Neutrophil depletion increases macrophage activation and CD4 ⁺ T cell numbers in lung lesions in TB-susceptible C3HeB/FeJ mice. (a) C3HeB/FeJ mice were aerosol infected with M. tuberculosis HN878 and received intraperitoneal injection of either anti-Ly6G (αLy6G) or isotype control three times per week between days 12 and 25. Tissues were analyzed at 20 and 26 days after infection. (b) Representative images of S100A9 immunohistochemistry in lung sections at 20 days after infection, confirming neutrophil depletion in αLy6G-treated mice. Scale bars = 100 μm. (c) Lung M. tuberculosis CFU counts. (d) Numbers of total, Ly6C⁺MHC-II⁻ and MHC-II⁺ MDMs (Siglec F⁻ Ly6G⁻ CD11b⁺CD64⁺MerTK⁺CD45⁺) in lung tissue as determined by flow cytometry. (e) Numbers of CD44⁺ CD62⁻ CD4⁺ T cells (CD3ε⁺CD45⁺) in lung tissue as determined by flow cytometry. (f) Representative images of lung lesions showing macrophage (CD68, magenta), CD4⁺ T cells (CD4, green), and neutrophil (Ly6G, white) staining at 20 and 26 days after infection. Scale bars = 100 μm. (g) Number of CD4⁺ T cells within lung lesions, normalized for the total area of all lesions across whole left lungs. (h) Numbers of CD4⁺ T cells in contact with a macrophage annotation (≤0 μm distance), normalized for the total area of all lesions across whole left lungs. Points show individual replicate mice with lines at the mean. Data shown are from a single experiment with N = 5 mice per group, representative of two independent experiments. Statistical analysis: (c–e) two-way ANOVA with Holm–Sidak post hoc test; (g and h) unpaired t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. See also: Fig. S3 d.