Fig 2: Pulmonary Th cell immune profile discriminates between active and relapse TB.a Schematic figure for the different markers used in flow cytometry for these experiments, created with BioRender.com. b Gating strategy utilized for Th cell analysis. c–h Percentage of specific-cytokine producers among Th cell subsets (CD3 + CD4+ lymphocytes) in lung after active TB (red, n = 10), post-drug treatment (blue, n = 10), and 4 and 8 weeks relapse (green, n = 5/group), compared to the uninfected controls (white, n = 10/group). c Th1 (IFN-γ + IL-4-) cells. d Th1Th17 cells that are double positive for IFN-γ and IL-17. e Th17 (IL-17 + ) cells. f Th2 (IL-4 + IFN-γ-) cells. g Th22 (IL-22 + IL-17-) cells. h Th cell percentages producing IL-10 (IL-10 + IFN-γ-). Open green symbols indicate animals that did not relapse. A Student’s t-test was used to determine differences between two groups within treatment phase. Comparisons in relapse, or across treatment stages were analyzed using one-way ANOVA followed by Tukey test for multiple comparisons. Data shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Polyfunctionality of lung Th cells changes with disease progression.Polyfunctional analysis of cytokine producing CD3 + CD4+ cells, determined by flow cytometry and post-acquisition Boolean gating, across active (n = 10), post-drug (n = 10), and relapse TB phases (4 and 8 weeks, n = 5/ group), by Pestle and SPICE. a Top panels represent the uninfected groups at ATB, PDTB, and relapse 8 weeks (n = 10/ group). Bottom panels represent Mtb-infected groups of each of the phases. Outside of the pie charts, the five arc colors are depicted in the arc legends showing overlap for one or more cytokines. The inside pie color legends (31 colors) are depicted at the right for each subgroup, showing positive or negative production of each of the combination for the five cytokines. b The fifteen populations demonstrating the most significant differences in Mtb-infected groups across disease stages are shown. Monofunctional populations are shown in the top panels while polyfunctional populations are shown in the middle and bottom panels. Open green symbols indicate animals that did not relapse. Comparisons across treatment stages were analyzed using one-way ANOVA followed by Tukey test for multiple comparisons. Data shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: Th cell dynamics of lung, spleen, and blood corresponding with disease presentation.a Summary bubble plot comparing the percentage change of each T cell subset in different organs versus its uninfected control. Changes were assessed subtracting the uninfected from the corresponding mean percentage of each phase, to visually assess increasing or decreasing populations due to infection. b Heat map comparing the observed cell percentages for each stage and organ. Variation is depicted across percentages of Th17, Th22, Th1, Th2, and Th IL-10+ cells among CD3 + CD4+ cells, in lung (Lg), spleen (Sp), or blood (Bl). Changes were assessed after 4 weeks of infection, post-drug administration, and at 4 and 8 weeks of TB relapse (Rel), for both uninfected (left) and Mtb-infected groups (right).