OX40 ligand expressed in glioblastoma modulates adaptive immunity depending on the microenvironment: a clue for successful immunotherapy.
BACKGROUND
Glioblastoma is the most malignant human brain tumor and has a dismal prognosis; however, some patients show long-term survival. The interaction between the costimulatory molecule OX40 and its ligand OX40L generates key signals for T-cell activation. The augmentation of this interaction enhances antitumor immunity. In this present study, we explored whether OX40 signaling is responsible for antitumor adaptive immunity against glioblastoma and also established therapeutic antiglioma vaccination therapy.


METHODS
Tumor specimens were obtained from patients with primary glioblastoma (n = 110) and grade III glioma (n = 34). Quantitative polymerase chain reaction (PCR), flow cytometry, and immunohistochemistry were used to analyze OX40L expression in human glioblastoma specimens. Functional consequences of OX40 signaling were studied using glioblastoma cell lines, mouse models of glioma, and T cells isolated from human subjects and mice. Cytokine production assay with mouse regulatory T cells was conducted under hypoxic conditions (1.5% O2).


RESULTS
OX40L mRNA was expressed in glioblastoma specimens and higher levels were associated with prolonged progression-free survival of patients with glioblastoma, who had undergone gross total resection. In this regard, OX40L protein was expressed in A172 human glioblastoma cells and its expression was induced under hypoxia, which mimics the microenvironment of glioblastoma. Notably, human CD4 T cells were activated when cocultured in anti-CD3-coated plates with A172 cells expressing OX40L, as judged by the increased production of interferon-γ. To confirm the survival advantage of OX40L expression, we then used mouse glioma models. Mice bearing glioma cells forced to express OX40L did not die during the observed period after intracranial transplantation, whereas all mice bearing glioma cells lacking OX40L died. Such a survival benefit of OX40L was not detected in nude mice with an impaired immune system. Moreover, compared with systemic intraperitoneal injection, the subcutaneous injection of the OX40 agonist antibody together with glioma cell lysates elicited stronger antitumor immunity and prolonged the survival of mice bearing glioma or glioma-initiating cell-like cells. Finally, OX40 triggering activated regulatory T cells cultured under hypoxia led to the induction of the immunosuppressive cytokine IL10.


CONCLUSIONS
Glioblastoma directs immunostimulation or immunosuppression through OX40 signaling, depending on its microenvironment.
Department of Neurosurgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Miyagi, Japan. shibahar@nsg.med.tohoku.ac.jp.
Ichiyo
I
Shibahara
Department of Neurosurgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Miyagi, Japan. ryuta@nsg.med.tohoku.ac.jp.
Ryuta
R
Saito
Department of Neurosurgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Miyagi, Japan. ctyouei@hotmail.co.jp.
Rong
R
Zhang
Department of Neurosurgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Miyagi, Japan. masashiko37@yahoo.co.jp.
Masashi
M
Chonan
Department of Neurosurgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Miyagi, Japan. natak525@gmail.com.
Takuhiro
T
Shoji
Department of Neurosurgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Miyagi, Japan. mkanamori@med.tohoku.ac.jp.
Masayuki
M
Kanamori
Department of Neurosurgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Miyagi, Japan. sono@nsg.med.tohoku.ac.jp.
Yukihiko
Y
Sonoda
Department of Neurosurgery, Kitasato University School of Medicine, Sagamihara, Kanagawa, 252-0374, Japan. kuma@kitasato-u.ac.jp.
Toshihiro
T
Kumabe
Department of Respiratory Oncology and Molecular Medicine, Tohoku University School of Medicine, Sendai, 980-8575, Miyagi, Japan. masahikokanehira@yahoo.co.jp.
Masahiko
M
Kanehira
Department of Respiratory Oncology and Molecular Medicine, Tohoku University School of Medicine, Sendai, 980-8575, Miyagi, Japan. kikuchi@idac.tohoku.ac.jp.
Toshiaki
T
Kikuchi
Department of Immunology, Tohoku University School of Medicine, Sendai, 980-8575, Miyagi, Japan. tso@med.tohoku.ac.jp.
Takanori
T
So
Department of Public Health, Tohoku University School of Medicine, Sendai, 980-8575, Miyagi, Japan. tkswatanabe@med.tohoku.ac.jp.
Takashi
T
Watanabe
Department of Pathology, Tohoku University School of Medicine, Sendai, 980-8575, Miyagi, Japan. acehsv@outlook.com.
Hiroaki
H
Takahashi
Department of Pathology, Tohoku University School of Medicine, Sendai, 980-8575, Miyagi, Japan. e-iwabuchi@med.tohoku.ac.jp.
Erina
E
Iwabuchi
Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan. yuetsu@s4.dion.ne.jp.
Yuetsu
Y
Tanaka
Department of Pathology, Tohoku University School of Medicine, Sendai, 980-8575, Miyagi, Japan. yukko-shiba@patholo2.med.tohoku.ac.jp.
Yukiko
Y
Shibahara
Department of Pathology, Tohoku University School of Medicine, Sendai, 980-8575, Miyagi, Japan. hsasano@patholo2.med.tohoku.ac.jp.
Hironobu
H
Sasano
Department of Immunology, Tohoku University School of Medicine, Sendai, 980-8575, Miyagi, Japan. ishiin@med.tohoku.ac.jp.
Naoto
N
Ishii
Department of Neurosurgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Miyagi, Japan. tomi@nsg.med.tohoku.ac.jp.
Teiji
T
Tominaga
D056704:Adaptive Immunity / Q000235:genetics* / Q000276:immunology; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D045744:Cell Line, Tumor; D018572:Disease-Free Survival; D005260:Female; D005909:Glioblastoma / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology; D016753:Interleukin-10 / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D053278:OX40 Ligand / Q000235:genetics* / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D050378:T-Lymphocytes, Regulatory / Q000276:immunology; D059016:Tumor Microenvironment / Q000235:genetics* / Q000276:immunology
D053278:OX40 Ligand; D012333:RNA, Messenger; D016753:Interleukin-10; D007371:Interferon-gamma