Fig 2: Pulmonary mucosal vaccination with MTBVAC induces immune signatures associated with protectionShown is an overview of BAL cell immune responses after mucosal or intradermal vaccination with BCG or MTBVAC.(A–D) Flow cytometry analysis over time of (A) IFN?, (B) TNF-a, (C) IL-2, and (D) IL-17A CD4+ T cell responses after vaccination.(E) Stacked bar graphs depicting CD4+ T cell cytokine polyfunctionality over time after PPD recall stimulation (by group median values).(F) PPD-specific cytokine production of CD4+ T cells in the lower right and lower left lung lobes at week 3 and week 8, indicating primary and disseminated vaccine responses.(G) Secretion of IL-10 by unfractionated BAL cells stimulated with PPD at week 8, plotted as culture medium control-corrected values.(H) Flow cytometry analysis of IL-10 production by CD4+ T cells over time after vaccination.All graphs show 6 animals per group. In (A)–(D) and (H), + indicates PPD-stimulated samples, and - indicates unstimulated, culture medium-incubated samples as controls. Horizontal lines within bars indicate group medians. Significance of group differences was determined by two-sided Mann-Whitney test adjusted for multiple comparisons. Holms-adjusted p = 0.05 is depicted. Color coding per individual is consistent throughout the paper.

Fig 3: Expression of mucosal homing markers after intradermal and pulmonary vaccination(A) Expression of CCR5, CD103, and CXCR3 on ex vivo CD4+ T cells from peripheral blood over time, depicted as group median values.(B) CCR5, CD103, and CXCR3 expression by PPD-specific (IFN?+ and/or IL-17A+) CD4+ T cells from PBMCs at week 8 after vaccination.(C–E) Comparison of PPD-specific T cells from BALs from pulmonary BCG-vaccinated, MTBVAC-vaccinated, and Mtb-infected animals.(C) Frequencies of IFN?+ and/or IL-17A+ CD4+ T cells after stimulation with PPD.(D and E) Expression of CCR5, CD103, and CXCR3 by cytokine+ CD4+ T cells, (D) depicted as group median values (stacked bar graph) or (E) as individual frequencies for separate markers.(F) Percentage of CD69 and PD1 double-positive cells of cytokine+ CD4+ T cells.For all graphs, n = 6 animals per group, with the exception of (B) where n = 5 for the BCG.muc group. Horizontal lines indicate group medians. Significance of group differences was determined by two-sided Mann-Whitney test adjusted for multiple comparisons. Holms-adjusted p = 0.05 is depicted. Color coding per individual is consistent throughout.