Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4(+) T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4(+) T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed approximately 1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of Mtb.

Figure 1.

Characterization of Foxp3-expressing cells in Mtb-infected mice. (A) PLNs from Foxp3-GFP mice 56 d after aerosol infection with Mtb were stained for TCRβ, CD4, and CD8. FACS plots are gated on live cells or live FoxP3-GFP⁺ cells. Similar data were obtained gating on cells in the spleen and lung and are representative of two mice in two different experiments. (B) The percentage of CD4⁺ cells that express Foxp3-GFP are shown for lung, pLN, spleen, and mLN 21, 64, and 127 d after infection, as indicated, for uninfected mice (open bars) and infected mice (shaded bars). Numbers represent the mean of three mice per group ± the SD for infected LNs and the mean of two mice for uninfected LNs. Lung numbers represent a pool of six lobes, two from each of three infected mice, and three each from each of two uninfected mice. Data is representative of four independent experiments. (C) Total numbers of CD4⁺ Foxp3-GFP⁺ cells are shown for lungs and pLNs at 21, 64, and 127 d after infection (black bars). Age-matched controls are shown in white bars. Numbers represent the mean of three mice per group ± the SD for infected LNs and the mean of two mice for uninfected LNs. Lung numbers represent a pool of six lobes, two from each of three infected mice, and three each from each of two uninfected mice. Data are representative of four independent experiments. (D) Mice infected with Mtb (or uninfected controls) were administered BrdU in drinking water beginning 21 d after infection. 14 d later, cells from pLN and mLN from Mtb-infected mice (black bars) or uninfected controls (white bars) were stained for CD4, and the percentage of CD4⁺, BrdU⁺ cells were compared between Foxp3-GFP⁺ and Foxp3-GFP–negative cells in pLNs and mLNs. Data represent the mean of three mice per group ± the SD.

Figure 2.

Localization of T reg cells in lungs of infected mice. (A) Representative sections showing perivascular areas from lungs of uninfected or Mtb-infected mice (115 d after infection) stained with anti-Foxp3 (green) and anti–E-cadherin (red) to stain respiratory epithelium. (B) Serial sections showing parenchymal granuloma of an Mtb-infected mouse (∼60 d after infection) stained with anti-Foxp3 (green) and CD4 (red; left) and hematoxylin/eosin (right). Section is representative of sections of eight mice examined, ranging from 40 to 120 d after infection. (C) Serial sections showing parenchymal granuloma of an Mtb-infected mouse stained with nonspecific rabbit IgG (green) and CD4 (red; left), and hematoxylin/eosin (right). Although the nonspecific green fluorescence of macrophages was similar to the section in B, the green pinpoint staining of nuclei within the CD4⁺ region of the granuloma (as seen in B using anti-Foxp3) was not seen using nonspecific polyclonal rabbit IgG as the primary antibodies. Bars, 50 μm.

Figure 3.

Foxp3⁺ cells do not make inflammatory cytokines after Mtb infection. 91 d after infection, cells from the lung and pLNs were stained for IFN-γ and TNF-α after stimulation for 4 h with anti-CD3 and -CD28 or media controls in the presence of monensin. Plots are gated on live CD4⁺ cells. The percentage of FoxP3-GFP–negative or Foxp3-GFP⁺ cells that stain positive for the cytokines are shown. Representative FACS plots are shown for five independent experiments with two to three mice per group.

Figure 4.

Foxp3⁺ cells up-regulate activation markers at the site of infection. (A) Cells from lung, pLNs, and mLNs of Mtb-infected mice (solid line) or uninfected controls (gray area) were stained for CD4 and ICOS (64 d after infection) or PD-1 (242 d after infection). Histogram plots are gated on live CD4⁺FoxP3-GFP⁺ cells. Representative FACS plots are shown for eight independent experiments with two to three mice per group. (B) FACS analysis of ICOS and PD-1 expression among CD4⁺FoxP3⁺ cells from indicated organs of Mtb-infected (shaded bars) or uninfected mice (open bars). The percentage of cells expressing ICOS was calculated from the gate indicated on graphs in A. PD-1 mean fluorescence intensity (MFI) was calculated from all CD4⁺ Foxp3⁺ cells. Increased ICOS expression at sites of infection was seen at all time points examined (between 21 and 242 d after infection) in all experiments (n = 8). In contrast, increased PD-1 expression was seen in 3 of 3 experiments performed 150 d after infection, but expression was not increased in 2 of 2 experiments at earlier time points.

Figure 5.

Depletion of T reg cells in bone marrow chimeric mice. (A) Schematic of mixed bone marrow chimeras and depletion of T reg cells. Mixed bone marrow chimeras were generated between Thy1.1⁺ B6 and Thy1.2⁺ Foxp3KO (designated [KO:WT]) or between Thy1.1⁺ B6 and Thy1.2⁺ B6 control (designated [WT:WT]), followed by a 7-d depletion of Thy1.1⁺ T cells, infection with Mtb on day 8, and sacrifice 23 d after infection (31 d after depletion). Mice were analyzed at time points indicated by encircled numbers. Chimeras were analyzed in the blood after reconstitution and before depletion with anti-Thy1.1 (time point 1) and 7 d after depletion of Thy1.1+ cells (preinfection; time point 2). They were subsequently killed 23 d after infection, and the lungs, pLNs, mLNs, and spleens were analyzed (time point 3). (B) FACS analysis of bone marrow chimeras at various time points, as designated in A. Plots are gated on live, TCRβ⁺ cells from the indicated tissue, and the percentage of total live cells is shown. Data shown are representative plots of three to five mice in each group from two independent experiments. (C) FACS analysis of CD44 and FoxP3 expression from CD4⁺ cells. CD4⁺ cells in lung or pLN from mock or Thy1.1-depleted, Mtb-infected, [KO:WT] chimeric mice were compared for CD44 and FoxP3 expression among Thy1.1⁺ and Thy1.2⁺ cells. FACS plots are gated on live CD4⁺ cells and separated into Thy1.1⁺ or Thy1.2⁺ events for analysis. (D) Comparison of frequencies of Foxp3⁺CD4⁺ T cells in different tissues from Mtb-infected mock or Thy1.1-depleted chimeric mice (KO:WT). Data shown are the percentages of Foxp3⁺ cells, as determined by intracellular staining for Foxp3, within the CD4⁺ populations from pooled samples from the lung, pLNs, or mLNs from each group (three to four mice per group), or the mean of three to four spleens per group ± the SD 23 d after infection. (E) Comparison of CD44^hi-expressing CD4⁺ cells in pLN and lung from uninfected mock or Thy1.1-depleted chimeric mice (KO:WT). Data shown represent the percentages of CD44^hi cells within the Thy1.2⁺CD4⁺ populations of Thy1.1-depleted (shaded bars) or mock-depleted (open bars) mice.

Figure 6.

Reduced Mtb CFU in lungs of T reg cell–depleted mice. (A and B) CFU analysis was performed 23 d after infection in Thy1.1-depleted (filled diamonds) or mock-depleted (open diamonds) mice in the spleen (A) or lung (B), as indicated. CFU counts from each organ were determined from 10-fold serial dilutions, and the numbers of viable bacteria are shown for each mouse from two independent experiments (**, P < 0.01; *, P < 0.05). The limit of detection for these experiments was 100 CFU per organ and is indicated on the graph by the dotted line. (C) Analysis of IFN-γ production by CD4⁺ cells from lungs or pLNs from mock or depleted [KO:WT] chimeras. Data represent analysis of pooled lung cells from all mice in each group, or, in the case of pLNs, from individual mice, representative of each group. Percentage of IFN-γ–producing cells (gated on CD4⁺ cells) is shown after in vitro restimulation with media alone, PMA/ionomycin, or ESAT6_1-20 peptide. (D) IFN-γ production by CD4⁺ cells in the pLN. The percentage of pLN CD4⁺ T cells producing IFN-γ after in vitro restimulation with media alone, ESAT6_1-20 peptide, or PMA/ionomycin. Using the same gates shown in C, data from each individual mouse 23 d after infection from Thy1.1-depleted (filled diamonds) or mock-depleted (open diamonds) mice are represented.