Fig 1.
B cell-deficient μMT mice exhibit diminished lung infiltrates relative to WT C56BL/6’s in chronic TB.
WT and B cell-deficient μMT mice were aerogenically infected with 100 CFU of M. tuberculosis Erdman and tissues analyzed by examination of H&E-stained lung sections at 12 weeks (A) and 24 weeks (C) post-infection. Quantification of the number of total lung cells was also carried out at the same time intervals post-inoculation (12 weeks, (B); 24 weeks (D)). These histological studies and enumeration of total lung cell numbers showed that in chronic TB, B cell-deficiency is associated with diminished granulomatous inflammatory response; n = 3–4 mice per group per time interval. The results depicted in (A) and (C) are representative of lung sections from 3 mice examined. The data shown in (B) and (D) denote the mean ± SEM (standard error mean). The results shown in (A, B, C, and D) are representative of 2 experiments. (E) Lung mycobacterial burden as measured by culturable bacilli 4, 12, and 24 weeks after a 100 CFU Erdman aerogenic challenge, demonstrating that bacillary loads in μMT and WT mice are comparable throughout the course of infection; n = 4 to 5 mice per group. The data depicted denote the mean ± SEM. The results shown are representative of two experiments. (F) B cell-deficient μMT mice exhibited increased median survival relative to WT controls after a 100 CFU aerosol Erdman infection (median survival time: 324 days versus 272 days); n = 10 mice per group. The result is demonstrable in three independent experiments.
Fig 2.
The presence of B cells promotes cellular expansion and CD4+ T cell proliferation in the lung of mice with chronic TB.
WT and μMT mice were infected with 100 CFU M. tuberculosis Erdman delivered via aerosolization. Dilution of CFSE by ex vivo lung cultures taken 5 months after infection. Cells from 3 mice per group were pooled for the analysis. Data in (B and C) show the frequencies of BrdU+ CD4+ T cells in the lungs of WT and μMT mice derived from in vivo BrdU labeling studies conducted at 5 months post-infection. The data shown in (B) depicts a representative dot plot targeting BrdU+ CD4+ T cells. Results shown in (C) are mean ± SEM of the frequencies of BrdU+ CD4+ T cells. Three to 4 mice per group were evaluated. (D) The absolute number of lung BrdU+ CD4+ T cells at 5 months after infection upon in vivo BrdU labeling. The data shown depict mean ± SEM of 3 to 4 mice per group. Data shown are representative of 2 to 3 experiments, except for that in (A), which are derived from 1 study.
Fig 3.
B cell-deficient μMT mice exhibit diminished levels of IFN-γ-producing CD4+ T cells during chronic TB.
Mice were infected with 100 CFU of M. tuberculosis Erdman by aerosolization. (A) Representative frequencies of IFN-γ+ CD4+ T cells detected by flow cytometry at 5 months after infection; n = 4 mice per group. (B) Absolute number of lung IFN-γ+ CD4+ T cells detected 5 months after infection; n = 4 mice per group. The results are expressed as mean ± SEM. Data in this figure is representative of 3 similar experiments. The data show revealed the B cell-deficient μMT mice, relative to WT, display an attenuated Th1 response during the chronic phase of tuberculous infection.
Fig 4.
Increased IL-10 production by the lung cells of B cell-deficient μMT mice during chronic infection with M. tuberculosis.
Mice were aerogenically infected with a low dose (100 CFU) of M. tuberculosis Erdman. Ex vivo IL-10 production by lung cells isolated from B cell-deficient μMT and WT control mice was evaluated at 12 weeks (A) and 24 weeks (B) after infection. Single-cell suspension of lung cells were cultured in complete RPMI with or without 10 μg/ml of PPD (Statens Serum Institute, Copenhagen) at 1.0 x 107 cells per ml. After 48 hours of culture, supernatants were collected and subjected to detection of IL-10 as described in “Materials and Methods” section. The results revealed that the levels of expression of lung IL-10 in tuberculous μMT mice are enhanced compared to that observed in WT animals. The data are depicted as mean ± SEM. The results shown in this figure are representative of two independent experiments; n = 4 to 5 mice per group per time interval.
Fig 5.
B cell-depleted WT C57BL/6 mice also exhibit diminished granulomatous inflammation and Th1 response and enhanced IL-10 levels in the lungs during chronic M. tuberculosis infection.
Mice were treated with the anti-CD20 mAb 5D2 beginning 2 days prior to a low-dose (100 CFU) aerogenic challenge with M. tuberculosis Erdman, as described in "Materials and Methods". B cell depletion was maintained throughout the duration of the experiment. The control mouse group (WT) received non-specific rat IgG. The lung tissues were examined at 5 months post-infection. The levels of granulomatous inflammation response were analyzed histologically by light microscopy on H&E-stained lung sections (A) and enumeration of total number of lung cells (B). The level of lung CD4+ T cell response was assessed by in vivo BrdU labeling to examine the proliferation capacity of this T cell subset (C), as well as by enumeration of IFN-γ-producing CD4+ T cells (D). Ex vivo evaluation of lung cells for the level of IL-10 production (E) was conducted as described in Fig 4. Four to 5 mice per group were evaluated per group. Data depicted in B, C, D, and E are presented as means ± SEM. The data shown are representative of two experiments. The results demonstrated that the inflammation, Th1 response, and IL-10 phenotypes observed in the μMT mice are recapitulated in mice depleted for B cells.
Fig 6.
IL-10R blockade reverses the inflammation and diminished CD4+ T cell response phenotypes observed in the lungs of B cell-depleted C57BL/6 mice during chronic M. tuberculosis infection.
C57BL/6 mice were depleted for B cells via administration of 5D2 beginning 2 days prior to infection with a low dose (100 CFU) of M. tuberculosis Erdman delivered by aerosol. B cell depletion was maintained for the duration of the experiment. At 3 months after the infection, IL-10R blockade was initiated using the anti-mouse IL-10R antibody clone 1B1.3A. The control group received non-specific rat IgG. The IL-10R blockade was continued for two months. At five months post-infection (2 months after initiation of IL-10R blockade), mice were sacrificed and analyzed for the levels of inflammation in the lungs, as assessed by histological examination (A) and enumeration of total lung cells (B), CD4+ T cells proliferation via BrdU labeling (C), and Th1 response (D). Data shown are representation of two experiments. Three to four mice were analyzed per group. Data depicted in (B), (C), and (D) denote mean ± SEM.
Fig 7.
Conspicuous B cell aggregates are present at the periphery of cavitary and necrotic lesions in human tuberculous lungs.
(A), (C), and (E) depict low power images demonstrating the histopathology of lung tissue from three patients infected with M. tuberculosis (see Table 1 for patient information). The medium and high-power images to the right of (A), (C), and (E) illustrate the corresponding B cell aggregates, as detected by anti-CD20 staining. The medium- and high-power images (B), (D), and (F) illustrate regions of tuberculous lung tissue from three patients with varied degree of consolidation (from non-consolidated to markedly consolidated areas) distal to cavitary or necrotic lesions, in which B cells aggregates are conspicuously absent. Photomicrographs of (G-J) show non-necrotic granulomas surrounded by CD20+ B cell aggregates, which may represent evolving lesions in transition to a necrotic and/or cavitary state as tissue-damage progresses. Tissue sections were prepared from formalin-fixed, paraffin-embedded lung specimens surgically removed from tuberculous patients. Ng: necrotic granuloma; Nng: non-necrotic granuloma; L: lumen of cavity. Arrows–Langhans giant cells.
Table 1.
Clinical characteristics of human subjects.