Figure 1

Analysis of antigen specific CD4⁺/CD8⁺ T cell responses induced by vaccination in the lung and spleen. (a) Schematic illustration of the gene deletion produced in the second-generation version of BCGΔBCG1419c compared with wild type BCG. (b) Experimental scheme for BCGΔBCG1419c and BCG vaccines testing. Each group of female mice (n = 10) was vaccinated with BCG or BCGΔBCG1419c via subcutaneous injection (1.0 × 10⁶ CFUs/mouse; blue arrow). Ten weeks after vaccination, mice were aerogenically infected with Mtb strain M2, achieving an initial infectious dose of ~ 200 CFUs (black arrow). Immunological analysis was conducted before and after Mtb infection (red arrow). Bacterial counts and histopathological analysis in each subset of mice were evaluated at the indicated time point after Mtb infection (red arrow). (c) Ten weeks after vaccination, mice from each group (n = 4) were sacrificed, and cells from lung and spleen were stimulated ex vivo with or without PPD (5 μg/ml) at 37 °C for 9 h in the presence of GolgiPlug. The frequencies of PPD-specific IFN-γ⁺TNF-α⁺- or IFN-γ⁺IL-2⁺-producing CD4⁺CD62L^-CD44⁺ T-cells were determined by intracellular cytokine staining in the lungs and spleen of each vaccinated mouse and presented as dot plots with bar graphs. (d) The frequencies of PPD-specific IFN-γ⁺TNF-α⁺- or IFN-γ⁺IL-2⁺-producing CD8⁺CD62L^-CD44⁺ T-cells were determined by intracellular cytokine staining in the lungs and spleen of each vaccinated mouse and presented as dot plots with bar graphs. The experimental results are presented as the mean ± SD from 4 mice from each group. The representative mean values are denoted in each FACS plot. One-way ANOVA with post hoc Tukey’s multiple comparison test was used to evaluate the significance. *p < 0.05, **p < 0.01, and ***p < 0.001. The experimental results of one representative experiment are shown.