative C3b(iC3b) staining of a section from an IDH-wildtype tumor (wtIDH) B, C3b(iC3b) staining of a section from an IDH-mutant tumor (mIDH). C, levels of complement activation semi-quantitated by C3b (iC3b) deposition intensity scores in anaplastic astrocytomas (AA) and glioblastomas (GBM). D, complement activation semi-quantitated by C3b (iC3b) deposition fraction scores in anaplastic astrocytomas and glioblastomas. E, F, representative C3b(iC3b) staining in small blood vessels (arrows) and necrotic areas (+) in sections of GBM samples. G, H, Complement activations in blood vessels and necrotic areas were semi-quantitated by C3b (iC3b) deposition intensity scores. I, J, C3b(iC3b)/OLIG2 double staining of sections from glioblastomas with wtIDH and mIDH. Each dot in panels C, D, G, H represents a single patient, *p<0.05; **, p<0.01; *** p<0.001. Scale bar 100 μm
Figure 2.
Figure 2.. D 2-HG inhibits both the classical and alternative pathways of complement activation through distinct mechanisms
A, D 2-HG reduces complement-dependent hemolysis as a function of complement concentration in normal human serum (NHS). Complement-mediated hemolysis by the classical pathway using EshA in the presence of varied concentrations of NHS in the absence or presence of 30 mM D 2-HG before hemolysis was quantitated. B, complement-mediated hemolysis by the classical pathway as a function of D 2-HG concentration using EshA in 5% NHS. C, effect of D 2-HG on assembly of C3 convertases of the classical pathway using EshA and C3-depleted serum. D, effect of D 2-HG on assembly of C5 convertases of the classical pathway using EshA and C5-depleted serum. E, effect of varied D 2-HG concentrations on the activity of pre-assembled C3 convertases of the classical pathway using EshA and C3-depleted serum. F, effect of varied D 2-HG concentrations on the activity of pre-assembled C3 convertases of the classical pathway using EshA and C5-depleted serum. G, effect of 30 mM D 2-HG on complement activation by the alternative pathway assessed by MAC-mediated Erabb hemolysis. H, concentration-dependent effect of D 2-HG in 20% NHS on alternative complement activation assessed by MAC-mediated Erabb hemolysis. I, effect of varied D 2-HG concentrations on assembly of C3/C5 convertases of the alternative pathway of complement activation using Erabb and C5-depleted serum. J, D 2-HG effect on the activity of pre-assembled C3/C5 convertases of the alternative pathway of complement activation using Erabb and C5-depleted serum as a function of D 2-HG concentration. K, concentration-dependent effect of D 2-HG in inhibiting different concentrations of complement(10% and 30% NHS)-mediated damage of antibody-sensitized T98 glioma cells. For all panels, PBS was used in all assays as control, and data are presented as mean ± SD that has been analyzed by Student’s t test and one-way ANOVA. *p<0.05. CP, classical pathway; AP, alternative pathway, conv, convertase, assy, assembly
Figure 3.
Figure 3.. D 2-HG reduces complement opsonization–induced phagocytosis and complement-mediated glioma cell line damage
A, representative flow cytometric histogram of C3b(i3Cb) deposition on the EshA cell surface in the presence of different concentrations of D 2-HG. For complement opsonization assays, EshA were incubated with 2% C5-depleted serum in the presence of the stated concentration of D 2-HG before C3b(iC3b) deposited on the cell surface was quantitated by flow cytometry. B, summarized results of the levels of C3b/i3Cb deposition on the cell surface in the presence of defined concentrations of D 2-HG as measured by mean fluorescence intensity (MFI). Pos Ctrl, positive control, cells were incubated with serum in the absence of D 2-HG; Neg Ctrl, negative control, cells were incubated with serum in the presence of EDTA (to completely inhibit complement activation), data are mean ± SD and analyzed by one-way ANOVA. * p<0.05. C, representative results of D 2-HG on phagocytosis. For complement-mediated phagocytosis assays, macrophages were prepared and labeled with CellTrace far-red fluorophore, while EshA were incubated with or without 2% C5-depleted serum in the absence or presence of 30 mM D 2-HG (for complement opsonization) and labeled with fluorescent DiI. Then the processed macrophages and EshA were co-cultured at a 1:10 ratio, and after either 30 or 120 min incubation the cells were analyzed by flow cytometry to measure the efficiency of phagocytosis (frequency of double-positive cells). w/o serum, EshA were incubated without serum (no opsonization, negative control), w/serum, EshA were incubated with serum in the absence of D 2-HG (maximum opsonization, positive control), w/serum+ D 2-HG, EshA were incubated with serum in the presence of 30 mM D 2-HG. D, summarized results of phagocytosis assays. Data are mean ± SD and analyzed by student t test. * p<0.05
Figure 4.
Figure 4.. IDH mutations associate with decreased numbers of infiltrating CD4+, CD8+ and FOXP3+ T cells in astrocytic brain tumors
A, representative CD4+ immunohistochemistry in tumor sections from patients with WHO grade III anaplastic astrocytoma (AA) or WHO grade IV glioblastoma (GBM) wildtype IDH (wtIDH). Arrows indicate CD4+ T cells. B, representative CD4+ immunohistochemistry in tumor sections from patients with WHO grade III anaplastic astrocytoma or WHO grade IV glioblastoma with mutant IDH (mIDH). C, statistical analysis of tumor-infiltrating CD4+ T cells in AA and GBM with wtIDH and mIDH. D, representative CD8 staining of tumor sections derived from a wtIDH tumor. E, representative CD8 staining of tumor sections derived from a mIDH tumor. F, statistical summary of tumor-infiltrating CD8+ T cells in AA and GBM tumors. G, representative FOXP3 immunostaining in sections obtained from a wtIDH astrocytoma. H, representative FOXP3 staining of tumor sections derived from with a mIDH astrocytoma. I, statistical summary of tumor-infiltrating FOXP3+ T cells and the ratio of FOXP3+ to total CD4+ T cells in AA and GBM sections. Each dot in all statistical analyses represents data from a single patient, **, p<0.01; *** p<0.001. Scale bar 100 μm
Figure 5.
Figure 5.. D 2-HG inhibits proliferation of activated T cells and their cytokine production
A, concentration-dependent inhibition of T cell proliferation by D 2-HG. T cells enriched from WT mice were labeled with CFSE and activated with anti-CD3 and anti-CD28 monoclonal antibodies in the presence of the stated concentration of D 2-HG. After 3 days of culture, CFSE dilution from the proliferation of the activated T cells was assessed by flow cytometry. B, statistical analysis of concentration-dependent inhibition of T cell proliferation by D 2-HG assessed by bromodeoxyuridine (BrdU) incorporation. C, statistical analysis of the concentration-dependent inhibition of IFNγ by D 2-HG assessed by ELISA. Pos Ctrl, positive control, T cells were activated in the absence of D 2-HG; Neg Ctrl, negative control, unactivated T cells; data were mean ± SD and analyzed by one-way ANOVA. * p<0.05 compared to the positive controls. D, histograms of concentration-dependent inhibition of proliferation of Th1 cells, Th17 cells and Tregs by D 2-HG. CD4+ T cells were purified from naïve WT mice, labeled with CFSE, activated with anti-CD3 and anti-CD28 monoclonal antibodies, and cultured under respective Th1, Th17 or Treg polarization conditions in the absence (blue peaks) or presence of 30 mM D 2-HG (orange peaks). Proliferations of these T cells were analyzed in three days by measuring the dilution of intracellular CFSE by flow cytometry. E, D 2-HG enhances CD4+CD25+ FOXP3+ Treg differentiation. Percentages of CD4+CD25+ FOXP3 + Tregs in the absence or presence of 30 mM D 2-HG were analyzed by flow cytometry. F, statistical analysis of the fraction of differentiated CD4+CD25+ FOXP3 + Tregs determined by flow cytometry. * p<0.05. G, D 2-HG inhibits IL-10 secretion by cultured CD4+CD25+ FOXP3 + Tregs determined by ELISA analysis of the cellular supernatants. H, D 2-HG inhibits T cell migration. CD4+ or CD8+ T cells were isolated from naïve WT mice, and migration of these cells in the presence of the stated concentrations of D 2-HG in response to CCL19 was assessed in a standard transwell-based migration assay. * p<0.05
Figure. 6
Figure. 6. D 2-HG has no significant effect on DC differentiation or the function of differentiated DCs
A, D 2-HG does not affect dendritic cell (DC) differentiation. DCs were differentiated from bone marrow cells in the absence or presence of 30 mM D 2-HG before surface markers on the surface of differentiated DCs were analyzed by flow cytometry. The top panel are untreated, and the bottom panel exposed to D 2-HG during differentiation. B, statistical analysis of D 2-HG effect on the fraction of cells expressing the stated surface antigen. C, D 2-HG fails to affect DC proliferation. DCs were differentiated in the presence of the defined concentrations of D 2-HG were cultured with CD4+ T cells from OT II mice together with OVA protein before the DC-activated T cells were assessed by measuring proliferation through BrdU incorporation. D, D 2-HG fails to affect DC function. IFNγ release in the forgoing panel was quantified by ELISA. E, D 2-HG has no effect on DC antigen processing/presentation, but inhibits antigen-specific CD4+ and CD8+ T cells. Differentiated bone marrow-derived DCs were incubated with OVA protein in the absence or presence of 30 mM of D 2-HG, then washed and cultured with CD4+ T cells from OTII mice, or CD8+ T cells from OT I mice without further additional D 2-HG (i). As control, marrow derived-DCs incubated with OVA protein in the absence of D 2-HG were washed and cultured with CD4+ T cells from OTII mice, or CD8+ T cells from OTI mice together with 30 mM D 2-HG (ii). Proliferation of the DC-activated antigen-specific T cells were analyzed by CFSE dilution using flow cytometry. F, D 2-HG inhibits OT II CD4+ T cell release of INFγ. Statistical analysis of INFγ quantified by ELISA after DC-activated antigen-induced release. * p<0.05. G, D 2-HG inhibits OT I CD8+ T cell release of granzyme B. Statistical analysis of released granzyme B defined by ELISA in the supernatants from cultures including OT I CD8+ T cells. * p<0.05.
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