摘要
Background Despite extensive research, the mechanism of immature dendritic ceils （DCs） induced immune hyporesponsiveness remains incompletely understood. Methods Recipient DCs from C3H mouse bone marrow cells were incubated with donor antigen from splenic lymphocytes of C57BL/6 mouse; these DCs were transfected with CD80/86 specific siRNA using lentiviral vectors. Flow cytometry was used to evaluate expression of CD80/86 on the antigen-pulsed recipient DCs. Immune regulatory activity was examined by mixed lymphocyte reaction, in which irradiated DCs were cultured with C3H spleen T cells. After the reaction, interleukin （IL）-2, IL-4, IL-10, and interferon （INF）-~ levels of mixed lymphocyte reaction culture supernatant were measured by enzyme-linked immunosorbent assay. The apoptotic T lymphocytes were identified by Annexin V and CD3 staining. Results There was a significant inhibition of CD80/86 expression in DCs transfected with CD80/86 lentiviral vectors compared with the control groups （P 〈0.05）, indicating the specificity of RNA interference. Enzyme-linked immunosorbent assay results showed a significant reduction of INF-γ, IL-2 and IL-10 in the CD80/86 lentivirus transfected group compared to the control groups （P 〈0.05）. There was no significant difference in IL-4 levels between the groups （P 〉0.05）. We also showed that CD80/86 low DCs loaded with alloantigen （1） stimulated low T cell proliferative responses via the indirect recognition pathway and （2） enhanced apoptotic activity （P 〈0.05） in co-cultured T cells. Conclusions Lentiviral vector transfection can effectively and specifically knock down target genes in DCs. The CD80/86 low DCs may show tolerogenic activity via induction of T-cell apoptosis, thereby modulating the activity of recipient-derived DCs. The use of this approach may potentially be clinicallv applicable.
Background Despite extensive research, the mechanism of immature dendritic ceils （DCs） induced immune hyporesponsiveness remains incompletely understood. Methods Recipient DCs from C3H mouse bone marrow cells were incubated with donor antigen from splenic lymphocytes of C57BL/6 mouse; these DCs were transfected with CD80/86 specific siRNA using lentiviral vectors. Flow cytometry was used to evaluate expression of CD80/86 on the antigen-pulsed recipient DCs. Immune regulatory activity was examined by mixed lymphocyte reaction, in which irradiated DCs were cultured with C3H spleen T cells. After the reaction, interleukin （IL）-2, IL-4, IL-10, and interferon （INF）-~ levels of mixed lymphocyte reaction culture supernatant were measured by enzyme-linked immunosorbent assay. The apoptotic T lymphocytes were identified by Annexin V and CD3 staining. Results There was a significant inhibition of CD80/86 expression in DCs transfected with CD80/86 lentiviral vectors compared with the control groups （P 〈0.05）, indicating the specificity of RNA interference. Enzyme-linked immunosorbent assay results showed a significant reduction of INF-γ, IL-2 and IL-10 in the CD80/86 lentivirus transfected group compared to the control groups （P 〈0.05）. There was no significant difference in IL-4 levels between the groups （P 〉0.05）. We also showed that CD80/86 low DCs loaded with alloantigen （1） stimulated low T cell proliferative responses via the indirect recognition pathway and （2） enhanced apoptotic activity （P 〈0.05） in co-cultured T cells. Conclusions Lentiviral vector transfection can effectively and specifically knock down target genes in DCs. The CD80/86 low DCs may show tolerogenic activity via induction of T-cell apoptosis, thereby modulating the activity of recipient-derived DCs. The use of this approach may potentially be clinicallv applicable.
基金
This study was supported by grants from the National Natural Science Foundation for Young Scholar of China （No. 81102238）, the Research Fund for the Doctoral Program of Higher Education （No. 20100071110066）, the Research Fund for the Doctoral Program of Higher Education （New Teacher, No. 20110071120069）.
