Research On NKG2D Mediated Cytotoxicity Of Allogeneic NK Cells Against Human Glioma Cells

Glioma, also known as neurogliocytoma, is originated from neural ectoderm. It is the most common primary intracranial tumor, accounting for about 36.26%～60.96% of all primary intracranial tumors. Glioma, especially high-grade glioma, is characterized by abnormal proliferation, infiltrative growth, tendency to relapse, abnormally vascular hyperplasia and immune tolerance. Therefore, even treated with comprehensive measures including constantly improved microsurgical resection, radiotherapy and chemotherapy, glioma is still easy to relapse with undesirable prognosis. For example, though treated with comprehensive therapy, the median survival time of glioblastoma (GBM) is still less than 15 months. So new methods for the treatment of glioma should be explored in clinical practice.Recent research has proved that allogeneic NK cells can effectively kill cells of nasopharyngeal carcinoma, gastric carcinoma, renal carcinoma, intestinal cancer and ovarian cancer. The cytotoxicity of NK cells against target cells is closely related to receptors on the surface of NK cells and ligands on the surface of target cells. Killer receptors on the surface of NK cells bind to HLA class I molecules on the surface of target cells, inhibiting the cytotoxicity of NK cells against target cells. When target cells lack HLA classⅠmolecules, NK cells are activated to kill target cells. NKG2D (natural killer group 2D) is the main killer activatory receptor on the surface of NK cells. Its ligands are MHC classⅠchain-related molecules (MIC) A and B and human cytomegalovirus UL16 binding proteins (ULBP) 1,2 and 3.What this study focuses on is whether allogeneic NK cells have cytotoxic effect on glioma cells or not. In this study, we isolated and cultured human glioma cells in vitro, observed their biological characteristics, detected the expression of HLA class I molecules and NKG2D ligands on human glioma cells, and observed the cytotoxicity of allogeneic NK cells against glioma cells and its molecular basis in vivo and in vitro, providing experimental and theoretical basis to further understand the feasibility and mechanism of cytotoxicity of allogeneic NK cells against glioma cells. Thus we explored a new method for the treatment of glioma.PartⅠ. Biological characteristics of primary cultured human glioma cellsObjective:To provide the necessary basis to explore cytotoxicity of allogeneic NK cells against human glioma cells by primary culture and identification human glioma cells and observe their biological property.Methods:Human glioma cells were isolated from tissue samples obtained during the surgery of patients who were clinically and pathologically diagnosed with glioma gradeⅣ, and the cells were primarily cultured and identified as human glioma cells by immunohistochemistry assay of glial fibrillary acidic portein (GFAP).The growth characteristics of glioma cells was observed under microscope. The growth curve was analyzed by MTT method. Human glioma cells of a certain amount were transplanted into BALB/c nude mice. Time of tumor formation, tumor formation rate and tumor growth curve were observed. Results:1. It was observed under tumor tissue HE stained slices that the nuclei were heavily stained with significant heteromorphism and that macronuclei and nuclear division were visible. The source tissue of cell culture was identified as glioma gradeⅣ.2. Human glioma cells cultured in vitro grew well, with a population density doubling time of 23.48 hr. Subcultured cells grew vigorously in a good status. The property was stable with an obvious exponential phase of growth.3. HE staining of cultured human glioma cells showed that the cells were in fusiform, starlike or irregular shapes with small prominences and with nucleus of variable sizes. Results of immunocytochemistry showed that most cells were positive for GFAP staining. There were yellow or golden brown positive substances of various concentrations in cytoplasm.4. The time of tumor formation was (9.17±1.68)d days after transplanting 0.1ml (1×106) suspension of glioma cells into BALB/c nude mice. Tumor formation rate was 100%. Tumor tissue was pathologically diagnosed as glioma gradeⅣ.Conclusions:1. Glioma cells can be cultured in vitro, and cell doubling time was 23.48 hr.2. Glioma cells can grow to form tumor. The time of tumor formation was about (9.17±1.68) days. Tumor grew rapidly with obvious pathological morphology.PartⅡ. Detection of the expression of HLA ClassⅠmolecules and NKG2D ligands in human glioma cellsObjective:To detect the expression of HLA classⅠmolecules and NKG2D ligands in primary culture human glioma cells, providing experimental and theoretical basis to further understand cytotoxicity of allogeneic NK cells against glioma cells.Methods:Reverse transcription PCR method was used to detect the expression of MICA, MICB, ULBP1, ULBP2 and ULBP3 in human glioma cells at mRNA level. Flow cytometry was used to detect the expression of MICA, MICB, ULBP1, ULBP2, ULBP3 and HLA classⅠmolecules. Western Blot technique was used to detect the expression of MICA, ULBP2 and ULBP3 proteins.Results:1. Results of RT-PCR showed that MICA, MICB, ULBP1, ULBP2 and ULBP3 were all expressed in human glioma cells at mRNA level.2. Results of flow cytometry confirmed NKG2D ligands MICA, ULBP2 and ULBP3 were expressed on the surface of human glioma cells while NKG2D ligands MICB and ULBP1 were not expressed on the surface of human glioma cells.3. The expression percentage of MICA, ULBP2, ULBP3 and HLA classⅠmolecules in human glioma cells were 63.26±6.71%,61.23±5.97%,51.47±2.43% and 89.67±7.28% respectively; Results of Western Blot confirmed that NKG2D ligands MICA, ULBP2 and ULBP3 proteins were expressed in glioma cells.Conclusions:1. NKG2D ligands ULBP1, ULBP2, ULBP3, MICA and MICB were expressed in human glioma cells at mRNA level.2. MICA, ULBP2 and ULBP3 are expressed on the surface of human glioma cells while MICB and ULBP1 are not expressed on the surface of human glioma cells.3. HLA classⅠmolecules are highly expressed on the surface of human glioma cells. PartⅢ. Study of cytotoxicity of allogeneic NK cells against human glioma cells in vitroObjective:To detect cytotoxicity of allogeneic NK cells against human glioma cells.Methods:Density gradient centrifugation was used to isolate mononuclear cells from peripheral blood and NK cells were purified through MACS; MTT method was used to detect cytotoxicity of NK cells against human glioma cells and cytotoxic activity of different effector-target ratios.Results:1. Among the isolated and purified positive cells, the proportion of CD3-CD 16+ CD56+ cells was 91.7%.2. Allogeneic NK cells showed cytotoxic activity against human glioma cells, which increased with the rise of effector-target ratio; when effector-target ratio was 5:1,20:1,50:1 and 100:1, the toxic activity was 23.64±1.73%,37.28±3.67%, 49.63±4.28% and 74.16±6.43% respectively.Conclusions:1. Human allogeneic NK cells have cytotoxic activity against glioma cells.2. HLA class I molecules, MICA, ULBP2 and ULBP3 play important roles in the cytotoxic effect of NK cells on glioma cells.PartⅣ. Inhibitory effect of allogeneic NK cells on human glioma cells in nude miceObjective:To provide experimental and theoretical basis for immunotherapy of glioma by observing cytotoxicity of allogeneic NK cells against human glioma cells in BLAB/c nude mice,Methods:32 BALB/c nude mice were randomly divided into 4 groups:group A: without cell transplantation; group B:Human glioma cells transplantation; group C: Human glioma cells+NK cells treatment; group D:NK cells transplantation, eight mice in each group. The time of tumor formation, tumor formation rate and changes of tumor size were observed for nude mice in each group, and tumor growth curves were drawn. Three weeks after tumor formation, mice were euthanized. Tumors were removed and weighted. Tumor inhibition rates were calculated and routine pathological examination was performed for tumor tissues.Results:1. There was no tumor in A and D group. In group B and group C, the times of tumor formation was (9.23±1.64) days and (15.26±1.53) days respectively.2. Growth curve showed NK cells could significantly slow down the growth of transplanted tumor from glioma cells in BALB/c nude mice. In group B and group C, the tumor masses were (1.74±0.13) g and (1.26±0.08) g respectively. Tumor size in group C was significantly smaller than that in group B. Tumor inhibition rate of group C was 27.59%.3. Through HE stainin, the specimen of tumors from nude mice were identified as glioma gradeⅣ. Under microscope, there were many necrotic tumor cells and NK cell infiltration on HE stained slices of tumor in group C.Conclusion:The human allogeneic NK cells can effectively inhibit the growth of human glioma cells which were transplanted in BALB/c nude mice.