Abstract: 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 could increase the γδ T cell purity, amplification efficiency and cytotoxicity to reach comparable levels to those of IL-2. IL-2 plus IL-15 up-regulated the expression of CD69 on γδ T cells and significantly increased their amplification efficiency (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 could significantly improve the cytotoxicity of γδ T cells but reduce their amplification efficiency. In addition, when IL-21 was applied for a short time, it could also enhance the cytotoxicity of γδ T cells (P<0.05). Conclusion 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.

Key words: γδT cell, in vitro amplification, IL-15, IL-21