Effect of CD3/CD28 activator on the cytotoxicity of human ex-vivo expanded γδ T cells against osteosarcoma cells

Abstract Background We investigated the effect of CD3/CD28 activator on the ex-vivo expansion of human γδ T cells and their cytotoxic effect against osteosarcoma cells. Methods Peripheral blood mononuclear cells from healthy donors were culture in three different conditions for 10 days: basal expansion media (COM media), COM media with zoledronate+hrIL-2 (EX media), and EX media with CD3/CD28 activator (EX28 media). Percentage of expanded γδ T cells was analyzed by flow cytometry. IFN-γ and TNF-α secreted in the culture media was measured using ELISA assay. Cytotoxicity of γδ T cells against KHOS/NP cells was measured by MTT assay. Results Compared with COM media, the fold increase of cell expansion were 33.6 in EX media and 368.2 in EX28 media. The population of γδ T cells expanded in COM media represented 4.9% of CD3+ T cells, while γδ T cells expanded in EX media reached 71% of CD3+ T cells. The population of γδ T cells expanded in EX28 media represented 18% of CD25+ T cells, but not CD3+ T cells. The concentration of IFN-γ and TNF-α in the culture media increased both in EX (1000-fold, 340-fold) and EX28 (2340-fold, 1849-fold) media, compared with unstimulated control. Unexpectedly, IFN-γ and TNF-α production per cells were higher in EX media than in the EX28 media. The γδ T cells expanded in EX media showed potent cytotoxicity against KHOS/NP osteosarcoma cells in a ratio-dependent manner, but γδ T cells expanded in EX28 media did not show significant cytotoxic effect. Conclusion CD3/CD28 activator were not as potent as the combination of zoledronate and hrIL-2, for expansion γδ T cells nor its cytotoxic activity against KHOS/NP cells, although it increased the total number of T cells.