The graft-versus-tumor （GVT） effect of T cells induced by tumor antigen-pulsed CD8α＋ dendritic cells （DCs） in vitro was investigated in this study. Immature CD8c（DCs were prepared from C57BL/6 （H-2b） bone mar...The graft-versus-tumor （GVT） effect of T cells induced by tumor antigen-pulsed CD8α＋ dendritic cells （DCs） in vitro was investigated in this study. Immature CD8c（DCs were prepared from C57BL/6 （H-2b） bone marrow cells by using a cytokine cocktail. On the 3rd day of culture, CD8α＋ DCs were pulsed by allogeneic （Balb/c, H-2d） EL9611 leukemia antigen, or RM-1 syngeneic prostate cancer antigen, with the concentration series of 0, 2.5, 5.0, 10.0, 20.0 μg/mL, respectively, then antigen-loaded immature CD8α＋ DCs were co-cultured with syngeneic T cells according to the DC/T ratio of 1：1, 2：1 and 4：1. T cell proliferation was measured by MTT assay. Cytokines including interferon gamma （IFN-γ） and interleukin-10 （IL-10） in CD8α＋ DCs and T co-culture supernatant were detected by using ELI SA. Cytotoxic effect of antigen-specific T cells was tested by LDH release assay. Conventional mature DCs （mDCs） induced, from C57BL/6 （H-2b） bone marrow cells by using granulocyte-macrophage colony stimulating factor （GM-CSF） and interleukin-4 （IL-4） served as a control. The results showed that the proliferative activity of T cells stimulated by CD8α＋ DCs loaded with allogeneic or syngeneic tumor antigen was augmented with the CD8＆ DC/T ratio increased （P〈0.05）. When antigen concentration 〈 5 μg/mL and CD8a＋ DC/T ratio 〈 2：1, the ability of CD8α＋ DCs to stimulate T cell proliferation was higher than mDC control in allogeneic tumor antigen-pulsed groups （P〈0.05）, but not in syngeneic tumor antigen-pulsed groups （P〉0.05）. The level of IFN-γ and IL-10 in CD8α＋DCs and T cell co-culture supernatant were increased in both allogeneic and syngeneic antigen-pulsed groups （P〈0.05）, and the cytokine level was higher in allogeneic antigen-pulsed groups than in syngeneic antigen groups when the CD8α＋ DC/T was 1：1 or 2：1 （P〈0.05）. There existed a negative correlation between the level of IL-10 and T cell proliferation. T cell cytotoxieity assay showed that when CD8α＋ DCs were pulsed with allogeneic tumor antigen, the maximal T cell killing efficiency could reach （100±7.7）%, whereas syngeneic tumor antigen-pulsed group had only （65.0±3.4）%. It was concluded that syngeneic and allogeneic tumor antigen-pulsed immature CD8α＋ DCs could stimulate T cells to exert the GVT effect in vitro, and the GVT effect was more obvious with allogeneic tumor antigen than with syngeneic tumor antigen. The optimal condition was low allogeneic tumor antigen pulsation （〈 5 μg/mL） and low CD8α＋ DC/T ratio （1：1 and 2：1）.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.81000230)the Natural Science Foundation of Guangdong Province of China(No.8451008901000213)Guangdong Science and Technology Project(No.2010B031600052andNo.2011B040300021)
文摘The graft-versus-tumor （GVT） effect of T cells induced by tumor antigen-pulsed CD8α＋ dendritic cells （DCs） in vitro was investigated in this study. Immature CD8c（DCs were prepared from C57BL/6 （H-2b） bone marrow cells by using a cytokine cocktail. On the 3rd day of culture, CD8α＋ DCs were pulsed by allogeneic （Balb/c, H-2d） EL9611 leukemia antigen, or RM-1 syngeneic prostate cancer antigen, with the concentration series of 0, 2.5, 5.0, 10.0, 20.0 μg/mL, respectively, then antigen-loaded immature CD8α＋ DCs were co-cultured with syngeneic T cells according to the DC/T ratio of 1：1, 2：1 and 4：1. T cell proliferation was measured by MTT assay. Cytokines including interferon gamma （IFN-γ） and interleukin-10 （IL-10） in CD8α＋ DCs and T co-culture supernatant were detected by using ELI SA. Cytotoxic effect of antigen-specific T cells was tested by LDH release assay. Conventional mature DCs （mDCs） induced, from C57BL/6 （H-2b） bone marrow cells by using granulocyte-macrophage colony stimulating factor （GM-CSF） and interleukin-4 （IL-4） served as a control. The results showed that the proliferative activity of T cells stimulated by CD8α＋ DCs loaded with allogeneic or syngeneic tumor antigen was augmented with the CD8＆ DC/T ratio increased （P〈0.05）. When antigen concentration 〈 5 μg/mL and CD8a＋ DC/T ratio 〈 2：1, the ability of CD8α＋ DCs to stimulate T cell proliferation was higher than mDC control in allogeneic tumor antigen-pulsed groups （P〈0.05）, but not in syngeneic tumor antigen-pulsed groups （P〉0.05）. The level of IFN-γ and IL-10 in CD8α＋DCs and T cell co-culture supernatant were increased in both allogeneic and syngeneic antigen-pulsed groups （P〈0.05）, and the cytokine level was higher in allogeneic antigen-pulsed groups than in syngeneic antigen groups when the CD8α＋ DC/T was 1：1 or 2：1 （P〈0.05）. There existed a negative correlation between the level of IL-10 and T cell proliferation. T cell cytotoxieity assay showed that when CD8α＋ DCs were pulsed with allogeneic tumor antigen, the maximal T cell killing efficiency could reach （100±7.7）%, whereas syngeneic tumor antigen-pulsed group had only （65.0±3.4）%. It was concluded that syngeneic and allogeneic tumor antigen-pulsed immature CD8α＋ DCs could stimulate T cells to exert the GVT effect in vitro, and the GVT effect was more obvious with allogeneic tumor antigen than with syngeneic tumor antigen. The optimal condition was low allogeneic tumor antigen pulsation （〈 5 μg/mL） and low CD8α＋ DC/T ratio （1：1 and 2：1）.
