Early type I IFN signaling promotes neutrophil swarming and limits CD4 ⁺ T cell numbers in TB lesions of both relatively resistant and highly TB-susceptible mice. Lung sections from experiments described in Fig. 8 were analyzed by multiparameter immunofluorescence. (a) Images of representative lesions from C57BL/6 mice treated with either early anti-IFNAR (αIFNAR) or isotype control, at 20 days after infection. Individual fluorescent channels and merged images are shown. (b) Stacked bar plots showing percentages of lesions across whole left lungs falling into low (≤20%), intermediate (Int, >20% ≤40%), or high (Hi, >40) bins for coverage with Ly6G staining. (c and d) Numbers of total CD4⁺ T cells (c) and CD4⁺ T cells in contact with a macrophage annotation (≤0 μm distance, d) within lung lesions at 20 days after infection, normalized for the total area of all lesions across whole left lungs. (e) Images of representative lesions from C3HeB/FeJ mice treated with either early anti-IFNAR (αIFNAR) or isotype control at 26 days after infection. Individual fluorescent channels and merged images are shown. (f) Stacked bar plots showing percentages of lesions across whole left lungs falling into low (≤20%), intermediate (Int, >20% ≤40%), or high (Hi, >40) bins for coverage with Ly6G staining. (g and h) Number of total CD4⁺ T cells (g) and CD4⁺ T cells in contact with a macrophage annotation (≤0 μm distance, h) within lung lesions at the indicated time points, normalized for the total area of all lesions across whole left lungs. (i–l) Immunofluorescence staining for CitH3 and Ly6G to detect NETs in lung lesions at the indicated time points. (i and k) Representative images showing all merged channels (top) or CitH3 and DAPI alone (bottom) in C3HeB/FeJ (i) and C57BL/6 (k) mice. (j and l) Quantification of CitH3 NET staining relative to Ly6G staining in lung lesions. Data shown in b and f are means ± standard error of N = 5 per group. Statistical analysis shown in b and f is Dirichlet-multinomial regression analysis of the effect of αIFNAR treatment on lesion composition. Symbols indicate significant differences in the proportion of Ly6G-low lesions. Plots in c, d, g, h, j, and l show individual replicate mice as points with lines at the mean or median (h). Statistical analysis in b and g: unpaired t test. Statistical analysis in h: Mann–Whitney test. Statistical analysis in j and l: unpaired t test with Welch’s correction. Actual P values are shown or: *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. Data shown are from single experiments with N = 5 mice per group that are representative of two independent experiments. Scale bars in a and e = 100 μm; scale bars in i and k = 50 μm. See also: Fig. S5, b–g.