NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors

Journal name:
Nature Medicine
Volume:
18,
Pages:
1827–1834
Year published:
DOI:
doi:10.1038/nm.3013
Received
Accepted
Published online
Corrected online

Abstract

The role of the immune response to oncolytic Herpes simplex viral (oHSV) therapy for glioblastoma is controversial because it might enhance or inhibit efficacy. We found that within hours of oHSV infection of glioblastomas in mice, activated natural killer (NK) cells are recruited to the site of infection. This response substantially diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on the NK cell natural cytotoxicity receptors (NCRs) NKp30 and NKp46, whose ligands are upregulated in oHSV-infected glioblastoma cells. We found that HSV titers and oHSV efficacy are increased in Ncr1−/− mice and a Ncr1−/− NK cell adoptive transfer model of glioma, respectively. These results demonstrate that glioblastoma virotherapy is limited partially by an antiviral NK cell response involving specific NCRs, uncovering new potential targets to enhance cancer virotherapy.

At a glance

Figures

  1. oHSV administration induces NK cell recruitment to the tumor-bearing brain.
    Figure 1: oHSV administration induces NK cell recruitment to the tumor-bearing brain.

    (a) Fluorescence-activated cell sorting (FACS) quantification of NK cells in the brains of athymic mice bearing U87dEGFR tumors 2 h after inoculation with rQNestin34.5 or vehicle (veh.) (n = 3 mice per group). (b) FACS quantification of the total number of NK cells in tumor-bearing hemispheres at 6, 24 or 72 h after inoculation with rQNestin34.5, heat-inactivated oHSV or vehicle in athymic mice bearing U87dEGFR tumors (n = 4–5 mice per group). (c) NK cell quantification in the brains of mice after rQNestin34.5 injection into intracranial U87dEGFR xenografts or WT HSV-1 in the brains of mice with no tumor, in tumor-bearing mice treated with vehicle or in untreated tumor-bearing mice (n = 3–5 mice per group). *P < 0.05, ***P < 0.001; Student's t test was used to calculate statistical significance in a and c, and two-way analysis of variance (ANOVA) and pairwise comparisons were used in b. Error bars, s.d.

  2. NK cells are activated after oHSV therapy.
    Figure 2: NK cells are activated after oHSV therapy.

    (a) FACS assessment of the mean fluorescent intensities (MFI) and percentage of NK cells (CD3DX5+) expressing various NK cell activation markers (CD69, CD62L, NKG2D, CD27 or Ly49d) 72 h after intracranial inoculation of rQNestin34.5 or vehicle (veh.) into athymic mice bearing U87dEGFR human glioblastomas. Additionally, FACS quantification of the same markers is shown in KR158dEGFR syngeneic tumors and after WT HSV-1 infection in the brains of athymic mice lacking glioblastoma (glioblastoma free). Supplementary Table 1 lists the average and ranges summarizing the total number of experiments. (b,c) FACS analysis of the NK cell markers CD11bhighCD27high (cytotoxic) or CD11blowCD27high (immature) compared to CD11bhighCD27low (senescent) 72 h after rQNestin34.5 or vehicle inoculation in both xenograft and syngeneic tumor models, in addition to WT HSV-1 infection in the brains of athymic mice lacking glioblastoma. These findings are presented as representative dot plots (b), where the percentage of cells in each quadrant is indicated by a red number and as a fold increase in the expression of each NK cell population compared to vehicle-treated mice (c). (n = 4–6 mice per group). **P < 0.01, ***P < 0.001 by two-way ANOVA. Error bars, s.d. (d) Percentage positivity and fold increase (in parentheses) of the degranulation marker CD107a in the CD11bhighCD27high and CD11bhighCD27low NK cell subsets 72 h after rQNestin34.5 or vehicle treatment of mice with glioblastoma xenografts.

  3. NK cells mediate macrophage and microglia activation after oHSV therapy.
    Figure 3: NK cells mediate macrophage and microglia activation after oHSV therapy.

    (a) FACS of time-dependent changes in CD115+CD45highCD11b+, CD115+CD45highCD11blow and CD115+CD45lowCD11b+ cells in glioblastomas after rQNestin34.5 treatment (n = 4–6 mice per group). (b) FACS of CD115+CD45highCD11b+ cells expressing macrophage activation markers (MHC-II, Ly-6C or CD86) 72 h after vehicle (veh.) or rQNestin34.5 infection as a function of NK cell presence (n = 4 mice per group). (c) FACS of CD115+CD45lowCD11b+ or CD115+CD45highCD11b+ cells 72 h after rQNestin34.5 inoculation as a function of NK cell presence. Asialo, antibodies to asialo-GM1. (d) Nos2 and Tnf expression in tumor-bearing hemispheres 72 h after vehicle or rQNestin34.5 inoculation in the presence or absence of NK cells (n = 4–5 mice per group). (e) Nos2 and Tnf expression as a function of NK cell depletion in FACS-sorted CD115+CD45lowCD11b+ or CD115+CD45highCD11b+ cells 72 h after rQNestin34.5 inoculation of U87dEGFR tumors. (f,g) Intracellular protein staining with FACS quantification (f) and Nos2 mRNA expression (g) in intracranial KR158dEGFR tumors 72 h after rQNestin34.5 inoculation as a function of NK cell presence and Ifng production. (h) FACS of intracellular Tnf 72 h after rQNestin34.5 treatment of U87dEGFR tumors as a function of NK cell presence. (i,j) Cxcl9, Cxcl10 and Cxcl11 expression 72 h after rQNestin34.5 treatment of xenograft (i) and syngeneic (j) tumors as a function of NK cell presence (n = 3–5 mice per group). The dependence on the expression of Ifng within the KR158dEGFR tumor was also assessed in j. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t test. Error bars, s.d.

  4. NK cell depletion enhances oHSV efficacy.
    Figure 4: NK cell depletion enhances oHSV efficacy.

    (a) Viral titers as a function of NK cell depletion 72 h after inoculation with rQNestin34.5 or rQNestin34.5 plus antibodies to asialo-GM1 (asialo) into athymic mice bearing U87dEGFR glioma (n = 4–5 mice per group). *P < 0.05 by Student's t test. Error bars, s.d. (b) Kaplan-Meier survival curves for athymic mice bearing U87dEGFR tumors treated with rQNestin34.5 or vehicle (veh.) and antibody to either asialo-GM1 or TMβ1 as a function of NK cell depletion (n = 5 mice per group). The P values shown indicate comparison of U87dEGFR + rQNestin34.5 to U87dEGFR + rQNestin34.5 + asialo or U87dEGFR + rQNestin34.5 + TMβ1. (c,d) Kaplan-Meier survival curves for the syngeneic 4C8 mouse glioblastoma model in which NK cell depletion was carried out with TMβ1-specific antibody (c) or NK1.1-specific antibody (d) before rQNestin34.5 or vehicle inoculation into brain tumors (n = 8–14 mice per group). For c, the P value shown indicates the comparison of 4C8 + rQNestin34.5 to 4C8 + rQNestin34.5 + TMβ1; for d, the P value shown indicates the comparison of 4C8 + rQNestin34.5 to 4C8 + rQNestin34.5 + NK1.1. (e,f) Differences in gene expression of 84 mouse inflammatory genes in the presence (e) or absence (f) of NK cells 72 h after intracranial inoculation of rQNestin34.5 into athymic mice bearing U87dEGFR cells (n = 4–5 mice per group). Each row and column is represented by a unique number denoting a different transcript. The identity of the transcript after tumor treatment is listed in Supplementary Table 2a (for vehicle compared to rQNestin34.5 treatment) and b (for antibody to asialo-GM1 plus rQNestin34.5 compared to rQNestin34.5 treatment).

  5. IL-15-stimulated NK cells preferentially lyse oHSV-infected human and mouse glioblastoma.
    Figure 5: IL-15–stimulated NK cells preferentially lyse oHSV-infected human and mouse glioblastoma.

    (a) NK cell–mediated cytotoxicity (measured as the percentage of glioblastoma lysis resulting from infection with rQNestin34.5 and coculture with IL-15–stimulated human NK cells compared to mock infection and coculture with IL-15–stimulated NK cells) in glioblastoma cell lines (U87dEGFR, Gli36dEGFR or U251) or primary human glioblastoma cells enriched for stem cell–like properties (X12). (b) IL-15–stimulated mouse NK cell–mediated cytotoxicity using splenocyte-derived mouse NK cells from athymic mice or from WT C57BL/6 mice, cocultured with KR158dEGFR or U87dEGFR glioblastomas, respectively. In a and b, NK cell–mediated killing was tested at varying effector-to-target ratios. Error bars, s.d.

  6. NKp30 and NKp46 orchestrate NK cell-mediated killing of oHSV-infected glioblastoma cells in vitro, and NKp46 mediates viral clearance in vivo.
    Figure 6: NKp30 and NKp46 orchestrate NK cell–mediated killing of oHSV-infected glioblastoma cells in vitro, and NKp46 mediates viral clearance in vivo.

    (a) NK cell–mediated killing of glioblastoma cell lines (U87dEGFR, Gli36dEGFR or U251) or primary human glioblastoma cells enriched for stem cell–like properties (X12) infected with rQNestin34.5 or vehicle before coculturing with IL-15–stimulated human NK cells at a ratio of 12.5:1. The effect of blocking antibodies to NKp30, NKp46 and NKp44 was also assessed. (be) FACS assessing ligand expression for NKp30 (b,c) and NKp46 (d,e) after infection with rQNestin34.5 or mock infection. Additionally, the expression of GFP by rQNestin34.5 was also assayed in virally infected cells as a function of NKp30 or NKp46 ligand expression (c,e). (f,g) NKp30 or NKp46 ligand expression in glioma cells in response to TMZ, radiation (10 Gy), hypoxia (f) or rQNestin34.5 infection (g). (h) WT HSV-1 titers in Ncr1−/− mice compared to WT C57BL/6 mice 72 h after infection (n = 3–4 mice per group). (i) Kaplan-Meier survival curves for U87dEGFR brain tumor–bearing SCID-γcnull mice adoptively transferred with either Ncr1−/− or WT NK cells and intracranially inoculated with rQNestin34.5 (n = 5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t test. Error bars, s.d.

Change history

Corrected online 07 October 2013

In the version of this article initially published, the Online Methods incorrectly stated that mouse NK cells were treated with a blocking antibody to mouse NKp46 called BAB281. The correct antibody used to treat the cells was 29A1.4. The error has been corrected in the HTML and PDF versions of the article.

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Author information

  1. These authors contributed equally to this work.

    • Christopher A Alvarez-Breckenridge &
    • Jianhua Yu

Affiliations

  1. Medical Scientist Training Program, Ohio State University Medical Center and James Cancer Hospital and Solove Research Institute, Columbus, Ohio, USA.

    • Christopher A Alvarez-Breckenridge &
    • Richard Price
  2. Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, Ohio State University Medical Center and James Cancer Hospital and Solove Research Institute, Columbus, Ohio, USA.

    • Christopher A Alvarez-Breckenridge,
    • Richard Price,
    • Jeffrey Wojton,
    • Jason Pradarelli,
    • Yan Wang,
    • Jayson Hardcastle,
    • Balveen Kaur,
    • Sean E Lawler &
    • E Antonio Chiocca
  3. Division of Hematology, Ohio State University Medical Center and James Cancer Hospital and Solove Research Institute, Columbus, Ohio, USA.

    • Jianhua Yu &
    • Michael A Caligiuri
  4. Comprehensive Cancer Center, Ohio State University, Ohio State University Medical Center and James Cancer Hospital and Solove Research Institute, Columbus, Ohio, USA.

    • Jianhua Yu,
    • Hsiaoyin Mao,
    • Min Wei,
    • Shun He,
    • Soledad A Fernandez,
    • Balveen Kaur,
    • Michael A Caligiuri &
    • E Antonio Chiocca
  5. Center for Biostatistics, Ohio State University Medical Center and James Cancer Hospital and Solove Research Institute, Columbus, Ohio, USA.

    • Soledad A Fernandez
  6. Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, INSERM, U631, Marseille, France.

    • Eric Vivier
  7. The Lautenberg Center for General and Tumor Immunology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

    • Ofer Mandelboim
  8. Dipartimento di Medicina Sperimentale e Centro di Eccellenza per le Ricerche Biomediche, Università degli Studi di Genova, Genova, Italy.

    • Alessandro Moretta
  9. Department of Neurosurgery, Brigham and Women's Hospital/Dana-Farber Cancer Institute/Harvard Medical School, Boston, Massachusetts, USA.

    • E Antonio Chiocca

Contributions

C.A.A.-B., J.Y., R.P., J.W., J.P., H.M., M.W., Y.W., S.H. and J.H. performed experiments. C.A.A.-B., J.Y., B.K., S.E.L., A.M., M.A.C. and E.A.C. conceived the experimental approach, directed experiments and interpreted data. S.A.F. performed statistical analysis. E.V., O.M. and A.M. provided reagents. C.A.A.-B., J.Y., M.A.C. and E.A.C. wrote the manuscript.

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