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Keywords:

  • B cells;
  • Chemokine receptors;
  • Immune responses;
  • Tuberculous pleuritis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

B-cell biology has been largely uncharacterized in the field of tuberculosis (TB). In this study, we investigated the immunophenotypical and functional characteristics of B cells obtained from the pleural fluid (PF) and peripheral blood of patients with tuberculous pleuritis (TP). Our results indicated that the total numbers of B cells, CD27+ memory B cells and plasmablasts were clearly lower in the PF than in peripheral blood. Furthermore, we found significantly higher expression of CXCR4 on B cells in the PF, and a chemotaxis assay showed that B cells in the PF were more responsive to stromal cell-derived factor-1 (SDF-1) than B cells from peripheral blood. In addition, SDF-1 levels in PF were remarkably high compared with SDF-1 levels in plasma, suggesting that the SDF-1/CXCR4 axis might facilitate the migration of circulating B cells into tuberculous pleural space. Importantly, we observed that significantly more antibodies were produced by B cells in the PF following stimulation with BCG, early secretory antigenic target (ESAT-6)/culture filtrate protein-10 (CFP-10) or ESAT-6 protein. Collectively, these data demonstrate that Mycobacterium tuberculosis-specific B cells exist at local sites of infection in TP patients and this localization might influence the immune response to M. tuberculosis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Tuberculosis (TB), one of the oldest infectious diseases associated with humans, is a chronic disease caused by infection with Mycobacterium tuberculosis 1, 2. The incidence of TB has increased during the past 20 years for reasons such as insufficient prevention efforts, incorrectly prescribed medication, the emergence of drug-resistant strains of M. tuberculosis and the prevalence of human immunodeficiency virus (HIV) infection 3, 4.

Traditionally, the immune response against M. tuberculosis infection is dominated by cell-mediated immunity (CMI), which relies primarily on CD4+ and CD8+ T cells. Therefore, many studies have been conducted in an attempt to augment cellular immunity, with an aim to develop new vaccine candidates against TB 5–7. In contrast to these investigations, the contribution of B cells and humoral immunity in the TB field remains largely unexamined and elusive. Fewer efforts to promote an effective humoral response against M. tuberculosis have been made compared with those devised to elicit a cellular response 8. However, it is worth noting that any successful, novel vaccine strategy against TB will have to properly invoke both humoral and cellular immunity, given that these responses are the two critical arms of adaptive immunity and always collaborate to repel infectious pathogens 9.

Numerous murine studies have shown that B cells and antibodies have pleiotropic activities and display previously underappreciated roles during M. tuberculosis infection 10–12. In contrast to the murine studies, there are limited data regarding any phenotypic and functional characterization of the B-cell compartment in TB patients, especially in patients with tuberculous pleuritis (TP), who are known to have a relatively strong immune defense against M. tuberculosis at the local site of disease 13. With this information, we carried out studies to compare the immunophenotypic and functional properties of B cells from the pleural fluid (PF) and the peripheral blood of TP patients.

In this study, we hypothesized that the stromal cell-derived factor-1 (SDF-1)/CXCR4 axis might be involved in the migration of circulating B cells into the tuberculous pleural space. Moreover, we reported that B cells in the PF actively responded to M. tuberculosis-specific antigens, which might influence the local immune response to M. tuberculosis in TP patients.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Populations of CD27+ memory B cells and plasmablasts are decreased in PFMCs from patients with TP

We analyzed the concentration of B cells among the pleural fluid mononuclear cells (PFMCs) and PBMCs of TP patients. As shown in Fig. 1A and B, the proportion of total B cells was significantly reduced in PFMCs (8.43±5.50%, range 2.80–16.21%) compared with PBMCs (11.57±3.68%, range 7.53–17.76%, p=0.0223).

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Figure 1. Decreased frequencies of CD27+ memory B cells and plasmablasts in PFMCs compared with PBMCs of TP patients. (A) Lymphocytes were gated and analyzed by flow cytometry for the expression of CD19 in PFMCs and PBMCs obtained from patients with TP. Data are representative of 20 separate experiments. (B) The statistical results of total CD19+ B cells in PFMCs and PBMCs. Data are expressed in box plots as medians, minimum and maximum values. (C) Lymphocytes were gated and analyzed by flow cytometry for the expression of CD19 and CD27. Data are representative of 12 separate experiments. (D) Statistical results of total CD19+CD27+ B cells in PFMCs and PBMCs. (E) CD19+ B cells were gated and analyzed by flow cytometry for the expression of CD20 and CD27. Data are representative of eight separate experiments. (F) Statistical results of CD19+CD27highCD20+/− plasmablasts in PFMCs and PBMCs. Statistical significance was determined with the Mann–Whitney test.

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We next investigated the pattern of CD27 expression on B cells from PFMCs and PBMCs. As shown in Fig. 1C and D, CD27+ memory B cells were less abundant in PFMCs compared with PBMCs (22.54±8.31% versus 42.48±10.80%, respectively, p=0.0002).

Furthermore, we also determined the population of plasmablasts by flow cytometry. There were fewer plasmablasts in PFMCs (0.14±0.12%) compared with PBMCs (0.85±0.43%, p=0.0050; Fig. 1E and F). Taken together, these results indicate that the levels of total B cells, CD19+CD27+ memory B cells and plasmablasts were decreased in PFMCs from patients with TP.

B-cell subsets are differentially distributed in PFMCs compared with PBMCs

When analyzed for the surface expression of IgD and CD27, we observed a distinct distribution of B cells in PFMCs in contrast with the distribution observed in PBMCs. As illustrated in Fig. 2A and B, the proportions of CD27+ IgD+ memory B cells and CD27+ IgD-switched memory B cells were dramatically decreased in PFMCs compared with PBMCs (p<0.0001 for both subsets), whereas the frequencies of CD27 IgD+ naïve B cells and CD27 IgD B cells in PFMCs were markedly higher than those in PBMCs (p=0.0002; p=0.0005, respectively).

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Figure 2. Identification of naïve and memory B cells in PFMCs and PBMCs of TP patients based on the expression of IgD/CD27. (A) CD19+ B cells were gated and analyzed by flow cytometry for the expression of IgD and CD27. A representative example of a profile was observed in PFMCs and PBMCs. “Isotype” refers to control staining. (B) Statistical results are shown for each B-cell subset in PFMCs and PBMCs from 13 TP patients. Data are expressed in box plots as medians, minimum and maximum values. Statistical significance was determined with the Mann–Whitney test.

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Expression of IgM and IgG on B cells from PFMCs and PBMCs and total Ig levels in PF and serum of TP patients

Subsequently, we evaluated whether there was a numerical deficit in the expression of surface Ig on B cells in tuberculous PF. Figure 3A and C shows a profound reduction of surface IgM expression on B cells in PFMCs relative to PBMCs (13.80±5.12% versus 26.37±5.05%, respectively, p=0.0003). Similarly, the expression of IgG on B cells was significantly lower in PFMCs than PBMCs (9.74±3.46% versus 13.67±2.22%, respectively, p=0.0263; Fig. 3B and D).

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Figure 3. Decreased expression of Ig in PFMCs and concentration of Ig in tuberculous PF compared with PBMCs and serum of TP patients. (A) CD19+ B cells were gated and analyzed by flow cytometry for the expression of IgM. Data are representative of seven separate experiments. Open histogram, IgM; filled grey histogram, isotype control. (B) Statistical results of IgM+ B cells in PFMCs and PBMCs. Each symbol represents the value for an individual patient, and horizontal bars represent the mean value of all data points. (C) CD19+ B cells were gated and analyzed by flow cytometry for the expression of IgG. Data are representative of seven separate experiments. Open histogram, IgG; filled grey histogram, isotype control. (D) Statistical results of IgG+ B cells in PFMCs and PBMCs. (E) Levels of total IgG, IgA and IgM in PF and serum from 12 TP patients were examined by ELISA. Data are expressed as mean+SD. Statistical significance was determined with the Mann–Whitney test.

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In addition, we also examined total Ig levels in the PF and serum using ELISA. The results shown in Fig. 3E indicated that the total amounts of secreted IgG and IgM in the PF were significantly reduced from those in serum (p=0.0004 and p=0.0015, respectively), but the levels of secreted IgA were comparable for the two sites (p=0.5441).

Activation markers and co-stimulatory molecules expressed on B cells from PFMCs and PBMCs

To understand the activation status of B cells at the local site of M. tuberculosis infection, we compared the expression levels of CD69, CD25, CD24, CD38, HLA-DR and CD80 on B cells obtained from the PFMCs and PBMCs of TP patients (Fig. 4A and B). Notably, the expression of CD69 was significantly up-regulated on B cells in PFMCs compared with that of PBMCs (p=0.0237), whereas there was no difference with regard to the expression levels of CD25, CD24, CD38, HLA-DR and CD80 on B cells from PFMCs and PBMCs (p>0.05).

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Figure 4. Increased frequency of CD69 expression on B cells from PFMCs compared with that from PBMCs of TP patients. (A) Either CD20+ or CD19+ B cells were gated and analyzed by flow cytometry for the expression of CD69, CD25, CD24, CD38, HLA-DR and CD80. Data are representative of more than six separate experiments. Open histograms represent surface marker staining, filled grey histograms represent isotype controls. (B) Statistical results for the expression of CD69, CD25, CD24, CD38, HLA-DR and CD80 on B cells from PFMCs and PBMCs. Data are expressed in box plots as medians, minimum and maximum values. Statistical significance was determined with the Mann–Whitney test.

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The expression of CXCR4 is dramatically up-regulated on B cells from PFMCs

To gain insight into the mechanisms of B-cell recruitment to the local sites of inflammation, we investigated the expression of a panel of chemokine receptors on B cells from the PF and the peripheral blood of TP patients (Fig. 5A and B). Interestingly, we found that both the percentage positivity and the MFI of CXCR4 were significantly increased on B cells from PFMCs (98.68±1.41%; MFI: 187.3±70.3) compared with PBMCs (74.51±11.33%, p=0.0024; MFI: 49.2±27.9, p=0.0097). In contrast, the expression levels of CXCR5, CCR6, CXCR3, CCR4 and CCR7 were not significantly different between the groups (p>0.05), except that the MFI of CCR6+ B cells from PFMCs was lower compared with the MFI of those from PBMCs (59.9±22.8% versus 152.8±65.45%, p=0.0360).

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Figure 5. The expression of chemokine receptors on B cells from PFMCs and PBMCs of TP patients. (A) Either CD19+ or CD20+ B cells were gated and analyzed by flow cytometry for the expression of chemokine receptors. Data are representative of seven separate experiments. Open histograms represent chemokine receptor staining, filled grey histograms represent isotype controls. (B) Percentage (left) and mean fluorescent index (MFI, right) of chemokine receptors on B cells from PFMCs and PBMCs. Data are expressed in box plots as medians, minimum and maximum values. (C) CD20+ CD27+ and CD20+ CD27 B cells were gated and analyzed by flow cytometry for the expression of CXCR4. Data are representative of five separate experiments. Open histograms represent CXCR4 staining, filled grey histograms represent isotype controls. (D) Statistical results of the expression of CXCR4 on subsets of CD20+ CD27+ or CD20+ CD27 B cells from PFMCs and PBMCs. Data are expressed as mean+SD. Statistical significance was determined with the Mann–Whitney test.

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To further assess whether the expression of CXCR4 was associated with a naïve and memory phenotype, we analyzed the CXCR4 expression on CD27+ and CD27 B cells obtained from PFMCs and PBMCs. The results in Fig. 5C and D demonstrated that most CD27 B cells were positive for the expression of CXCR4 compared with CD27+ B cells from PBMCs, whereas CXCR4 was expressed on almost all CD27+ and CD27 B cells from PFMCs.

B cells from PFMCs show enhanced migration in response to SDF-1

Based on the expression of CXCR4 on B cells from PFMCs, the chemotactic properties of SDF-1 on B cells in PFMCs were analyzed in a trans-well migration assay. SDF-1 induced B-cell migration in a dose-dependent manner, with the maximum migration observed at a SDF-1 concentration of 100 ng/mL (Fig. 6B). Since B cells in the PF showed enhanced expression of CXCR4, this study was extended to determine whether these B cells were more responsive to SDF-1 than were B cells from PBMCs. As demonstrated in Fig. 6C, B cells from PFMCs migrated more efficiently toward SDF-1 compared with those from PBMCs. Moreover, we measured the SDF-1 levels in the PF and plasma of TP patients. As shown in Fig. 6D, the SDF-1 levels in PF were significantly higher than those in plasma (p=0.0004).

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Figure 6. SDF-1 and its receptor CXCR4 may be responsible for B-cell migration to tuberculous PF. (A) Purification of CD19+ B cells from PFMCs and PBMCs of one representative TP patient. (B) SDF-1 induced concentration-dependent B-cell migration. Purified B cells from PFMCs of TP patients were incubated with the indicated concentrations of SDF-1 (0–100 ng/mL) for 4 h in a trans-well migration assay. The number of cells migrating to the lower chamber was determined by counting under microscopy. Data are expressed as mean+SD, whereas error bars represent triplicates within the same experiment. One of two separate experiments is shown. (C) Comparison of the migration activities of B cells from PFMCs and PBMCs. Purified B cells from PFMCs and PBMCs were cultured for 4 h in the presence or absence of SDF-1 (100 ng/mL) in a trans-well migration assay. The number of cells migrating to the lower chamber was determined by counting under microscopy. Data are expressed as mean+SD, and error bars represent triplicates within the same experiment. One of two separate experiments is shown. (D) Comparison of SDF-1 levels between PF and plasma of 10 TP patients by ELISA. Each symbol represents the value for an individual person and horizontal bars represent the mean value of all data points. Statistical significance was determined with the Mann–Whitney test.

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M. tuberculosis-specific antibody are persistent in PFMCs but not in the PBMCs of TP patients

To compare the production of antibodies following TB-specific antigen stimulation, PFMCs and PBMCs of TP patients were co-cultured with BCG, early secretory antigenic target (ESAT-6)/culture filtrate protein-10 (CFP-10) or the ESAT-6 protein. Supernatants were collected on day 7, and the levels of IgG, IgA and IgM were detected by ELISA (Fig. 7A). The results showed that PFMCs secreted remarkably high levels of IgG, IgA and IgM following stimulation with BCG. PFMCs also elicited large secreted amounts of IgG and IgM in response to the ESAT-6/CFP-10 protein, although the production of IgA to ESAT-6/CFP-10 protein was not induced. In addition, ESAT-6 protein could induce PFMCs to mount a significant IgG response. In contrast, PBMCs from TP patients did not respond to BCG, ESAT-6/CFP-10 or ESAT-6 protein. To confirm that the antibody response was indeed TB specific, PFMCs from TP patients were also stimulated with hepatitis B virus surface antigen (HBsAg) and tetanus toxoid (TT). We found that PFMCs did not respond to these irrelevant antigens (Fig. 7B). Taken together, these results indicate that M. tuberculosis-specific B cells were present in the tuberculous PF.

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Figure 7. The production of Ig in response to antigens specific for M. tuberculosis is induced from PFMCs but not from PBMCs of TP patients. (A) PFMCs and PBMCs from TP patients were cultured with or without BCG (2 μg/mL), ESAT-6/CFP-10 (0.5 μg/mL) or ESAT-6 protein (0.5 μg/mL) for 7 days. Cell culture supernatants were harvested and the production of IgG, IgA and IgM was assessed by ELISA. Data are expressed as mean+SD, and error bars represent triplicates within the same experiment. One of six separate experiments is shown. (B) PFMCs from TP patients were cultured alone or in the presence of 2 μg/mL BCG, 0.5 μg/mL ESAT-6, 0.2, 1, 5 μg/mL HBsAg or 0.04, 0.2, 1 μg/mL TT protein for 7 days. Cell culture supernatants were harvested and the production of IgG, IgA and IgM was assessed by ELISA. Data are expressed as mean+SD and are representative of four separate experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

To date, studies regarding the role of humoral immunity in the containment of M. tuberculosis or the development of tissue damage vary from study to study 14–16. These discrepancies are probably due to the fact that different models and parameters of mAbs were used in these different studies for assessing the outcome of infection 17. As one of the most frequent types of PF, TP develops when M. tuberculosis releases antigenic proteins into the pleural space and involves the migration of immune cells to the site of disease 18, 19. Therefore, TP is a suitable model for understanding the localized and systemic characteristics of B cells that are observed during M. tuberculosis infection.

In this study, we found that the numbers of total CD19+ B cells, CD27+ memory B cells and CD19+CD27highCD20+/− plasmablasts were decreased in PFMCs. Considering the cellular composition of the tuberculous PF, several studies have indicated that PF contains increased numbers of T lymphocytes, particularly in the numbers of CD4+ T cells compared with those in peripheral blood 20, 21. Therefore, the reduced percentage of total B cells in PF might be explained by the relative predominance of T cells in the pleural space.

CD27 is widely used as a marker of memory B cells in humans 22. Studies in a TB mouse model and pulmonary TB patients have shown a reduced level of CD27 expression on antigen-specific CD4+ T cells associated with the persistence of active TB, suggesting a high ratio of fully differentiated effector CD4+ T cells and the presence of antigens in vivo 23, 24. Similarly, we also noted an elevated frequency of CD27 antigen-specific CD4+ T cells in tuberculous PF (data not shown). Therefore, we speculate that the reduced numbers of CD27+ B cells might be associated with the downregulation of CD27 expression on antigen-specific CD4+ T cells because CD27 is expressed on B cells only after antigen-induced activation 25. Moreover, in human B cells, the triggering of CD27 mainly promotes Ig synthesis and the differentiation into plasma cells 26. Therefore, the reduced numbers of plasmablasts, IgG+ and IgM+ B cells in tuberculous PF might be the consequence of the reduction of CD27 expression on B cells at the disease site.

In contrast to PBMCs, the number of CD69+ B cells was higher in PFMCs, suggesting that CD69 is involved in the process of B-cell activation. We speculate that antigen-specific B cells might be present specifically on the CD69+ subset of B cells. However, it remains to be formally proven whether the CD69+ B-cell subset could produce higher levels of antigen-specific antibodies than the CD69 B-cell subset by exclusively purifying these cells in sufficient quantities to perform functionality studies.

In mice, CD5 expression identifies B 1 cells, which are primarily located in the peritoneal and pleural cavities 27. We found, conversely, that the number of CD5+ B cells in tuberculous PF was small (Supporting Information Fig. 1). The characteristics of human equivalent of murine B 1 cells in this disease model remains to be accurately defined because of the very heterogeneous nature of human CD5+ B cells 28.

In the present study, we noticed that B cells in the PF exhibited a strong up-regulation in CXCR4 expression, which is a specific chemokine receptor that only interacts with SDF-1 29. We propose two mechanisms that likely contribute to this phenomenon: (i) elevated levels of SDF-1 at the site of infection regulate CXCR4 expression on B cells in situ as a result of potent induction of chemokine production by M. tuberculosis 30, 31 and (ii) B cells migrate selectively to the affected tissues 32. Therefore, SDF-1 and CXCR4 might play a role in the trafficking of B cells to the site of infection, given that almost all B cells in the PF were CXCR4+ and the chemotaxis assay indicated that SDF-1 was a functional chemokine for the attraction of CXCR4+ B cells. In addition, our group found that the level of CXCR4 expression on CD4+ T cells was comparable between the PFMCs and PBMCs of TP patients (data not shown). Therefore, we hypothesize that the migration of CD4+ T cells from the blood to the site of infection might be mediated mainly by other chemokine–chemokine receptor axes and more efficient than B cells during a TB infection 33, 34. The communication between chemokines and chemokine receptors might aid T cells, B cells and other immune cells in the PF functioning coordinately to fight against M. tuberculosis.

A most remarkable observation in this study was that the PFMCs but not the PBMCs of TP patients actively responded to M. tuberculosis-specific antigens in vitro. We surmise that this finding might be due to the recruitment of antigen-activated B cells to the site of infection where they are no longer accessible in the peripheral blood 35, 36. Despite the decreased total number of B cells in the PF, the functional capacity of B cells increased when challenged with TB antigens. The difference between the systemic and localized immune response in TP patients suggested that amplified specific responses, including the humoral response, were required for antigen elimination at the site of M. tuberculosis infection.

Our results demonstrated that PFMCs responded to BCG more vigorously than to ESAT-6/CFP-10 or ESAT-6 protein stimulation, which might be due to the fact that BCG contains many complex antigens that can activate more abundant M. tuberculosis-specific antibodies than the other TB-specific antigens 37. We also observed a predominant TB-specific IgG response in PFMCs, which appears to be associated with a strong local response in the PF. In contrast, the levels of IgA antibodies to TB-specific antigens were reduced, although they were presumed to be prominent in the pleural environment 38. Two factors may explain this phenomenon. First, IgA reactivity was restricted by antigen components. To our knowledge, only a few well-defined subsets of mycobacterial antigens can elicit an IgA response 39, 40. Second, it has been documented that IgA provides protection from early tuberculous pulmonary infection and that the protection is of short duration 41. As TP onset is insidious, IgA-producing cells might already have been stimulated in vivo and unresponsive to further stimulation when patients sought medical care.

In summary, our studies provide important data concerning the phenotype and functional capacity of B cells from tuberculous PF and the peripheral blood of TP patients. At present, this study needs further strengthening by comparing more samples from TP and non-TP populations and determining whether B cells and the humoral response are essential in protecting the body from an M. tuberculosis infection at the local pleural space. Nevertheless, our preliminary data provide further rationale for the evaluation of the B-cell profiles of TB patients.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Subjects

This investigation was approved by the Medical School Review Board at Sun Yat-sen University, Guangzhou, China. A group of 21 patients with TP, consisting of 13 men and 8 women aged 15–69 years, were enrolled at the Chest Hospital of Guangzhou. The patients were newly diagnosed as having PF by clinical symptoms, physical examination and radiographical evidence. PF and peripheral blood were collected after obtaining informed consent from each patient and before the start of anti-tuberculosis treatment. All patients were seronegative for HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) and were without a history of autoimmune diseases (AD). Clinical data related to patients under study are shown in Table 1.

Table 1. Demographics and clinical characteristics of patients examined in this study
IDSexAgeStageMedicationTuberculin skin testLymphocytes (%) in PF
  1. a

    ID: patient identification number; M: male; F: female; ND: newly diagnosed; lymphocytes (%) in PF were counted by FACS.

1M23NDNonePositive65
2M15NDNonePositive55
3F36NDNonePositive80
4M48NDNoneNegative100
5M37NDNonePositive75
6F30NDNoneNegative85
7F24NDNoneNegative56
8F30NDNonePositive95
9F30NDNonePositive79
10M46NDNonePositive77
11M26NDNonePositive32
12M20NDNonePositive80
13M35NDNoneNegative70
14F25NDNonePositive88
15M33NDNonePositive58
16F25NDNonePositive74
17M23NDNonePositive69
18M69NDNonePositive93
19M27NDNonePositive64
20F19NDNoneNegative90
21M66NDNonePositive78

Preparation of mononuclear cells

PFMCs and PBMCs were obtained by Ficoll-Hypaque (Tianjin Hao Yang Biological Manufacture, Tianjin, China) density-gradient centrifugation within 24 h of sampling. Cells were washed twice in Hank's balanced salt solution. The viability of the cells was assessed using Trypan blue exclusion dye. The cells were finally suspended at a concentration of 2×106/mL in complete RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Sijiqing, China), 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μM 2-mercaptoethanol and 2 mM L-glutamine (Gibco). The PF supernatants, serum and plasma of TP patients were cryopreserved at −80°C until assay.

Purification of B cells

B cells were magnetically purified from fresh PFMCs and PBMCs using a biotin-antibody cocktail (anti-CD2, anti-CD14, anti-CD16, anti-CD43, anti-CD36 and anti-glycophorin A) and anti-biotin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The unlabeled B cells were negatively selected from the column. The purity of B cells (>98%) was assessed by flow cytometry using the anti-CD19 antibody.

mAbs

The following mAbs were used for cell surface staining. Fluourescein isothiocyanate (FITC)-labeled anti-CD20, anti-CD24, anti-IgD, anti-IgM, anti-CD25, anti-CD80, anti-CXCR5; phycoerythrin(PE)-labeled anti-CD69, anti-HLA-DR, anti-CXCR4, anti-CCR4, anti-CCR6, anti-CCR7; allophycocyanin (APC)-labeled anti-CD38, anti-IgG; PE-cy7-conjugated anti-CD69, anti-CCR7 and isotype-matched control antibodies were purchased from BD Biosciences Pharmingen (San Jose, CA, USA). PE-labeled CD19 was obtained from eBioscience (San Diego, CA, USA), APC-labeled CD27 and PE-labeled anti-CXCR3 was purchased from Biolegend (San Diego, CA, USA) and R&D Systems (Minneapolis, MN, USA), respectively.

Cell culture conditions

For antigen-specific stimulation, PFMCs and PBMCs were stimulated with or without 2 μg/mL BCG (purchased from Chengdu Institute of Biological Products, Chengdu, China), 0.5 μg/mL ESAT-6/CFP-10 or 0.5 μg/mL ESAT-6 protein (obtained from Shanghai H&G Biotechnology, Shanghai, China) in a round-bottom 96-well plate. The cells were cultured at a final concentration of 4×105/well in triplicate and incubated for 7 days at 37°C with 5% CO2.

Flow cytometry

For surface staining, PFMCs and PBMCs were washed with PBS buffer containing 0.1% BSA and 0.05% sodium azide and incubated with the respective mAbs for 30 min in 4°C. The cells were thereafter washed twice and resuspended in PBS. Flow cytometry was performed on a BD FACSCalibur (Becton Dickinson, San Jose, CA, USA) and analyzed using the Flowjo software (TreeStar, San Carlos, CA).

ELISA

Cell-free supernatants were harvested and assayed by ELISA for the detection of IgG, IgA and IgM according to manufacturer's protocol (Bethyl, Montgomery, TX, USA). Levels of IgG, IgA and IgM in PF and serum of TP patients were also analyzed using ELISA kits. An ELISA kit that recognizes SDF-1 (R&D Systems) was purchased for the detection of SDF-1 concentrations in PF and plasma of TP patients.

Trans-well migration assay

Freshly isolated B cells from PFMCs and PBMCs were allowed to rest at 37°C for 2 h to equilibrate in migration medium (containing RPMI-1640 plus 1% BSA, 100 U/mL penicillin and 100 μg/mL streptomycin) before being subjected to a trans-well migration assay. After this incubation period, a total of 200 μL containing 2×105 cells was added to the upper chamber of 5-μm pore size trans-well inserts (Millipore, USA). The lower chamber of each well contained 1.2 mL of migration medium alone or in the presence of SDF-1 (R&D Systems). The chambers were incubated for 4 h at 37°C in 5% CO2. The numbers of cells migrating to the lower chamber were counted by microscopy. The assay was performed in triplicate for each condition.

Statistical analysis

Data were reported in terms of medians, minimum and maximum values or as mean±SEM. The Mann–Whitney U-test was used to compare quantitative parameters between two groups of observations. p-Values less than 0.05 were considered significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by a grant from the National Key Basic Research Program of China (973; No. 2007CB512404) and the National Nature Science Foundation of China (No. 30872300).

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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