(A) The number of IFN-γ spot-forming units (SFU) per million popliteal lymph node (pLN) cells of BCG-vaccinated B6 and CC042 mice after incubation with the P300 peptide pool (left) or peptide Ag85B (right) was determined following 44-hour coculture via ELISPOT. Each dot represents 1 subject; n = 4–5 mice per strain. Data are from 1 of 2 independent experiments with similar results. (B) Experimental scheme. CC042 mice were vaccinated or not vaccinated in the flank with BCG and rested for 12 weeks. FTY720 (4 mg/kg) was administered to half the mice in each group, starting 1 day before infection and continuing for 4 weeks. All mice were infected with Rv.YFP and analyzed at 4 wpi. (C) Analysis of FTY720-treated CC042 mice. Frequency of T and B cells, as a percentage of total live cells, in blood of different groups of control and vaccinated mice, untreated or treated with FTY720. (D) CFU in lung (left) and spleen (right) of CC042 mice at 4 wpi. (E) Naive (left, CD44^–CD62L⁺) and antigen-experienced (right, CD44⁺CD62L^–) T cell counts in lungs of CC042 mice at 4 wpi. (F) Analysis of T cells from lungs of CC042 mice, 12 weeks after BCG vaccination. Control (top row) or BCG-vaccinated (bottom row) mice were injected i.v. with anti-CD90–AF647, euthanized, and analyzed by flow cytometry. First plot is gated by scatter, live cells, and CD90, and shows CD4 expression of cells in the vasculature (IV⁺) versus in the parenchyma (IV^–). The second plot shows CD44 and CD69 expression of cells that are CD4⁺IV⁺. (G) Lung parenchymal CD4⁺CD44^hi T cells with a Trm phenotype (CD69⁺) were enumerated 12 weeks after vaccination with or without BCG. (A, C–E, and G) Two-way ANOVA with Šidák’s multiple-comparison test (A and C–E) or t test (G). Box-and-whisker plots indicate median (middle line), 25th and 75th percentiles (box), and minimum and maximum (whiskers). Each point represents an individual subject; n = 3–5 mice per group. Data are from 1 of 2 independent experiments with similar results.