Article Figures & Data
Figures
- FIGURE 1.
M. tuberculosis–specific CD4 T cells are highly enriched in both the lung parenchyma and lung vasculature. (A) iv staining of M. tuberculosis–infected mice. (B) Representative FACS plots of CD4 T cells on day 30 p.i. Data are representative of at least three independent experiments. (C) Immunofluorescence microscopy of day-30 lung sections from anti-CD45 (red)-injected mice. CD4 is green, and CD31 is blue. Scale bar, 100 μm. Green arrows indicate the parenchymal and orange arrows indicate iv CD4 T cells. (D) Frequency and total number of iv− CD4 T cells after M. tuberculosis infection. Data are pooled from at least two independent experiments (n ≥ 3/experiment). (E) Quantification of I-AbESAT-64–17– and I-AbEsxG46–61–specific cells by MHC class II tetramer staining on day 30 p.i. (left panels). Each connecting line represents an individual animal (right panels). Data are pooled from two independent experiments (n = 5/experiment). BAL, bronchoalveolar lavage fluid; Mtb, M. tuberculosis.
- FIGURE 2.
Lung parenchymal and iv M. tuberculosis–specific CD4 T cells are functionally and phenotypically distinct subsets. (A) IFN-γ staining after in vitro stimulation with ESAT-61–20 peptide on day 30 p.i. Plots are gated on lung CD4 T cells, and data are pooled from three independent experiments (n = 4/experiment). (B) Direct ex vivo ICS for IFN-γ–producing I-AbESAT-64–17–specific CD4 T cells in the lung at 30 d p.i. (left panels). Each connecting line represents an individual animal (n = 5). Data are representative of three independent experiments. T-bet (C) and PD-1, CD69, and KLRG1 (D) expression in total naive (CD44loFoxp3−) and iv+ or iv− I-AbESAT-64–17 tetramer+ lung CD4 T cells on day 30 p.i. Numbers represent the MFI or percentage positive for the iv− (top line) and iv+ (bottom line) cells. (E) CXCR3 or CX3CR1 staining gated on total lung CD4 T cells on day 30 p.i. (left panels) and expression of the chemokine receptors and KLRG1 gated on iv− and iv+ I-AbESAT-64–17 tetramer+ cells (middle and right panels). Cells in (C), (D), and (E) were pooled from n ≥ 4 mice/experiment for FACS analysis. Data are representative of three independent experiments.
- FIGURE 3.
KLRG1− parenchymal effector CD4 T cells migrate most efficiently into the lung, whereas iv KLRG1+ cells are the least efficient. FACS-purified iv− and iv+ CD4 T cells from lungs of day-30 infected mice (CD45.1) were transferred into infection-matched congenic recipient mice (CD45.2). After 1.5 d, migration of the donor cells into the lung tissue was monitored with a second iv stain in the recipient mice. (A) Migration of I-AbESAT-64–17 tetramer+ donor CD4 T cells into the parenchyma. Plots are electronically concatenated from each group of mice (n = 5). Data are representative of two independent experiments. (B) Gating strategy to identify naive, KLRG1hi, and KLRG1lo effector donor CD4 T cells. Plots are concatenated from each group of mice (n = 5). (C) Summary of donor cells migrating into the lung parenchyma of recipients. Data are pooled from two independent experiments (n = 5/experiment). ns, not significant.
- FIGURE 4.
Parenchyma-homing CD4 T cells display greater control of M. tuberculosis infection compared with iv cells. The iv− and iv+ CD4 T cells were FACS purified from day-30 infected mice and adoptively transferred into TCRα−/− mice that had been infected with M. tuberculosis 7 d earlier; lungs were harvested on day 28 p.i. (A) Absolute number of CD44hiFoxp3− effector and I-AbESAT-64–17 tetramer+ donor CD4 T cells in the recipient lungs. (B) Direct ex vivo IFN-γ staining of donor CD4 T cells. (C) Bacterial loads in the lungs of recipient mice. All data are representative of two independent experiments (n = 5/experiment). ns, not significant.
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