Multifunctional profile of CD4⁺ T cells in the lung of infant and adult mice following BCG immunization.

<p>Infant and adult mice were immunized s.c. with BCG and sacrificed at 4 (A), 8 (B), or 16 (C) weeks following immunization. Cells from the lung were stimulated with <i>M</i>.<i>tb</i> CF + crude BCG for 24h or left unstimulated as a control. Cells were stained and analyzed by flow cytometry. Average proportions displayed in pie chart are of the CD4 T cells expressing specific cytokine combinations. Absolute numbers of CD4⁺ T cells in the tissues were calculated and displayed in bar graphs. Results are from one independent experiment per timepoint, n = 4-5/group/timepoint. Data are expressed as Mean ± SEM. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.</p

Total # of IFN-γ⁺ T cells in BAL in mice boosted at 16 weeks post-BCG (x 10³).

<p>Infant and adult mice were immunized s.c. with BCG, and the AdHu5Ag85A booster vaccine was administered intranasally (i.n.) at 16 weeks post-BCG. The mice were sacrificed 4 or 8 weeks after boosting. Cells isolated from the BAL were stimulated with <i>M</i>.<i>tb</i> CF + crude BCG, or unstimulated as a control. Cells were stained with extracellular antibodies for T cell markers, followed by intracellular staining for IFN-γ, and analyzed by flow cytometry. Absolute numbers of IFN-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ T cells were calculated (unstimulated subtracted from stimulated). Results are from one experiment per timepoint, n = 4-5/group/timepoint. Data are expressed as Mean ± SEM.</p><p>*, p < 0.05.</p><p>Total # of IFN-γ⁺ T cells in BAL in mice boosted at 16 weeks post-BCG (x 10³).</p

Effect of elapsed time between BCG priming and AdHu5Ag85A boost immunization on Ag-specific responses boosted by AdHu5Ag85A boost immunization.

<p>Infant and adult mice were BCG immunized, and boosted with AdHu5Ag85A at 8 or 16 weeks post-BCG (A). The mice were sacrificed 4 weeks after boosting. Cells isolated from the lung were stimulated either with <i>M</i>.<i>tb</i> CF + crude BCG (B), Ag85A-specific CD4 T cell peptide (C), or CD8 T cell peptide (D), or left unstimulated as a control. Cells were stained and analyzed by flow cytometry. Absolute numbers of IFN-γ⁺CD4⁺ (B, C) and IFN-γ⁺CD8⁺ (D) T cells were calculated (unstimulated subtracted from stimulated). Ag85A CD8 peptide tetramer staining was performed on lung cells, and analyzed by flow cytometry (E). Absolute numbers of tet⁺CD8⁺ T cells were calculated. Results are from one experiment per timepoint, n = 4-5/group/timepoint. Data are expressed as Mean ± SEM. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. All other comparisons (not indicated) were not significant.</p

Protection against <i>M</i>.<i>tb</i> challenge in mice immunized with BCG alone or primed with BCG and boosted with AdHu5Ag85A.

<p>Infant and adult mice were BCG immunized, and boosted with AdHu5Ag85A i.n. at 16 weeks post-BCG (BCG/AdHu5Ag85A) (A). The control group was not boosted (BCG). The mice were then challenged i.n. 4 weeks after boosting with <i>M</i>.<i>tb</i> H37Rv. Bacterial burden in the lungs was assessed at 4 weeks post-challenge by a cfu assay with lung homogenates, expressed as log₁₀ cfu per lung (B). Histopathological changes were examined by H&E staining of the lungs (C). Magnification: 5x. Results are from one experiment, n = 5/group. Data are expressed as Mean ± SEM. *, p < 0.05; **, p < 0.005.</p

Ag-specific T cell responses in the lung after boost AdHu5Ag85A immunization in BCG primed mice.

<p>Infant and adult mice were BCG immunized, and the AdHu5Ag85A booster vaccine was administered i.n. at 16 weeks post-BCG (A). The mice were sacrificed 4 weeks (B, C, D) or 8 weeks (E, F, G) after boosting. Lung cells were stimulated either with <i>M</i>.<i>tb</i> CF + crude BCG for 24h (open bar), or Ag85A-specific CD4 or CD8 T cell peptide for 6h (black bar), or left unstimulated (B, C, E, F). Cells were stained and analyzed by flow cytometry. Absolute numbers of IFN-γ⁺CD4⁺ (B, E) and IFN-γ⁺CD8⁺ (C, F) T cells were calculated (unstimulated subtracted from stimulated). Ag85A CD8 peptide tetramer staining was performed on lung cells, and analyzed by flow cytometry (D, G). Absolute numbers of tet⁺CD8⁺ T cells were calculated. Results are from one experiment per timepoint, n = 4-5/group/timepoint. Data are expressed as Mean ± SEM. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. All other comparisons (not indicated) were not significant.</p

Memory T cells in the lung and spleen of infant and adult mice following BCG immunization.

<p>Infant and adult mice were BCG immunized and sacrificed at 8 or 16 weeks. Cells from the lung (A) and spleen (B) were stained with extracellular antibodies for memory CD4 T cell markers and analyzed by flow cytometry. Absolute numbers of CD4⁺CD44⁺ cells that are T_eff/T_EM (CD127^-/+CD62L^-) or T_CM (CD127⁺CD62L⁺) in the tissues were calculated. Results are from one experiment per timepoint, n = 4/group/timepoint. Data are expressed as Mean ± SEM. *, p < 0.05.</p

IFN-γ⁺CD4⁺ T cell kinetics in the lung and spleen of infant and adult mice following BCG immunization.

<p>Infant and adult mice were immunized s.c. with BCG and sacrificed at 4, 8, or 16 weeks following immunization. Cells from the lung (A) and spleen (B) were isolated and stimulated with mixed <i>M</i>.<i>tb</i> culture filtrate (CF) and crude BCG (cBCG) for 24h or with media only as a control (unstimulated). Cells were stained with extracellular cell markers, followed by intracellular staining for IFN-γ, and analyzed by flow cytometry. Absolute numbers of IFN-γ⁺CD4⁺ T cells in the tissues were calculated (unstimulated numbers were subtracted from stimulated), and representative dot plots are shown. Results are from one to two independent experiments per timepoint, n = 4-8/group/timepoint. Data are expressed as Mean ± SEM. *, p < 0.05; **, p < 0.005; ****, p < 0.0001.</p