| γδT cells in human peripheral blood mediate non-MHC-restricted cytolytic effect against many tumor cells. The immunological properties of γδT cells enable them to be used in adoptive immunotherapy.Heat-shock proteins (HSP), which are expressed constitutively in all cells, perform a multitude of house keeping function that is essential for cellular survival. They are super antigens for y5T cells. They also have been found to be remarkably potent in their ability to elicit T-cell-mediated immune response to tumors, viruses and minor histocompatibility antigens.The HSP-peptide complexes derived from cancer cells have been shown to have immunogenicity, and can elicit cancer-specific immunity, induce immunological responsiveness of CTL. Several clinical trials with cancer patients have shown that the patients are vaccinated with the HSP-peptide complexes derived from their own rumors, the size of residual rumors and survival of time to recurrence of tumors wre controlled. But the dose of HSP-peptide complexes come from tumor cells is too little to attain the effective dose.In order to seek a new method for obtaining a large number of γδT cells which may become a new effector in the adoptive immunotherapy, we use heterologous HSP70 and heterologous HSP70-tumor peptide complexes to selectively expand human γδT cells in vitro from peripheral blood mononuclear cells (PBMC). We examined the subtypes and cytotoxic activities of expanded y5T cells. We built up HSP70BCG-Daudi peptide complexes to active y8T cells, then examined the level of proliferation, cytotoxicity and cytokines. The results were as follows:The first part: We used anti-TCRγδ-antibody , HSP70BCG and HSP70-peptide complexes abstracted from EC-TCV( elemene combo-murine Hca-F hepatic carcinoma cell vaccine) to proliferate y5T cells. The main results were as follows:1 After 14 days of culture, in the antibody system, the total cell number increased about 30-40 fold, and the ratio of y6T cells reached to 86.7%±5.7%; In the HSP70BCG culture system, the total cell number increased about 7-8 fold, and the ratio of γδT cells reached to 71.23%±2.7%; In the HSP70-peptide complexes culture system, the total cell number increased about 4-5 fold, and the ratio of γδT cells reached to 27.26% ±4.24%. The ratio of proliferation is:anti-TCRy5 antibody> HSP70BCG> HSP70-peptide complexes >1640.2 Subtypes of the expanded γδT cells were analysed with FACS and RT-PCR. The results showed that the antibody and HSP70BcG activated γδT cells possessed a whole repertoire and mainly expressed V 9/V 2 subtype.3 The cytotoxicity studies indicated that: When the E/T ratio was 10:1, cytotoxic activities of y5T cells against Daudi was: anti-TCRγδ antibody 98.8%±0.02%> HSP70BCG 71.1%±2.04%>HSP70-pepude complexes 52.83%±11.67%>1640 45.57%±9.94%(P<0.05); When the E/T ratio was 5:1, the cytotoxic activities was: anti-TCRy6 antibody 85.2%±5.79%>HSP70BCG 50.29%±7.33%>HSP70-peptide complexes 39.49%±2.49%, 1640 34.98% ±6.32%. The last two groups had no difference(P>0.05).The second part: The y5T cells elicited by anti-TCRyS antibody were being treated with resting, and then were elicited with Daudi antigen peptide. HSP70BCG and HSP70BCG-Daudi peptide complexes for 24 or 48 h. The proliferation TNF-a bioactivity and cytotoxicity were studied. The main results were as follows:1 Cytotoxiciry: When the ratio of E/T was 10:1, the cytotoxicity was:HSP70BCG- Daudi peptide complexes 88.36%±8.21%> HSP70BCG 71.44%±13.7%> Daudi antigen peptide 69.61%±11.53%>1640 50.47%±8.76%(P<0.05). When the ratio of E/T was 5:1, the cytotoxicity was:HSP70BCG-Daudi peptide complexes 75.23% ± 16.36%> HSP70BCG 58.13%±21.78%, Daudi antigen peptide 53.16%±18.96% and 1640 39.43%±7.74%, The last three groups had no difference (P>0.05).2 > Proliferation: The 24 and 48 h proliferation among each group had no difference (P> 0.05). 3, Cytokines: We found no difference of the bioactivity of TNF-a among eachgroup.In our study, we confirmed that heterol... |
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