Cytotoxic Effect And Molecular Mechanism Of γδT Cells Ex Vivo For Osteosarcoma Cells

Background:Osteosarcoma is the one of the most common primary malignant bone tumor. Currently there is still 30%-40%of osteosarcoma patient occured lung metastasis metastatic after using treatment programs of surgery and neoadjuvant chemotherapy. And immune therapy is another comprehensive treatment tool. In the current research, CD8+CTL cells and classical CD3 NK cells, such as tumor-infiltrating lymphocytes, cytokine-induced killer cells are less effective for osteosarcoma. So in a longer period, the basic and clinical research of adoptive immunetherapy is in a relatively stagnant. While yδT cells as the new immune have been paid attention in the potential role of anti-tumor.In the past y8 T cells were considered to be immature T cells, and its function may be related to the removal of necrotic tissue. But more and more research shows yδT cells have the function of killing tumor cells and viral infection cells like CTL and NK cells. So it plays a very important role in immune surveillance. The research finds thatγδT cells have the significant anti-tumor effects for multiple myeloma and melanoma and kill the tumor cells through infiltrating to metastatic renal tumors after generation in vivo. More significantly, the studies have been proved that yδT cells after proliferation in vitro had inhibition for osteosarcoma cell lines. But so far, yδT cells how to kill osteosarcoma cells and its specific Molecular mechanism has not been reported.Objectives:To study the different proportion ofγδT cell in peripheral blood mononuclear cells between osteosarcoma patients and normal, and the cytotoxic effect and molecular mechanism of y8 T cells ex vivo for osteosarcoma cells.Methods:After the isolation of peripheral blood mononuclear cells from peripheral blood of 10 osteosarcomar patients,5 osteosarcomar patients with lung metastases and 10 health adult respectively, the count of y8 T cells was analyzed by Flow Cytometry. And yδT cell of peripheral blood mononuclear cells was specific proliferated through stimulation of zoledronic acid and interleukin-2. Following 14 days culture, it was determined the percentage again.yδT cells and osteosarcomar cells (MG63, U2OS, SAOS) were co-cultured for 4 hours at different effector/target ratios. Then the cytotoxic effect of y8 T cells was determined using LDH release assay kit. Pre-treatment of y8 T cells with anti-human NKG2D antibody and anti-pan TCR-y8-PE antibodies to block the relevant pathways for evaluating the mechanism of its cytotoxicity. Using ELISA assay to determine IFN-y and TNF-a levels in supernatant of y8 T cells and osteosarcoma cells (E/T=20:1) after co-culture 8,12,24,48,72 hours.Statistical analysis was done using SPSS software (version 15.0 for Windows). The data was expressed as mean±SD and the difference between the groups was determined by a two-tailed Student's t test. P-value<0.05 was considered as significant.Results:The proportion of yδT cells in peripheral blood mononuclear cells of osteosarcoma patients and osteosarcoma patients with lung metastases were significantly lower than normal. yδT cells of peripheral blood mononuclear cells were specific proliferated through stimulation of zoledronic acid and interleukin-2. Following 14 days culture, y8 T cells ratio can reach 95%and the proportion of y8 T cells among groups was no significant difference.y8 T cells had significant cytotoxic effect for different osteosarcoma cell lines in vitro. The cytotoxic effect in osteosarcoma patients group was no significantly difference with the normal group. Then pre-treatment of yδT cells with anti-human NKG2D antibody and anti-pan TCR-yδ-PE antibodies to block the relevant pathways, it found the cytotoxic effect was decreased. After y8 T cells and osteosarcoma cells had co-cultured for 24 hours, IFN-y in the supernatant was significantly increased, while TNF-a was not significantly increase.Conclusions:The proportion of y8 T cells in peripheral blood mononuclear cells of osteosarcoma patients and osteosarcoma patients with lung metastases were significantly lower than normal. It may related with the proliferation of y8 T cells in osteosarcoma peripheral blood is inhibited.In vitro yδT cells have a stronger immune cytotoxic effect for osteosarcoma cells. There is no significant difference between osteosarcoma patients and normal. The molecular mechanism is related with NKG2D and TCRy8 receptor pathway.