Fig 2: T cell response to purified protein derivative (PPD) in male and female mice vaccinated with Bacillus Calmette-Guérin (BCG) or BCGΔBCG1419c cultured as biofilms. BALB/c mice (male and female) were subcutaneously vaccinated with biofilm-derived BCG Pasteur or BCGΔBCG1419c. Sixty days post-vaccination, lung and spleen leukocytes were stimulated ex vivo with PPD and analysed by flow cytometry. (A) Frequency of CD3⁺CD8a⁺ interferon gamma (IFN-γ)⁺ T cells in CD3⁺CD8a⁺ cells. (B) Frequency of CD3⁺CD8a⁺ tumour necrosis factor alpha (TNF-α)⁺ T cells in CD3⁺CD8a⁺ cells. (C) Frequency CD3⁺CD4⁺IFN-γ⁺ T cells in CD3⁺CD4⁺ cells. (D) Frequency of CD3⁺CD8a⁺ interleukin-2 (IL-2)⁺ TNF-α⁺ T cells in CD3⁺CD8a⁺ cells. (E) Frequency of CD3⁺CD4⁺ IL-2⁺ T cells in CD3⁺CD4⁺ cells. Each dot represents an individual animal; horizontal lines indicate group means and vertical lines standard deviation. Statistical analyses were performed using two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Significant p-values are indicated in the panels.