(A) At four months p.i., the formalin fixed and paraffin embedded lungs sections from Mtb-infected BCG-vaccinated and BCG-vaccinated T2DM were analyzed by confocal microscopy for Foxp3⁺IL13⁺ cells. Foxp3 (red), IL-13 (green), and DAPI (blue) expression is shown. Scale bar: 20 μm. BCG-vaccinated T2DM mouse splenocytes were isolated and cultured at 1 × 10⁶ cells/well in RPMI-1640 containing penicillin (Invitrogen) and 10% heat-inactivated FCS, with or without γ-irradiated Mtb H37Rv (10 µg/mL) at 37°C and 5% CO₂. In some wells, Treg-depleted splenocytes were stimulated with γ-irradiated Mtb (10 µg/mL). After 72 hours, the cells were collected and phenotypically characterized for M1/M2 macrophages, and the supernatants were used to measure levels of the cytokine IL-13. (B) Mean fluorescence intensity (MFI) values showing the CD45⁺F4/80⁺iNos⁺ and CD45⁺F4/80⁺Arg1⁺ cells, and IL-13 levels in culture supernatants, were measured by ELISA. (C) BCG-vaccinated nondiabetic and BCG-vaccinated T2DM mouse splenocytes were stimulated with γ-irradiated Mtb H37Rv (10 µg/mL) and treated with anti–IL-13R (10 µg/mL) for 72 hours. After 72 hours, flow cytometry was performed to determine the M2 macrophage phenotype (CD45⁺F4/80⁺Arg1⁺ cells). (D) MFI values and histogram plot showing IL-13R expression gated on CD45⁺F4/80⁺Arg1⁺ cells. (E) Macrophages isolated from BCG-vaccinated mice were cocultured with autologous Tregs or Tregs isolated from BCG-vaccinated T2DM mice and stimulated with γ-irradiated Mtb (10 µg/mL). After 72-hour incubation, the macrophages were analyzed for M1 and M2 phenotype by flow cytometry. MFI values showing the CD45⁺F4/80⁺iNos⁺ cells and CD45⁺F4/80⁺Arg1⁺ cells. (F) For neutralization studies, BCG-vaccinated T2DM mouse macrophages were stimulated with γ-irradiated Mtb (10 µg/mL) with or without autologous Tregs, and in some wells, IgG control or anti–IL-13R (10 µg/mL) antibodies were added. After 72 hours of incubation, the M1 and M2 phenotypes of cultured macrophages were determined by flow cytometry, and TNF-α and IL-6 levels were measured by ELISA in the culture supernatants. rIL-13 (10 ng/mL) was added to the macrophages from BCG-vaccinated T2DM mice and stimulated with γ-irradiated Mtb (10 µg/mL). (G) After 72 hours of incubation, the M1 and M2 phenotypes of cultured macrophages were determined by flow cytometry, and MFI values for CD45⁺F4/80⁺iNos⁺ cells and CD45⁺F4/80⁺Arg1⁺ cells are shown. Experiments were performed 2 times, and each time, 2–3 mice per group were used. The data are shown as mean ± SDs of n = 5 mice per group. The statistical analysis was performed by 1-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001.