Fig. 1.

Reduced uptake of soluble antigen by BMDC in the presence of TLR agonists. Granulocyte macrophage colony-stimulating factor (GM-CSF) BMDC were cultured for 4 h with OVA–Alexa555 (5 μg ml⁻¹) in the presence of polyI:C (50 μg ml⁻¹), CpG (0.5 μg ml⁻¹), R837 (1 μg ml⁻¹), LPS (1 μg ml⁻¹) or left untreated. Cells were stained with anti-CD11c antibody and samples were acquired by flow cytometry. (A) The mean fluorescent intensity of cells for Alexa555 was determined after gating on the CD11c⁺ cell population. The data of two independent representative experiments were pooled (n = 4) and the standard deviation is indicated. (B) The frequency of OVA–Alexa555⁺ cells was determined after gating on the CD11c⁺ cell population. The relative percentage of OVA–Alexa555⁺ BMDC in samples stimulated with TLR agonists is depicted in relation to the frequency of OVA–Alexa555⁺ BMDC in the absence of TLR stimulation (unst), which was set to 100%. The results of eight independent experiments were compiled and the standard deviation is indicated. (C) Mice were vaccinated intravenously with GM-CSF BMDC that had been pulsed overnight with OVA in the presence or absence of various TLR ligands. Seven days after vaccination, CFSE-labelled target cells were injected and antigen-specific lysis of peptide-pulsed targets cells versus unpulsed target cells was determined by flow cytometry the following day. The graph compiles data from two independent experiments (n = 10). For statistical analysis, one-way analysis of variance in combination with Dunnett's (A and B) or Tukey's (C) multiple comparison test was used (**P < 0.01, ***P < 0.001).