BCG induces antigen-specific CD4+ T cells in the lungs, spleen and peripheral blood, with highest frequencies in the lung parenchyma and vasculature. Following BCG immunisation, intravascular staining and ICS identified populations of antigen- (TB10.4 peptide) specific (cytokine+) CD4+ T cells in the lungs, spleen and peripheral blood producing IFN-γ, IL-2 or TNF-α alone or in combination. (a) Frequencies of antigen-specific CD4+ T cells as a % of the total number of CD4+ T cells in the same compartment. Statistical comparison is between BCG and control at each time point. (b) Frequencies of antigen-specific CD4+ T cells from all compartments in BCG-vaccinated animals expressed as a % of the total number of CD4+ T cells in the same compartment. Statistical comparison is between frequency of BCG-induced antigen-specific CD4+ T cells in all compartments at each time point. For all graphs, points represent mean ± SEM (n = 6). Two-way ANOVA with Sidak’s post-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.