Objective To optimize in vitro amplification of human γδ T cells with cytokines for tumor adoptive immunotherapy.Methods On the basis of the immobilized anti-TCR γδ antibody plus IL-2 system, other γ chain receptor family cytokines, including IL-7, IL-15 and IL-21, were tested to amplify human peripheral blood γδ T cells either alone or in diversity combination.The percentage of γδ T cells was measured by flow cytometry, and the proliferation efficiency of γδ T cells was calculated.The expression of proliferation-or cytotoxicity-related molecules on γδ T cells was examined by flow cytometry in order to explore the relevant mechanisms.The cytotoxicity of γδ T cells to Daudi cells was detected by lactate dehydrogenase.Results IL-15 alone but not IL-7 or IL-21 increases the γδ T cell purity, amplification efficiency and cytotoxicity to reach comparable levels to those of IL-2.IL-2 plus IL-15 up-regulates the expression of CD69 on γδ T cells and significantly increases their amplificationefficiency (P<0.05).IL-2 plus IL-21 enhanced the cytotoxicity of γδ T cells against Daudi cells by increasing the expression of granzyme A (P<0.001).The combination of IL-2, IL-15 and IL-21 significantly improves cytotoxicity of γδ T cells but reduces their amplification efficiency.In addition, when IL-21 was applied for a short time, it also enhanced the cytotoxicity of γδ T cells (P<0.05).Conclusions The combination of IL-2 and IL-15 as well as a short time addition of IL-21 is the best cytokine recipe to amplify human peripheral blood γδ T cells in vitro with immobilized anti-TCR γδ antibody, which can increase both the proliferation efficiency and the cytotoxicity to tumor cells of γδ T cells.

Objective To construct human lung cancer cell model with human MutS homologous protein 2 (hMSH2) overexpression for exploring the effect of hMSH2 molecule in the cytotoxicity of γδ T cell against lung cancer cells.Methods hMSH2 coding sequence was cloned by PCR for construction of recombinant vector which over expressed hMSH2-EGFP fusion protein using homologous recombination.The recombinant vector was transfected to lung cancer cell line NCI-H520 to construct human lung cancer cell model overexpressing hMSH2 molecule.The expression of hMSH2 molecule in NCI-H520 was detected by Western blot.The expression of hMSH2 on the cell membrane was measured by flow cytometry.Cytotoxicity of expanded γδ T cells from peripheral blood mononuclear cells against NCI-H520 cells was detected by LDH release assay in vitro.Results hMSH2 coding sequence (2805 bp) was cloned and the result of restriction endonuclease digestion of Fugw-hMSH2 recombinant vector was accordance with the anticipated objective strip size.Exogenous hMSH2-EGFP fusion protein was expressed in NCIH520 cells.The level of hMSH2 molecule on the surface of NCI-H520 cells with overexpression of hMSH2 was significantly increased (P＜0.001).Cytotoxicity of γδ T cells against NCI-H520 cells with overexpression of hMSH2 was significantly increased compared to the wild type NCI-H520 cells (P＜0.05).Conclusions Lung cancer cell model that overexpresses hMSH2 molecule is successfully constructed,hMSH2 molecule on the cell membrane is increased and the cytotoxicity of γδ T cells against lung cancer cells is enhanced.