Figure 2. TLR3 knockdown has a considerable effect on OK432 induced IL-12p70 production by DC.

A) DC were electroporated with either control siRNA that has no specific target in human cells or with two siRNA specific against TLR3. The level of siRNA mediated TLR3 knockdown was found to be around 40 percent after 24 hours of OK432 stimulation (48 h after electroporation). Open histogram: unstained control; shaded histogram: mock treated; black histogram: TLR3 siRNA treated; one representative experiment shown of five. B) After resting for 24 hours, the DC were incubated with OK432 for 24 hours and the supernatant was collected and analyzed for IL-12p70 production by ELISA. OK432 is critically dependent on TLR3 signaling to induce IL-12p70 production. Immature cells (Imm) received only IL-4 and GM-CSF, the Cyto group were matured with IL-1β, IL-6, TNF-α and PGE₂. Mean+SEM are shown for four donors. C) To further verify the specificity of the TLR3 siRNA knock down, DC were electroporated with escalating concentrations of TLR3 siRNA and rested for 24 hours. Subsequently, after 24 hour stimulation with OK432, IL-12p70 production was analyzed. One representative donor is shown out of three. D) TLR3 siRNA knockdown has a marked effect on the phenotype of OK432 matured DC. Percent MFI of CD1a, HLA-DR, CD86 and CD40 after TLR3 siRNA knock down and OK432 stimulation compared to OK432 plus control siRNA (set to 100%). Mean of two donors shown. E) TLR3 siRNA inhibit IL-12p70 production upon polyI∶C stimulation of DC. IL-12p70 production of polyI∶C treated DC were set to 100%. One representative donor is shown.