Abstract
The anatomic location and immunologic characteristics of brain tumors result in strong lymphocyte suppression. Consequently, conventional immunotherapies targeting CD8 T cells are ineffective against brain tumors. Tumor cells escape immunosurveillance by various mechanisms and tumor cell metabolism can affect the metabolic states and functions of tumor-infiltrating lymphocytes. Here, we discovered that brain tumor cells had a particularly high demand for oxygen, which affected γδ T cell-mediated antitumor immune responses but not those of conventional T cells. Specifically, tumor hypoxia activated the γδ T cell protein kinase A pathway at a transcriptional level, resulting in repression of the activatory receptor NKG2D. Alleviating tumor hypoxia reinvigorated NKG2D expression and the antitumor function of γδ T cells. These results reveal a hypoxia-mediated mechanism through which brain tumors and γδ T cells interact and emphasize the importance of γδ T cells for antitumor immunity against brain tumors.
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Data availability
The scRNA-seq data are deposited in the Gene Expression Omnibus database under accession number GSE159542. Public data from human patients were obtained from the CGGA (https://cgga.org.cn)47,48. We obtained messenger RNA-seq data from a total of 325 patients (mRNAseq_325) whose data were stored in the CGGA database. TCGA (https://portal.gdc.cancer.gov) data were obtained through the University of California, Santa Cruz Xena platform (http://xena.ucsc.edu/)49. Patient data from the GBM/LGG, LGG and GBM studies were hosted by TCGA Genomic Data Commons. Other data that support the findings of this study are available from the corresponding author upon request.
References
Salmon, H., Remark, R., Gnjatic, S. & Merad, M. Host tissue determinants of tumour immunity. Nat. Rev. Cancer 19, 215–227 (2019).
Thorsson, V. et al. The immune landscape of cancer. Immunity 48, 812–830.e14 (2018).
Arvanitis, C. D., Ferraro, G. B. & Jain, R. K. The blood–brain barrier and blood–tumour barrier in brain tumours and metastases. Nat. Rev. Cancer 20, 26–41 (2020).
Lim, M., Xia, Y., Bettegowda, C. & Weller, M. Current state of immunotherapy for glioblastoma. Nat. Rev. Clin. Oncol. 15, 422–442 (2018).
Barnes, T. A. & Amir, E. HYPE or HOPE: the prognostic value of infiltrating immune cells in cancer. Br. J. Cancer 117, 451–460 (2017).
Zhai, L. et al. IDO1 in cancer: a Gemini of immune checkpoints. Cell. Mol. Immunol. 15, 447–457 (2018).
Rivadeneira, D. B. & Delgoffe, G. M. Antitumor T-cell reconditioning: improving metabolic fitness for optimal cancer immunotherapy. Clin. Cancer Res. 24, 2473–2481 (2018).
Scharping, N. E. & Delgoffe, G. M. Tumor microenvironment metabolism: a new checkpoint for anti-tumor immunity. Vaccines 4, 46 (2016).
Najjar, Y. G. et al. Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma. JCI Insight 4, e124989 (2019).
Scharping, N. E., Menk, A. V., Whetstone, R. D., Zeng, X. & Delgoffe, G. M. Efficacy of PD-1 blockade is potentiated by metformin-induced reduction of tumor hypoxia. Cancer Immunol. Res. 5, 9–16 (2017).
Zheng, X. et al. Mitochondrial fragmentation limits NK cell-based tumor immunosurveillance. Nat. Immunol. 20, 1656–1667 (2019).
Jayaprakash, P. et al. Targeted hypoxia reduction restores T cell infiltration and sensitizes prostate cancer to immunotherapy. J. Clin. Invest. 128, 5137–5149 (2018).
Henze, A. T. & Mazzone, M. The impact of hypoxia on tumor-associated macrophages. J. Clin. Invest. 126, 3672–3679 (2016).
Jensen, R. L. et al. Preoperative dynamic contrast-enhanced MRI correlates with molecular markers of hypoxia and vascularity in specific areas of intratumoral microenvironment and is predictive of patient outcome. Neuro Oncol. 16, 280–291 (2014).
Lee, M. et al. Preferential infiltration of unique Vγ9Jγ2-Vδ2 T cells into glioblastoma multiforme. Front. Immunol. 10, 555 (2019).
Bryant, N. L. et al. Preclinical evaluation of ex vivo expanded/activated γδ T cells for immunotherapy of glioblastoma multiforme. J. Neurooncol. 101, 179–188 (2011).
Beck, B. H. et al. Dynamics of circulating γδ T cell activity in an immunocompetent mouse model of high-grade glioma. PLoS ONE 10, e0122387 (2015).
Varn, F. S., Wang, Y., Mullins, D. W., Fiering, S. & Cheng, C. Systematic pan-cancer analysis reveals immune cell interactions in the tumor microenvironment. Cancer Res. 77, 1271–1282 (2017).
Lee, J. H. et al. Human glioblastoma arises from subventricular zone cells with low-level driver mutations. Nature 560, 243–247 (2018).
Becht, E. et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat. Biotechnol. 37, 38–44 (2019).
Parajuli, P. & Mittal, S. Role of IL-17 in glioma progression. J. Spine Neurosurg. https://doi.org/10.4172/2325-9701.S1-004 (2013).
Kuen, D. S., Kim, B. S. & Chung, Y. IL-17-producing cells in tumor immunity: friends or foes? Immune Netw. 20, e6 (2020).
Pernicova, I. & Korbonits, M. Metformin—mode of action and clinical implications for diabetes and cancer. Nat. Rev. Endocrinol. 10, 143–156 (2014).
Duan, W. et al. Metformin mitigates autoimmune insulitis by inhibiting TH1 and TH17 responses while promoting Treg production. Am. J. Transl. Res. 11, 2393–2402 (2019).
Brill-Karniely, Y. et al. Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability. Sci. Adv. 6, eaax2861 (2020).
Sesen, J. et al. Metformin inhibits growth of human glioblastoma cells and enhances therapeutic response. PLoS ONE 10, e0123721 (2015).
Dowling, R. J. et al. Metformin pharmacokinetics in mouse tumors: implications for human therapy. Cell Metab. 23, 567–568 (2016).
Suzuki, T. et al. The antitumour effect of γδ T-cells is enhanced by valproic acid-induced up-regulation of NKG2D ligands. Anticancer Res. 30, 4509–4513 (2010).
Ribot, J. C. et al. CD27 is a thymic determinant of the balance between interferon-γ- and interleukin 17-producing γδ T cell subsets. Nat. Immunol. 10, 427–436 (2009).
Sumaria, N., Grandjean, C. L., Silva-Santos, B. & Pennington, D. J. Strong TCRγδ signaling prohibits thymic development of IL-17A-secreting γδ T cells. Cell Rep. 19, 2469–2476 (2017).
Han, J. S. et al. Hypoxia restrains lipid utilization via protein kinase A and adipose triglyceride lipase downregulation through hypoxia-inducible factor. Mol. Cell. Biol. 39, e00390 (2019).
Simko, V. et al. Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII. Sci. Rep. 7, 10121 (2017).
Aspelund, A. et al. A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J. Exp. Med. 212, 991–999 (2015).
Louveau, A., Harris, T. H. & Kipnis, J. Revisiting the mechanisms of CNS immune privilege. Trends Immunol. 36, 569–577 (2015).
Medawar, P. B. Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br. J. Exp. Pathol. 29, 58–69 (1948).
Taggart, D. et al. Anti-PD-1/anti-CTLA-4 efficacy in melanoma brain metastases depends on extracranial disease and augmentation of CD8+ T cell trafficking. Proc. Natl Acad. Sci. USA 115, E1540–E1549 (2018).
Belcaid, Z. et al. Focal radiation therapy combined with 4-1BB activation and CTLA-4 blockade yields long-term survival and a protective antigen-specific memory response in a murine glioma model. PLoS ONE 9, e101764 (2014).
Pang, D. J., Neves, J. F., Sumaria, N. & Pennington, D. J. Understanding the complexity of γδ T-cell subsets in mouse and human. Immunology 136, 283–290 (2012).
Chauvin, C. et al. NKG2D controls natural reactivity of Vγ9Vδ2 T lymphocytes against mesenchymal glioblastoma cells. Clin. Cancer Res. 25, 7218–7228 (2019).
Lyssiotis, C. A. & Kimmelman, A. C. Metabolic interactions in the tumor microenvironment. Trends Cell Biol. 27, 863–875 (2017).
Li, L. et al. Metformin-induced reduction of CD39 and CD73 blocks myeloid-derived suppressor cell activity in patients with ovarian cancer. Cancer Res. 78, 1779–1791 (2018).
Eikawa, S. et al. Immune-mediated antitumor effect by type 2 diabetes drug, metformin. Proc. Natl Acad. Sci. USA 112, 1809–1814 (2015).
Nakae, S. et al. Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses. Immunity 17, 375–387 (2002).
Kim, I. K. et al. Sox7 promotes high-grade glioma by increasing VEGFR2-mediated vascular abnormality. J. Exp. Med. 215, 963–983 (2018).
Oh, D. S. et al. Intratumoral depletion of regulatory T cells using CD25-targeted photodynamic therapy in a mouse melanoma model induces antitumoral immune responses. Oncotarget 8, 47440–47453 (2017).
Oh, D.S., Park, J.H., Jung, H.E., Kim, H.J. & Lee, H.K. Autophagic protein ATG5 controls antiviral immunity via glycolytic reprogramming of dendritic cells against respiratory syncytial virus infection. Autophagy https://doi.org/10.1080/15548627.2020.1812218 (2020).
Bao, Z. S. et al. RNA-seq of 272 gliomas revealed a novel, recurrent PTPRZ1–MET fusion transcript in secondary glioblastomas. Genome Res. 24, 1765–1773 (2014).
Zhao, Z. et al. Comprehensive RNA-seq transcriptomic profiling in the malignant progression of gliomas. Sci. Data 4, 170024 (2017).
Goldman, M. et al. The UCSC Xena platform for public and private cancer genomics data visualization and interpretation. Preprint at bioRxiv https://doi.org/10.1101/326470 (2019).
Park, J. H. & Lee, H. K. Re-analysis of single cell transcriptome reveals that the NR3C1–CXCL8–neutrophil axis determines the severity of COVID-19. Front. Immunol. 11, 2145 (2020).
Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902.e21 (2019).
Acknowledgements
The authors thank the members of the Hyehwa forum for helpful discussions and J. Y. Kim at the BioMedical Research Center for technical service. This work was supported by the National Research Foundation of Korea (NRF-2018M3A9H3024611). This study was also supported by the Samsung Science and Technology Foundation (SSTF-BA1902-05), Republic of Korea.
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J.H.P., H.-J.K., C.W.K., H.C.K. and H.K.L. designed and conducted the experiments. Y.J., H.-S.L. and S.-H.P. performed the MRI. Y.L. and Y.S.J. converted the raw scRNA-seq data. J.E.O. analyzed the data and edited the revised manuscript. J.H.L. provided vectors containing a p53/PTEN-targeting single-guide RNA, Cas9 and Cre recombinase and LSL-EGFRviii mice. S.K.L. provided human bloods recruited from healthy volunteers. J.H.P. and H.K.L. conceived of the study, analyzed the data and wrote the manuscript.
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Peer review information Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 γδ T cells, but not conventional T cells, are a positive prognosis factor for survival in brain cancer patients.
Analysis of brain cancer patient survival according to TCGA data. a, Overall survival of brain cancer patients grouped according to high or low expression compared to median value of CD4 (n = 347 (low), 349 (high)) and CD8A (n = 348 (low), 347 (high)) of GBMLGG. b, Overall survival of LGG (Low-grade glioma) patients grouped by high or low expression compared to quartile value of CD4 (n = 132 (low), 130 (high)), CD8A (n = 130 (low), 131 (high)) and TRGC1 (n = 130 (low),130 (high)). c, Overall survival of GBM (Glioblastoma multiforme, one of high-grade glioma, HGG) patients grouped by quartile value of CD4 (n = 38 (low), 42 (high)), CD8A (n = 42 (low), 40 (high)) and TRGC1 (n = 43 (low), 37 (high)). (c). Survival data were analyzed by log-rank test. d, Gating scheme for analysis of flow cytometry for engineered HGG mice model. FVD450, Fixable viability dye 450.
Extended Data Fig. 2 TILs are not functional in brain tumors because of immunosuppression.
a,b, After 16 weeks post-transfection into LSL-EGFRviii mice, cells were isolated from brain. CD107a expression of γδ T cells (n = 2 per group) (a) and CD8 T cells (n = 2 per group) (b) was analyzed by flow cytometry. The data are representative of three independent experiments. c, C57BL/6J mice received antibodies on days 1 and 2 prior to injection and then every 7 days post-injection. d–g, Survival of mice injected with anti-CD8 antibody (d), anti-CD4 antibody with or without anti-CD8 antibody (e), anti-NK1.1 antibody (f), or anti-γδTCR antibody (g). IgG was used as an isotype control. Survival data were analyzed by log-rank test (d-g). The data presented are representative of five (d), three (e), two (f), and one (g) independent experiments. h, C57BL/6 mice were injected with GL261 tumor cells into the brain cortex. At 20 days post-injection, immune cells were isolated from tumor tissues and analyzed by flow cytometry. i–k, Percentage of splenic and tumor-infiltrating T cells expressing PD-1, TIM-3 and Annexin V. PD-1+ CD4 (n = 5), CD8 (n = 5) and γδ T cell (n = 4) (i), TIM-3+ CD4 (n = 5) and CD8 (n = 5) T cell (j) and Annexin V+ CD4 (n = 4), CD8 (n = 4) and γδ T cell (n = 4) (k). Data are representative of three (i), two (j-k). Data were analyzed by a two-tailed unpaired Student’s t test (i-k). n.s., not significant (p > 0.05). Error bars show mean ± SEM.
Extended Data Fig. 3 IL-17 is dispensable for HGG progression.
a, Schematic image about work flow of scRNA-seq. b, 7-AAD was used to differentiate live and dead cells. Among live cells, CD45hi cells were sorted as TILs. c, GSEA of differentially expressed genes between IL-17A+ γδ T cells and cytotoxic γδ T cells using gene set about regulation of immune effector function. d, WT and IL-17AF−/− mice were injected GL261 cells into brain cortex. Data is representative of three independent experiments. Survival was monitored. Survival data were analyzed by log-rank test. FDR, False discovery rate.
Extended Data Fig. 4 The antitumor effect of metformin is not mediated by direct cytotoxicity.
a, Flow cytometry analysis of GL261 cells after 24 h treatment with indicated concentrations of metformin in vitro (n = 3 per group). Data are representative of two independent experiments. b, Volumes of tumors from ctrl-treated orthotopic HGG mice at days 20 (n = 6) and 25 (n = 5) post-injection and metformin-treated mice at 20 (n = 6) and 25 (n = 6) days post-injection. c, Weights of tumors from ctrl-treated group at 20 (n = 5) and 25 (n = 4) days post-injection and metformin-treated group at 20 (n = 6) and 25 (n = 6) days post-injection. Data are representative of three independent experiments. Data were analyzed by a two-tailed unpaired Student’s t test. Error bars show mean ± SEM in all panels.
Extended Data Fig. 5 The antitumor effect of metformin is not mediated by conventional T cells.
a,b, Survival of ctrl- and metformin-treated C57BL/6J mice depleted for CD8 T cells (a) or CD4 T cells (b) following injection with GL261 cells into the brain cortex. For T cell depletion, mice were injected intraperitoneally with antibody 1 and 2 days prior to tumor injection and every 7 days post-tumor injection. Data are representative of three independent experiments. Survival data were analyzed by log-rank test. c,d, Confocal microscopy of brain tumor tissue from Met-treated mice at 20 days post-tumor injection. Filled white arrows indicate perivascular γδ T cells. Empty white arrows indicate tumor-infiltrating γδ T cells (c). Cleaved Caspase-3 (Asp175) of tumor area with γδ T cells were shown (d). The images are representative of three independent experiments. Scale bar indicates 20 μm. e, Gating scheme of flow cytometry analysis for orthotopic HGG mice model. f-g, Flow cytometry quantification of CD4 T cells (f) and CD8 T cells (g) from ctrl- and metformin-treated C57BL/6 mice following 0 (n = 3), 10 (n = 5), 15 (n = 5) and 20 (n = 4) days after injection with GL261 cells into the brain cortex. Data are representative of four independent experiments. Error bars show mean ± SEM. Data were analyzed by a two-tailed unpaired Student’s t test. n.s., not significant (p > 0.05).
Extended Data Fig. 6 Phenotypic analysis of γδ T cells using GSEA from metformin-treated mice.
a, IL-17A protein levels in brain tumor tissues (n = 5), measured by bead-based cytokine immunoassays. b-c, After 20 days of tumor inoculation, brain cells were isolated. Cells were stimulated with PMA and ionomycin. Fixed and permeabilized cells were stained by antibodies and acquired into flow cytometry. Frequency of IL-17A+ γδ T cells was analyzed (n = 5 per group) (b). Contour plot of IL-17A-, IFN-γ- and CD107a-producing γδ T cells (c). d, U87MG cells were co-cultured with human PBMC-derived γδ T cells pretreated with 10 μg/ml of anti-NKG2D or 30 μM of HIF-1α inhibitor (Cay10585). Cells were stained with 7-AAD and analyzed by flow cytometry (n = 3 (white dots) and 2 (black dots) per group). Data are representative of two independent experiments. Data were analyzed by a two-tailed unpaired Student’s t test (a,b,d). n.s., not significant (p > 0.05). Error show mean ± SEM. e-f, Analysis of gene set enrichment among the genes differentially expressed in IL-17A+ γδ T cells between metformin-treated and ctrl-treated mice. e, GSEA of central nervous system development. f, GSEA of innate immune response. g-k, Analysis of gene set enrichment among the genes differentially expressed in cytotoxic γδ T cells between metformin-treated and ctrl-treated mice. g, GSEA of innate immune response genes. h, GSEA of T cell activation genes. i, GSEA of upregulated genes of induced T cells to natural killer cells. j, GSEA of citric acid (TCA) cycle and respiratory electron transport genes. k, GSEA of IL-2-STAT5 signaling genes.
Extended Data Fig. 7 The effects of metformin on conventional T cells and tumor cells.
a, IL-10 protein levels were measured by ELISA in tumor tissues from ctrl- and metformin-treated C57BL/6 mice at 20 days post-tumor injection (n = 4 per group). b–h, Flow cytometry analysis of immune cells isolated from ctrl- and metformin-treated C57BL/6J mice at indicated dates (b-c) or 20 days post-tumor injection (d-g). b-c, CD44-expressing CD4 (b) and CD8 (c) T cells after 0 (n = 3 per group), 10, 15 and 20 (n = 5 per group) days post-injection. d-e, IFN-γ or TNF-expressing CD4 (d) and CD8 (e) T cells 20 days after tumor injection (n = 5 per group). f-g, PD-1 or TIM-3-expressing CD4 (f) and CD8 (g) T cells after 20 days of tumor injection (n = 5 per group). h-i, GL261 cells were treated with indicated concentrations of metformin for 24 h and then analyzed by flow cytometry for expression of H-2Kb (h) and Rae-1 (i) (n = 3 per group). j, Flow cytometry analysis of γδ T cells showing that NKG2D-expressing γδ T cells are CD44int. k, Quantification of CD107a expression from γδ T cells of engineered HGG mice using flow cytometry. Data are representative of more than two independent experiments. Data were analyzed by a two-tailed unpaired Student’s t test. Error bars show mean ± SEM in all panels.
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Park, J.H., Kim, HJ., Kim, C.W. et al. Tumor hypoxia represses γδ T cell-mediated antitumor immunity against brain tumors. Nat Immunol 22, 336–346 (2021). https://doi.org/10.1038/s41590-020-00860-7
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DOI: https://doi.org/10.1038/s41590-020-00860-7
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