Figure 5. Vaccine-promoted CD8⁺ T cell responses to TB10.3/4_3-11 do not confer protection to aerosol challenge.

(A) CB6F1 mice (n = 4) were immunized three times with 10 μg TB10.3 P1_1-20 + CAF05 (i.p.) or left unimmunized. Three weeks after the last immunization, splenocytes from individual mice were analyzed by intracellular flow cytometry and the frequency of IFN-γ-producing CD4⁺ and CD8⁺ T cells determined. Cells were stimulated with 2 μg/mL of the TB10.3 P1_1-20 peptide and mean frequencies ± SEM following background subtraction are presented from one experiment representative of 3 independent experiments. **p < 0.01, (Student’s T-test).

(B) Groups of CB6F1 mice were immunized three times with 10 μg TB10.3 P1_1-20 + CAF05 either by the i.p. route or through pulmonary vaccine delivery using a Penn-Century Microsprayer aerosolizer. Non-immunized mice were included as controls. Three weeks post-immunization, pooled cells from lungs, spleen and PBMCs were isolated and non-stimulated cells subjected to ex vivo staining using PE-conjugated MHC class I dextramer H2-K^b-IMYNYPAM and surface markers. Bars show the frequency of IMYNYPAM⁺CD8⁺CD62L⁻ cells out of CD8⁺ T cells in the different compartments.

(C) Seven weeks post-immunization, mice from naïve (n = 11), BCG (n = 6), pulmonal (n = 5) and i.p. (n = 6) groups were subjected to aerosol challenge (~100 CFU/mouse). Mice were euthanized at week 7 post-infection and CFU levels were determined by serial dilutions of individual lung homogenates onto Middlebrook 7H11 agar plates. Each symbol represents the Log₁₀ CFU level of one mouse and means ± SEM are shown. *** p < 0.001 (ANOVA with Tukey’s post-test). Experiment was repeated once with similar results.