H56/CAF01 sustains population of IL-2 and IL-17-producing vaccine-specific CD4 T cells in lung parenchyma during Mtb infection.\r\nCB6F1 mice were assayed before and after aerosol Mtb infection using i.v. staining to identify parenchymal (IV−) T cells. Lung cells were stimulated ex vivo with ESAT-61-15 (A) or TB10.471-88 (B) and stained by ICS to determine the total number of antigen-specific CD4 cells (IFNγ, TNFα, IL-2, or IL-17-producing). (C) The total number of lung parenchyma (IV−) ESAT-6-specific CD4 T cells expressing the indicated cytokine from adjuvant control (Ctrl, ○) and H56/CAF01-vaccinated (●) mice is shown. Symbol, mean±SEM of 3–6 mice. * p< 0.05, **p<0.01 ***p<0.001 vs Ctrl. (D) Frequency of IV− ESAT-6-specific Lung CD4 T cells expressing each cytokine combination, based on combinatorial Boolean gating analysis, in adjuvant control (Ctrl, □) and H56/CAF01 -vaccinated (■) mice 42 days post Mtb infection. Bar, mean±SD of 5(□) or 6(■) mice\r\n