The PD-1/B7-H1 pathway modulates the natural killer cells versus mouse glioma stem cells.
OBJECTIVE
Glioblastoma multiforme (GBM) is the most malignant primary type of brain tumor in adults. There has been increased focus on the immunotherapies to treat GBM patients, the therapeutic value of natural killer (NK) cells is still unknown. Programmed death-1 (PD-1) is a major immunological checkpoint that can negatively regulate the T-cell-mediated immune response. We tested the combination of the inhibiting the PD-1/B7H1 pathway with a NK-cell mediated immune response in an orthotopic mouse model of GBM.


METHODS
Mouse glioma stem cells (GL261GSCs) and mouse NK cells were isolated and identified. A lactate dehydrogenase (LDH) assay was perfomed to detect the cytotoxicity of NK cells against GL261GSCs. GL261GSCs were intracranially implanted into mice, and the mice were stratified into 3 treatment groups: 1) control, 2) NK cells treatment, and 3) PD-1 inhibited NK cells treatment group. Overall survival was quantified, and animal magnetic resonance imaging (MRI) was performed to determine tumor growth. The brains were harvested after the mice were euthanized, and immunohistochemistry against CD45 and PCNA was performed.


RESULTS
The mouse NK cells were identified as 90% CD3- NK1.1+CD335+ by flow cytometric analysis. In the LDH assay, the ratios of the damaged GL261GSCs, with the E:T ratios of 2.5:1, 5:1, and 10:1, were as follows: 1) non-inhibited group: 7.42%, 11.31%, and 15.1%, 2) B7H1 inhibited group: 14.75%, 18.25% and 29.1%, 3) PD-1 inhibited group: 15.53%, 19.21% and 29.93%, 4) double inhibited group: 33.24%, 42.86% and 54.91%. In the in vivo experiments, the mice in the PD-1 inhibited NK cells treatment group and IL-2-stimulated-NK cells treatment group displayed a slowest tumor growth (F = 308.5, P<0.01) and a slower tumor growth compared with control group (F = 118.9, P<0.01), respectively. The median survival of the mice in the three groups were as follows: 1) conrol group: 29 days, 2) NK cells treatment group: 35 days (P = 0.0012), 3) PD-1 inhibited NK cells treatment group: 44 days (P = 0.0024). Immunologic data of PCNA-positive cell ratios and CD45-positive cell ratios of the tumor specimens in the three groups were as follows: 1) control group: 65.72% (PCNA) and 0.92% (CD45), 2) NK treatment group: 27.66% (PCNA) and 13.46% (CD45), and 3) PD-1 inhibited NK cells treatment group: 13.66% (PCNA) and 23.66% (CD45) (P<0.001).


CONCLUSIONS
The results demonstrated that blockade of PD-1/B7H1 pathway could promote mouse NK cells to kill the GL261GSCs, and the PD-1-inhibited NK cells could be a feasible immune therapeutic approach against GBM.
Department of Neurosurgery, Beijing San Bo Brain Hospital, Capital Medical University, Beijing, China.
Bo Yuan
BY
Huang
Department of Neurosurgery, Beijing San Bo Brain Hospital, Capital Medical University, Beijing, China.
Yi Ping
YP
Zhan
College of traditional Chinese Medicine, Capital Medical University, Beijing, China.
Wen Jing
WJ
Zong
Department of Neurosurgery, Beijing San Bo Brain Hospital, Capital Medical University, Beijing, China.
Chun Jiang
CJ
Yu
Department of Neurosurgery, Brain Sciences Institute of Beijing, Capital Medical University, Beijing, China.
Jun Fa
JF
Li
Department of Neurosurgery, Beijing San Bo Brain Hospital, Capital Medical University, Beijing, China.
Yan Ming
YM
Qu
Department of Neurosurgery, Brain Sciences Institute of Beijing, Capital Medical University, Beijing, China.
Song
S
Han
D000818:Animals; D060890:B7-H1 Antigen / Q000037:antagonists & inhibitors / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation / Q000235:genetics; D064987:Cell- and Tissue-Based Therapy; D005434:Flow Cytometry; D005909:Glioblastoma / Q000235:genetics* / Q000276:immunology / Q000628:therapy; D005910:Glioma / Q000235:genetics / Q000276:immunology; D006801:Humans; D007167:Immunotherapy; D007694:Killer Cells, Natural / Q000276:immunology*; D051379:Mice; D009374:Neoplasms, Experimental / Q000235:genetics* / Q000276:immunology / Q000628:therapy; D014411:Neoplastic Stem Cells / Q000276:immunology; D061026:Programmed Cell Death 1 Receptor / Q000037:antagonists & inhibitors / Q000235:genetics*
D060890:B7-H1 Antigen; C498919:Cd274 protein, mouse; C491383:Pdcd1 protein, mouse; D061026:Programmed Cell Death 1 Receptor