Analysis of lung and lymph node early during M. tuberculosis infection. C57BL/6 and C3HeB/FeJ mice were aerosol infected with M. tuberculosis HN878, 6C4, or 4I2, and infection and immune parameters were assessed. (a) Lung CFU counts taken immediately after infection of C57BL/6 and C3HeB/FeJ mice with HN878 in five independent experiments. (b) Lung CFU counts at different time points after HN878 infection, represented as fold increases from the mean CFU in the respective age-, sex-, and strain-matched mice analyzed immediately after infection. Panels a and b show data from three to five independent experiments with N = 5 mice per group are overlaid per time point with lines at the grand mean. Statistical analysis in a and b shows mouse strain effects across pooled experiments as determined by two-way ANOVA. (c–e) Representative flow cytometry dot plots showing the gating strategies used to identify: (c) myeloid cell populations; (d) MDMs specifically in 6C4 and 4I2 experiments; (e) T cell populations. (f) Numbers of total CD4+ T cells (CD3ε+CD45+) in lung tissue as determined by flow cytometry. Points show individual mice with lines at the mean. Data are from single experiments with three to five mice per group that are representative of two independent experiments. (g) CFU counts in lung-draining lymph node (LN) tissue. Data are from a single experiment with five mice per group, showing individual points with lines at the mean. Data are representative of two independent experiments. Statistical analysis in f and g: two-way ANOVA with Holm–Sidak post hoc test. (h) Total live cells in lung-draining lymph nodes. (i) Numbers of CD44+CD62L− CD4+ T cells (CD3ε+CD45+) in lung-draining lymph nodes as determined by flow cytometry. Panels h and i show points from N = 5 mice per group from a single experiment with lines at the median. Data are representative of three independent experiments. Statistical analysis in h and i: aligned ranks transformation two-way ANOVA analysis, with post hoc comparisons between groups shown following Holm’s correction. (j) UMAP (Uniform Manifold Approximation and Projection) of integrated and clustered lung leukocyte scRNA-seq data as shown in Fig. 3 a, broken down into individual experimental groups (N = 3 per group); cDC, conventional dendritic cell; pDC, plasmacytoid dendritic cell. (k) Stacked bar plots showing the relative abundance of each scRNA-seq cluster in each mouse, grouped by broad cell types, with major groups of clusters highlighted. (l) Differences in relative abundance of key scRNA-seq clusters between C57BL/6 and C3HeB/FeJ mice at the two infection time points. Differential abundance with false discovery rate <0.1 was taken as statistically significant. (m) Reference-based analysis of macrophage and monocyte scRNA-seq clusters against references from the ImmGen database for lung macrophages and blood monocytes. ImmGen contributing investigators for each reference are listed by surname. (n) Dot plots showing relative expression of selected marker genes in T and NK cell scRNA-seq clusters. Circle sizes represent the abundance of cells expressing the marker gene, as a percentage of all cells in the cluster within all samples in the analysis. Circle color is proportional to the mean expression of the gene within all cells in the cluster. Panels j–n show data from a single scRNA-seq experiment, and plots show combined data from cells from N = 3 mice per group, except for panel k, which shows data from individual mice. Actual or adjusted P values are shown or: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.
Figure S1.

Analysis of lung and lymph node early during M. tuberculosis infection. C57BL/6 and C3HeB/FeJ mice were aerosol infected with M. tuberculosis HN878, 6C4, or 4I2, and infection and immune parameters were assessed. (a) Lung CFU counts taken immediately after infection of C57BL/6 and C3HeB/FeJ mice with HN878 in five independent experiments. (b) Lung CFU counts at different time points after HN878 infection, represented as fold increases from the mean CFU in the respective age-, sex-, and strain-matched mice analyzed immediately after infection. Panels a and b show data from three to five independent experiments with N = 5 mice per group are overlaid per time point with lines at the grand mean. Statistical analysis in a and b shows mouse strain effects across pooled experiments as determined by two-way ANOVA. (c–e) Representative flow cytometry dot plots showing the gating strategies used to identify: (c) myeloid cell populations; (d) MDMs specifically in 6C4 and 4I2 experiments; (e) T cell populations. (f) Numbers of total CD4+ T cells (CD3ε+CD45+) in lung tissue as determined by flow cytometry. Points show individual mice with lines at the mean. Data are from single experiments with three to five mice per group that are representative of two independent experiments. (g) CFU counts in lung-draining lymph node (LN) tissue. Data are from a single experiment with five mice per group, showing individual points with lines at the mean. Data are representative of two independent experiments. Statistical analysis in f and g: two-way ANOVA with Holm–Sidak post hoc test. (h) Total live cells in lung-draining lymph nodes. (i) Numbers of CD44+CD62L− CD4+ T cells (CD3ε+CD45+) in lung-draining lymph nodes as determined by flow cytometry. Panels h and i show points from N = 5 mice per group from a single experiment with lines at the median. Data are representative of three independent experiments. Statistical analysis in h and i: aligned ranks transformation two-way ANOVA analysis, with post hoc comparisons between groups shown following Holm’s correction. (j) UMAP (Uniform Manifold Approximation and Projection) of integrated and clustered lung leukocyte scRNA-seq data as shown in Fig. 3 a, broken down into individual experimental groups (N = 3 per group); cDC, conventional dendritic cell; pDC, plasmacytoid dendritic cell. (k) Stacked bar plots showing the relative abundance of each scRNA-seq cluster in each mouse, grouped by broad cell types, with major groups of clusters highlighted. (l) Differences in relative abundance of key scRNA-seq clusters between C57BL/6 and C3HeB/FeJ mice at the two infection time points. Differential abundance with false discovery rate <0.1 was taken as statistically significant. (m) Reference-based analysis of macrophage and monocyte scRNA-seq clusters against references from the ImmGen database for lung macrophages and blood monocytes. ImmGen contributing investigators for each reference are listed by surname. (n) Dot plots showing relative expression of selected marker genes in T and NK cell scRNA-seq clusters. Circle sizes represent the abundance of cells expressing the marker gene, as a percentage of all cells in the cluster within all samples in the analysis. Circle color is proportional to the mean expression of the gene within all cells in the cluster. Panels j–n show data from a single scRNA-seq experiment, and plots show combined data from cells from N = 3 mice per group, except for panel k, which shows data from individual mice. Actual or adjusted P values are shown or: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

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