NCan mice control Mtb replication with low Th1 cytokines and reduced CD4 T cell polyfunctionality. (A–C and E) (A) Lung and (B) spleen bacterial burden (log₁₀ transformed) at 2 and 5 wk after infection. LOD, limit of detection. Percent of lung CD4 T cells staining for IFNγ by flow cytometry after stimulation with (C) MTB300 peptide pool or (E) polyclonally stimulated with anti-CD3 and anti-CD28 antibodies. (D) Percent of total lung cells staining for IFNγ by flow cytometry after stimulation with MTB300 peptide pool. (A–E) Each point depicts an individual mouse (n = 2–4 mice per sex per strain per time point from one experiment). Canonical mice are represented by blue symbols with solid connecting line. NCan mice are indicated by purple symbols with dotted connecting line. Significance was determined by two-way ANOVA with Sidak multiple comparisons test. (F) Polyfunctionality score calculated by COMPASS for lung CD4 T cells after restimulation with MTB300 peptide pool. Box-and-whisker plots depict bounds from the 25th to 75th percentile, median line, and whiskers extending from min to max. Statistical significance assessed via Mann–Whitney U test. (G) Percentage of responding lung CD4 T cells after restimulation with MTB300 peptide pool. Data are split over two graphs to accommodate visualization of the lowly expressed populations. P values are derived from Mann–Whitney U test with Benjamini–Hochberg correction (ns = not significant [P > 0.05], *P < 0.05, **P < 0.01, ***P < 0.001). Mean ±1 SE are visualized as crossbars.