Fig. 3
CFP-10 and ESAT-6-derived peptides activate APCs through MyD88 and TLR-2-dependent pathways. a–d APCs from wild-type C57Bl/6, MyD88, or TLR-2-deficient mice were tested using in vitro antigen presentation (peptides tested at 0.5, 1, and 2 µM; one of two similar experiments shown; values are mean of IL-2 concentration for triplicate wells; *p < 0.01, **p < 0.006 vs. BCG alone; one-way ANOVA). IL-2 levels secreted by BB7 T cells overlaid on knockout APCs are not significantly different. e, f APCs were activated or not with C5 and BCG and TH1 cytokines measured using sandwich ELISA. IL-12, and TNF-α cytokine responses are comparable with peptide activation followed by BCG vaccine infection or BCG infection alone among MyD88 or TLR-2 deficient APCs. g MΦs were infected with BCG or BCG with C5 peptide followed by western blot of lysates for phosphorylated MAPKs at 30 min. Densitometry shows that BCG + C5 peptide induce a stronger phosphorylation of p38 and ERK compared with native MAPK. Upper lanes show phosphorylated proteins, and lower lanes indicate whole protein. One of two similar experiments averaged for densitometry (t test). h, i MΦs from wild-type and TLR-2 knockout mice were incubated 4 h with C5 or E3 peptide or non-immunogenic peptides from CFP-10 protein followed by BCG infection for 90 min, followed by lysis and western blot assay for phosphorylated (P) MAPKs or c-Jun/AP-1 and CREB transcription factors. Densitometry (shown below blots) indicate that C5 peptide induces better phosphorylation in the presence of TLR-2 (phosphorylated bands marked (p); upper lanes have native proteins). One of two similar experiments is shown, which were averaged for densitometry (t test)
