Figure 2.

TLR3 and TLR7 are required for the acquisition of suppressive properties by pDCs upon RNA-gag oligonucleotide treatment. (A) Skin test reactivity of mice sensitized with a combination of splenic HY-pulsed immunostimulatory wild-type cDCs and a minority fraction (5%, indicated) of pDCs purified from either Tlr7^−/− (Tlr7^−/− pDC) or Tlr3^−/− (Tlr3^−/− pDC) mice. pDCs were left untreated (untr.) or stimulated with 1.5 μM RNA-gag before being HY-pulsed together with the cDC majority fraction, and i.p. transferred into syngeneic C57BL/6 recipient female mice. Two weeks after the immunization, mice were assayed by footpad challenge with HY peptide. Skin test reactivity of the recipient mice to the eliciting peptide (n = 6 per group) is represented as the change in weight of the treated footpad vs. the vehicle-receiving counterpart. Data are reported as the mean value ± S.D. of three experiments. Significance is referred to as a paired Wilcoxon test (experimental vs. control footpads) in each group of mice; * p < 0.05. (B) Co-immunoprecipitation assay in HEK cells transfected with HA-tagged Tlr7 or/and Myc-tagged Tlr3. TLR7-HA protein complexes were immunoprecipitated with a specific anti-HA antibody and protein A-agarose (anti-HA + beads); samples immunoprecipitated with protein A-agarose alone were used as controls (beads). Membranes were immunoblotted with an anti-HA antibody as an immunoprecipitation control or with an anti-Myc antibody to reveal TLR7-HA/TLR3-Myc co-immunoprecipitated complexes.