Figure 3. Endosomal TLRs are highly expressed in CD8α ⁺ DCs and upregulated upon T. gondii infection.

(A) Real-time PCR was performed to determine the relative levels of TLR3, TLR7, TLR9, TLR11 and TLR12 mRNA expressed by CD11b⁺, CD11c⁺/CD8α⁺ and CD11c⁺/CD8α ⁻ cells sorted from splenocytes from uninfected controls as well as infected (5 days post-infection) WT mice. TLR mRNA levels were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Data are represented as mean ±SD of three experiments. (B) HEK 293T cells were transfected with different pairs of plasmids, total lysates immunoprecipitated (IP) with anti-hemagglutinin (anti-HA, top) or anti-Flag (bottom), and analyzed by immunoblot (IB) with anti-Flag (top and bottom). The top membrane was then stripped and re-probed with anti-HA to ensure expression of hemagglutinin-tagged TLR11 (middle). (C) CD11c⁺ cells were purified from spleen of WT, 3d, TLR7/TLR9, TLR3/TLR7/TLR9 and TLR11 KO mice and stimulated with LPS (100 ng/ml) ODN CpG 1826 (1 μm), R848 (2 μm) or infected with ME49 tachyzoites (MOI 3:1). (D) CD11c⁺ cells purified from WT, TLR4, TLR7/TLR9 and TLR12 KO mice were stimulated with LPS (100 ng/ml), ODN CpG 1826 (1 μm), STAg (10 μg/ml), or rTgPRF (10 ng/ml) left untreated, or treated with DNAse (100 U/ml), RNAse (10 μg/ml) or Proteinase K (10 μg/ml). IL-12 levels were measured in the supernatant at 24 h after stimulation. (C–D) Data are represented as mean ± SD of four experiments. Asterisks indicate that difference is statistically significant, when comparing cytokines levels from WT to different KO mice, infected or not infected with T. gondii (*0.01 < p < 0.05, **0.001 < p < 0.01, ***p < 0.001). See also Figure S1.