Role for both TLR9 and TLR2 in the MyD88-dependent IL-12p40 response of splenic DCs to M. tuberculosis. (A) Purified CD11c+ spleen cells from WT or MyD88−/− mice were exposed to live (MOI = 1:1 [M. tuberculosis 1] or 3:1 [M. tuberculosis 3]) or heat-killed (MOI = 1:1 H-K M. tuberculosis 1]) M. tuberculosis for 24 h. (B) WT DCs were treated with 5 μg/ml cytochalasin D or vehicle (DMSO) for 30 min and then incubated with live M. tuberculosis (MOI = 1:1) or 10 μg/ml LPS for 24 h. (C) DCs from WT or TLR2−/− mice were treated with 5 μg/ml chloroquine or vehicle (saline) for 30 min and then stimulated with live M. tuberculosis (MOI = 1:1) for 24 h. (D) DCs from WT, MyD88−/−, TLR2−/−, TLR9−/−, and TLR2/9−/− were exposed to live M. tuberculosis (MOI = 1:1) or TLR agonists as described in A. In all experiments, supernatants were harvested and IL-12p40 was determined by ELISA. Results are means ± SE of triplicate measurements. Experiments shown are representative of at least three performed. *, P < 0.05 between experimental and control groups in A, B, and C. **, P < 0.05 between TLR2−/− versus TLR9−/− values in D.