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 explore the role of N-cadherin and β-catenin in the formation of spinal commissural axon projection during chicken embryonic development.Methods:Fertilized eggs were cultured for three days(stage22),N-cadherin orβ-catenin inter-ference plasmid was injected into the neural tube and in vivo electroporation was performed.Three days after the electroporation, embryos were collected,fixed with 4%PFA,embeded with OCT,and cut into frozen sections.Four groups ( knockdown of N-cadherin orβ-catenin or both of them,and control) were included in this study.Immunohistochemistry method was used to analyze the protein expression result of N-cadherin or β-catenin.The changes of spinal commissural projections were observed with GFP fluorescence.Results:During chicken embryonic development,knockdown of N-cadherin inhibited the expression of β-catenin in the spinal cord.The commissural nerve fibers projecting to the contralateral side of the spinal cord was impaired after knockdown of N-cadherin or β-catenin;this phenotype was similar after knocking down both of them.Conclusion: N-cadherin is implicated in the formation of spinal commissural projection in the developing spinal cord,possibly viaβ-catenin.