| T cells can be divided into two subgroups: TCRαβlymphocytes and TCRγδlymphocytes based on their expression of T cell receptors (TCR) on the cell surfaces. AlthoughγδT cells account for no more than 10% of the peripheral blood mononuclear cells (PBMC), they display unique nature of antigen recognition and appear to play an important role in host defense against tumor growth.γδT cells could recognize and bind to antigen molecules directly, including peptides and lipoid proteinosis molecules and so on.γδT cell invokes great interests of many immunologists for its unique nature of antigen recognition and cytotoxic activity in recent years.In order to evaluate their functional activity against tumors, large numbers of cells are required. In our laboratory, the research on the tumor immunology ofγδT cells has already been performed and the solid-phase monoclonal anti-TCRy5 antibody was used to isolate and expand theγδT cells from PBMC and obtainγδT cells with high purity. In the present study, we tried to find the best culture conditions and obtain the most effectiveγδT cells for the clinical adoptive immunotherapy . The conditions and processes forγδT cells expanded in vitro were optimized and the relative functional studies were performed to validated the results. We hope to establish experiment foundation and provide theory instruction for the clinical application ofγδT cells. The research mainly included:1.γδT cells preparation for adoptive therapy. According to the request of "Human cell therapy research and quality control technical guidelines" (abbreviated as guidelines) published by State Drug Administration on 22th March, 2003, we performed the normalization and standardigation research ofγδT cells preparation for adoptive therapy. It showed thatγδT cells with high purity could be obtained from PBMC of healthy individuals and patients with malignant carcinoma using the solid-phase monoclonal anti-TCRγδAb methods.2. Safety investigation ofγδT cells preparation. The safety test mainly included tumorigenicity test, systemic and local safety tests such as acute toxicity, allergy, hemolytic and local irritation which were related to local or systemic administration. The results indicated thatγδT cells preparation had neither acute toxicity nor tumorigenicity to the acceptor.3. Effectiveness evaluation ofγδT cells preparation. The study included detecting the biological characteristic ofγδT cells through phenotype analysis and cytotoxic experiments in vitro. In this study, we validated the cytotoxic effects ofγδT cells to tumor cells, determinated the suitable propotion of effective cells to target cells and find out the suitable taget tumor cells. The results demonstratedγδT cells expanded from human PBMC could kill various solid tumor cells, such as human ovarian carcinoma (SKOV3), lung carcinoma (NCI-H520), pancreatic carcinoma (PANC-1), and hematological B lymphoma (Daudi) cell lines within a wide range of effector to target ratios. Furthermore,γδT cells expanded from healthy individual PBMC could kill SKOV3 cells and cis-platinum resistant SKOV3 cells within a wide range of effector to target ratios, suggesting thatγδT cells might be used in future clinical treatment combined with chemotherapy.4. Anti-tumor effect on tumor-bearing animals in vivo was observed , including the effects of adoptive immunotherapy with healthy mice spleenγδT cells using mice tumor model, the effects of adoptive immunotherapy with healthy humanγδT cells and the relation between adoptive immunotherapy and chemotherapy using nude mice xenograft tumor model and the effects of adoptive immunotherapy with healthy humanγδT cells xenograft tumor model in immune-reconstituted SCID mice with healthy human PBMC . The results demonstrated that adoptive transferring ofγδT cells was effective in inhibiting SKOV3 ovary tumor cells growth in Hu-PBMC-SCID mice and nude mice; adoptive transferring ofγδT cells was effective in inhibiting NCI-H520 lung cancer cells growth in nude mice;γδT cells from spleen of mice can effectively inhibited tumor growth of OV2944 in vivo.In conclusion, the present study demonstrated thatγδT cells with high purity could be obtained using this solid-phase method. The purity ofγδT cells expanded could reach 90% and 70% from normal controls and patients with malignant carcinoma, respectively. The methods was proved to be safe, effective and simple. TheseγδT cells displayed significant cytotoxicities against a variety of tumor cell lines both in vivo and in vitro. The results demonstrated thatγδT cells might be suitable for clinical trials of adoptive immunotherapy.
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