Figure 4

Modification of DCs by LV-siRNA targeting IL-10. (A) LV-siRNA targeting IL-10. LV siRNAs targeting two different sites of IL-10 mRNA were illustrated. The predicted hairpin siRNA structure is shown. (B) Illustration of efficient down-regulation of IL-10 in B lymphocytes after LV IL-10 siRNA transduction. Epstein-Barr virus (EBV) transformed B cells were transduced with LV siRNA targeting IL-10 (#1 and #2) or GFP gene. The siRNA LVs also carry a lacZ reporter gene which could be labeled with fluorescein di-b-D-galactopyranoside (FDG) to separate the transduced from un-transduced cells by FACS sort. (C) Immature DCs were transduced with mock, empty LVs, LV-nlacZ, or LV-nlacZ plus LV-siIL-10 #1 or #2, treated with LPS, and analyzed by ICCS and FACS using anti-IL-10 antibody. (D) Enhanced Th1 response by DCs transduced with LV-siRNA targeting IL-10. DCs were transduced with LVs and either co-transduced with LV-siRNA targeting IL-10 (LV-IL10i#2) or GFP (GFPi) or treated with soluble IFN-γ as controls, and the DCs were then assayed for T-cell activation function by DC:T cell co-culture. The T cells were fully rested before reactivation with PMA and ionomycin after 10 days of co-culture. The numbers shown in the FACS quadrants are percentages of the total gated cell population. Results are representative of three independent experiments.