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Toll-like receptor 9 (TLR9) is expressed intracellularly by dendritic cells (DCs) and specifically recognizes unmethylated CpG motif. Recognition of TLR9 to CpG DNA can induce DC maturation followed by the subsequent immune responses. Here, RNA interference (RNAi) was used to identify the effect of CpG DNA signaling on DC function. The results showed that transfection of DCs with siRNA specific for TLR9 gene significantly down-regulated TLR9 expression. Immature DCs trausfected with TLR9 siRNA did not differentiate into mature DCs with exposure to CpG, TLR9 siRNA-treated DCs expressed low levels of MHC II and CD40 without reducing endocytosis. Furthermore, TLR9 siRNA-transfected DCs exhibited a decreased allostimulatory capacity in a lymphocyte proliferation assay and attenuated Thl responses by decreasing IL-12p70 production. Our findings indicate that siRNA in silencing TLR9 gene in DCs 5 offer a potential tool to study the TLR9-CpG pathway. Cellular & Molecular Immunology. 2009;6(3):199-205.