Fig. 4.

ttRNA–DC ex vivo-expanded human T cells commit specific recognition and lytic activity. a The analysis of HLA-A2 on PBMC and U87 line. HLA-A2 expression in human U87 tumor line and PBMC was measured by fluorescent-conjugated HLA-A2 antibody. For abbreviation, positive was labeled as pos and negative as neg. One representative PBMC from HLA-A2 pos/neg donors was shown. HLA-A2 expression was denoted on left of the image. b The phenotype of ex vivo ttRNA–DC-expanded T cells. Human DC were electroporated with ttRNA from U87 tumor line followed by overnight maturation as described in “Materials and methods” section; the T cells from HLA-A2+/− donors were co-cultured with ttRNA–DC in the presence of IL-2 or a cocktail for 12 days. The phenotype of T cells was analyzed using fluorescent-conjugated CD62L and CD45RO antibodies by flow cytometry. c The specific IFNγ induction. DC from both donors was electroporated with ttRNA or pseudo-electroporated, which were co-cultured with T cells from autologous donors. After 12 days expansion, T cells (2.5 × 10⁵/well) were co-cultured with U87 at a ratio of 10:1, and the levels of IFNγ were measured by ELISA kit. d The specific lysis of U87 tumor line. T cells cultured with IL-2 (left) and a cocktail (right) were co-cultured with U87 cells at different ratio of effector to target (E:T) for 5 h before the lytic assay was performed using CytoTox 96 non-radioactive cytotoxicity assay