Reduced DTH response, but normal IFN-γ secretion, to mycobacterial antigens stimulation in MyD88^–/– mice. (A) Cutaneous DTH response was performed 3 weeks after vaccination with M. bovis BCG (10⁵ CFU s.c.; v) by assessing the footpad swelling in response to PPD injection as described. Paw swelling is defined as the difference in thickness between the left paw injected with PPD and the right paw injected with saline. DTH responses of nonvaccinated MyD88^–/– and MyD88^+/+ mice are shown as control (nv). Data are expressed as mean ± SD (n = 7–8) and are from 1 representative experiment out of 2. **P – 0.01. (B) For the T cell response, spleen cells from M. bovis BCG–vaccinated MyD88-deficient and control mice were harvested 4 weeks after inoculation (10⁵ CFU s.c.) and were restimulated in vitro in the presence of soluble BCG antigens (SupBCG, 10 μg/ml) or unrelated antigen (HKLM, 100 bacteria per cell). Naive MyD88^–/– mice and wild-type mice were used as negative control. IFN-γ production was quantified in the supernatants after 48 hours of incubation. Results are mean ± SD from n = 2 mice per genotype and are representative of 3 independent experiments. (C) Intracellular IFN-γ staining of CD4⁺ or CD8⁺ splenocytes from BCG-infected MyD88-deficient and control mice 4 weeks after infection, restimulated for 18 hours in the presence of soluble BCG antigens (SupBCG, 10 μg/ml) or unrelated antigen (HKLM, 100 bacteria per cell). Typical dot blots are shown for CD4⁺ T cells (numbers indicate percentage of IFN-γ–positive CD4⁺ cells).