Recipient dendritic cells modified by RNA interference targeting CD80 and CD86 elicit T cell hyporesponsiveness via enhanced T cell apoptosis.

BACKGROUND: Despite extensive research, the mechanism of immature dendritic cells (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)-γ 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 clinically applicable.

[Inducing liver-derived dendritic cell proliferation by plasmid-IL-3 and CD40L genes in mice in vivo].

OBJECTIVES: Try to induce liver-derived dendritic cells proliferation by plasmid-IL-3 and CD40L genes in mice in vivo. METHODS: Rapid tail-vein injection of large amounts of plasmid-carrying genes was performed to transfect genes in mice livers. Liver nonparenchymal cells were isolated by Percoll gradient centrifugation. Dendritic cell markers were detected and dendritic cells were sorted out by flow cytometry. Morphology of dendritic cells was studied microscopically (with Giemsa staining) and under scanning electron microscopy. RESULTS: Liver nonparenchymal cells dramatically increased in the liver lobules, portal and periportal areas in the treated group, but not in the control group. B220+/DEC205+ dendritic cells were detected and sorted by flow cytometry. There were more B220+/DEC205+ dendritic cells (16.0%) in the experiment group than those in the control group (1.1%). Morphologically, the sorted B220+/DEC205+ cells showed irregular shaped nuclei, paucity of cytoplasmic granules and extensive cytoplasmic processes. CONCLUSION: B220+/DEC205+ dendritic cells were expanded in vivo in mice livers by rapid tail-vein injection of plasmid-carrying genes encoding IL-3 and CD40L in a large amount. Inducing liver dendritic cell proliferation in vivo provides a more convenient way for studying the biology of these cells.