title: Recipient dendritic cells modified by RNA interference targeting CD80 and CD86 elicit T cell hyporesponsiveness via enhanced T cell apoptosis.
abstract: 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.
authors.affiliation: Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Hong
authors.forename: Xiao-dong
authors.initials: XD
authors.lastname: Gu
authors.forename: Jian-bin
authors.initials: JB
authors.lastname: Xiang
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhang
authors.forename: Yi-wen
authors.initials: YW
authors.lastname: Zang
authors.forename: Qun-hua
authors.initials: QH
authors.lastname: Zhang
authors.forename: Shi-guang
authors.initials: SG
authors.lastname: Qian
authors.forename: Zong-you
authors.initials: ZY
authors.lastname: Chen
mesh_terms: D000818:Animals; D017209:Apoptosis; D018122:B7-1 Antigen / Q000235:genetics / Q000502:physiology*; D051940:B7-2 Antigen / Q000235:genetics / Q000502:physiology*; D003713:Dendritic Cells / Q000276:immunology*; D016086:Lentivirus / Q000235:genetics; D008213:Lymphocyte Activation; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D034622:RNA Interference; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*
chemical_list: D018122:B7-1 Antigen; D051940:B7-2 Antigen