[Show abstract][Hide abstract] ABSTRACT: In the current study of Mycobacterium tuberculosis (MTB)-specific T and B cells, we found that MTB-specific peptides from early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) induced the expression of IL-21 predominantly in CD4+ T cells. A fraction of IL-21-expressing CD4+ T cells simultaneously expressed Th1 cytokines but did not secrete Th2 or Th17 cytokines, suggesting that MTB-specific IL-21-expressing CD4+ T cells were different from Th1, Th2 and Th17 subpopulations. The majority of MTB-specific IL-21-expressing CD4+ T cells co-expressed IFN-γ and IL-21+IFN-γ+CD4+ T cells exhibited obviously polyfunctionality. In addition, MTB-specific IL-21-expressing CD4+ T cells displayed a CD45RO+CD62LlowCCR7lowCD40LhighICOShigh phenotype. Bcl-6-expression was significantly higher in IL-21-expressing CD4+ T cells than IL-21-CD4+ T cells. Moreover, IL-12 could up-regulate MTB-specific IL-21 expression, especially the frequency of IL-21+IFN-γ+CD4+ T cells. Taken together, our results demonstrated that MTB-specific IL-21+IFN-γ+CD4+ T cells from local sites of tuberculosis (TB) infection could be enhanced by IL-12, which have the features of both Tfh and Th1 cells and may have an important role in local immune responses against TB infection.
[Show abstract][Hide abstract] ABSTRACT: Natural killer T (NKT) cells from mouse and human play an important role in the immune responses against Mycobacterium tuberculosis. However, the function of CD3+TCRvβ11+ NKT cells at the local site of M. tuberculosis infection remains poorly defined. In the present study, we found that after stimulation with M. tuberculosis antigens, NKT cells isolated from tuberculosis (TB) pleural fluid mononuclear cells (PFMCs) produced IL-21 and other cytokines including IFN-γ, TNF-α, IL-2 and IL-17. IL-21-expressing NKT cells in PFMCs displayed effector memory phenotype, expressing CD45ROhighCD62LlowCCR7low. Moreover, NKT cells expressed high levels of CXCR5 and all of IL-21-expressing NKT cells co-expressed CXCR5. The frequency of BCL-6-expression was higher in IL-21-expressing but not in non-IL-21-expressing CD3+TCRvβ11+ NKT cells. Sorted CD3+TCRvβ11+ NKT cells from PFMCs produced IFN-γ and IL-21 after stimulation, which expressed CD40L. Importantly, CD3+TCRvβ11+ NKT cells provided help to B cells for the production of IgG and IgA. Taken together, our data demonstrate that CD3+TCRvβ11+ NKT cells from a local site of M. tuberculosis infection produce IL-21, express CXCR5 and CD40L, help B cells to secrete IgG and IgA, and may participate in local immune responses against M. tuberculosis infection.
[Show abstract][Hide abstract] ABSTRACT: Natural killer T (NKT) cells from mouse and human play a protective role in the immune responses against the infection of Mycobacterium tuberculosis. However, the characteristic of CD3(+)TCRvβ11(+) NKT cells at the local site of M. tuberculosis infection remains poorly defined. In the present study, we found that the numbers of CD3(+)TCRvβ11(+) NKT cells in pleural fluid mononuclear cells (PFMCs) were significantly lower than those in peripheral blood mononuclear cells (PBMCs). However, CD3(+)TCRvβ11(+) NKT cells from PFMCs spontaneously expressed high levels of CD69 and CD25 and effector memory phenotypes of CD45RO(high)CD62L(low)CCR7(low). After stimulation with the antigens of M. tuberculosis, CD3(+)TCRvβ11(+) NKT cells from PFMCs produced high levels of IFN-γ. Sorted CD3(+)TCRvβ11(+) NKT cells from PFMCs cultured with antigen presenting cells (APCs) produced IFN-γ protein and mRNA. The production of IFN-γ could be completely inhibited by AG490 and Wortmannin. In addition, CD3(+)TCRvβ11(+) NKT cells from PFMCs expressed higher levels of Fas (CD95), FasL (CD178) and perforin but lower levels of granzyme B compared with those from PBMCs. Taken together, our data demonstrated for the first time that M. tuberculosis-specific CD3(+)TCRvβ11(+) NKT cells participated in the local immune responses against M. tuberculosis through the production of IFN-γ and the secretion of cytolytic molecules.
No preview · Article · Sep 2014 · Journal of Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: Increasing evidences in animals and humans suggest that CD8+ T cells contribute significantly to immune defenses against Mycobacterium tuberculosis (Mtb). In the present study, we found that without any stimulation, CD8+ T cells in pleural fluid cells (PFCs) expressed significantly higher levels of CD69 than PBMCs from patients with tuberculous pleurisy (TBP). CD8+CD69+ T cells expressed significantly higher levels of CD45RO and HLA-DR and lower levels of CD45RA than CD8+CD69− T cells, demonstrating that CD8+CD69+ T cells were activated memory cells. Furthermore, we found higher expression of CCR6 and lower expression of CCR7 and CD62L on CD8+CD69+ T cells compared with CD8+CD69− T cells, suggesting that the expression of CCR6 and reduced expression of CCR7 and CD62L might facilitate the migration of circulating CD8+CD69+ T cells into tuberculous pleural space. Importantly, following stimulation with culture filtrate protein of 10 kDa (CFP10) peptides, CD8+CD69+ T cells but not CD8+CD69− T cells expressed CD107a/b, IFN-γ and TNF-α, demonstrating that CD8+CD69+ T cells were MTB-specific cells. In addition, the majority of CD8+CD69+ T cells were dominated by polyfunctional T cells. In summary, we demonstrated that CD69 as a useful marker for MTB-specific CD8+ T cells in PFCs from patients with TBP enabled a direct ex vivo estimation of the quantity, as well as the quality, of MTB-specific CD8+ responses.
No preview · Article · May 2014 · Tuberculosis (Edinburgh, Scotland)
[Show abstract][Hide abstract] ABSTRACT: The mechanism by which IFN-α regulates the host response to Mycobacterium tuberculosis (M.tb) infection in humans is poorly understood. In the present study, we found that freshly isolated pleural fluid mononuclear cells (PFMCs) from tuberculous pleural effusion but not peripheral blood mononuclear cells (PBMCs) spontaneously expressed IFN-α and IL-1β in vivo. In addition, exogenous IFN-α significantly inhibited production of IL-1β in PFMCs after stimulation with Bacillus Calmette-Guérin (BCG). To further evaluate the effect of endogenous IFN-α on BCG-induced IL-1β production, a neutralizing antibody to IFN-α was added to the cultures of BCG-stimulated PFMCs. As expected, neutralization of IFN-α by antibody significantly enhanced the production of IL-1β. Notably, we showed that IFN-α inhibited production of IL-1β through 2 distinct mechanisms: IFN-α signaling, via the STAT1 transcription factor, suppressed caspase-1-dependent IL-1β maturation, and IFN-α induced the production of IL-10 in a STAT1-dependent manner in which IL-10 reduced the abundance of IL-1β. In contrast, we found that IFN-α enhanced the production of IFN-γ, and IFN-γ also suppressed IL-1β production in the PFMCs during BCG stimulation. Our findings demonstrate that IFN-α employs distinct pathways for regulating IL-1β production and reveal that in the case of M.tb infection, the induction of IFN-α and IFN-γ might be associated with M.tb immune escape and disease progression in infected humans.-Ma, J., Yang, B., Yu, S., Zhang, Y., Zhang, X., Lao, S., Chen, X., Li, B., Wu, C. Tuberculosis antigen-induced expression of IFN-α in tuberculosis patients inhibits production of IL-1β.
[Show abstract][Hide abstract] ABSTRACT: CD8(+) T cells are essential for host defense to Mycobacterium tuberculosis (Mtb) infection and identification of CD8(+) T cell epitopes from Mtb is of importance for the development of effective peptide-based diagnostics and vaccines. We previously demonstrated that the secreted 10-KDa culture filtrate protein (CFP10) from Mtb is a potent CD8(+) T cell antigen but the repertoire and dominance pattern of human CD8 epitopes for CFP10 remained poorly characterized. In the present study, we undertook to define immunodominant CD8 epitopes involved in CFP10 using a panel of CFP10-derived 13-15 amino acid (aa) peptides overlapping by 11 aa. Four peptides in CFP10 were observed to induce significant CD8(+) T cell responses and we further determined the size of the epitopes involved in each individual peptide tested. Four 9 aa CD8 epitopes were finally identified and deleting a single amino acid from the N or C terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimum of CD8 epitopes. In the individuals tested, each epitope represented a single immunodominant response in CD8(+) T cells. The epitope-specific CD8(+) T cells displayed effector or effector memory phenotypes and could upregulate the expression of CD107a/b upon antigen stimulation. In addition, we found that epitope-specific CD8(+) T cells shared biased usage of T cell receptor (TCR) variable region of β chain (Vβ) 12, 9, 7.2 or Vβ4 chains. As judged from HLA-typing results and using bioinformatics technology for prediction of MHC binding affinity, we found that the epitope-specific CD8(+) T cells are all restricted by HLA-B alleles. Our findings suggest that the four epitopes in CFP10 recognized by CD8(+) T cells might be of importance for the development of Mtb peptide-based vaccines and for improved diagnosis of TB in humans.
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that human memory-like NK cells were persistent in tuberculous pleurisy but it was unclear how NK cells migrated into the pleural fluids. At present, we found that NK cells from TB pleural fluid cells (PFCs) expressed significantly higher levels of CXCR3 and CXCR4 than NK cells from PBMCs. Migration assay demonstrated that IP-10 and SDF-1 induced more migration of NK cells from PFCs than PBMCs. CD45RO(+) or CD45RO(-) NK cells from PFCs were co-cultured with autologous monocytes and stimulated with BCG. The results showed CD45RO(+) but not CD45RO(-) NK cells produced significantly higher levels of IFN-γ, which was IL-12-dependent since anti-IL-12Rβ1 mAbs could significantly inhibit the IFN-γ by NK cells. Collectively, our data demonstrated that human Mycobacterium tuberculosis-specific NK cells were migrated into the local site of TB infection mainly via IP-10/CXCR3 and SDF-1/CXCR4 axis, memory-like NK cells might display an important role against M. tuberculosis infection.
No preview · Article · Apr 2013 · Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: We recently reported that pleural fluid mononuclear cells (PFMCs) from tuberculous pleurisy stimulated with Bacillus Calmette-Guérin (BCG) or TB antigens produced high levels of cytokines. However, it was still unclear what mechanism of the PFMCs used to migrate into the pleural fluids in TB infection. In the present study, we found that CD3+CD4+ and CD3+CD8+ T cells from PFMCs expressed significantly high levels of CXCR3 compared to PBMCs. In addition, the levels of CXCL10 (the ligand for CXCR3) in pleural fluids were significantly higher than those in normal serum and cancerous fluids. After stimulation with BCG, PFMCs produced high levels of CXCL10. Importantly, the synthesis of CXCL10 was mainly dependent on the BCG-induced production of IFNs, because the neutralization of endogenous IFN-α or IFN-γ with mAbs significantly reduced the production of CXCL10 from BCG-stimulated PFMCs. In addition, the tubercular pleural fluid (TBPF) or exogenous CXCL10 induced the migration of PFMCs, indicating that IFN-α or IFN-γ modulated the immune response through the expression of CXCL10 to aid the recruitment and selective homing of activated/effector cells to the site of Mycobacterium tuberculosis (M.tb) infection. Taken together, the levels of CXCL10 in pleural fluids were high and BCG-stimulated PFMCs expressed high levels of CXCL10, and CXCL10 induced the migration of PFMCs into the pleural fluids in TB infection.
No preview · Article · Jan 2013 · Tuberculosis (Edinburgh, Scotland)
[Show abstract][Hide abstract] ABSTRACT: Tuberculous pleurisy (TBP) is a frequent extrapulmonary manifestation characterized by the accumulation of inflammatory cells that can sometimes be spontaneously self-cured. To achieve a greater insight into T cells at a local site, we systematically characterized and compared the numbers of antigen-specific T cells responding to BCG- or MTB-specific antigens. Our results showed that significantly higher levels of Th1 cytokines were produced by pleural fluid cells (PFCs) than PBMCs following stimulation with BCG or peptides from Mycobacterium tuberculosis (MTB)-specific antigens, ESAT-6 and CFP-10. The proportions of Th1 cells producing IL-2 alone or IL-2 and TNF-α were higher than those producing IFN-γ alone, following stimulation with ESAT-6 or CFP-10 peptides. The cells responding to BCG, ESAT-6 and CFP-10 displayed a CD45RA(-)CCR7(-)CD62L(-)CD27(-) effector/effector memory phenotype. The percentages and median fluorescence intensity (MFI) of polyfunctional CD4(+) T cells were significantly higher following stimulation with peptides from ESAT-6 or CFP-10 than BCG. Our results demonstrated that significantly higher levels of polyfunctional CD4(+) T cells for the epitopes of ESAT-6 or CFP-10 in PFCs may play an important role in the local control of tuberculosis (TB) infection.
No preview · Article · Dec 2011 · Tuberculosis (Edinburgh, Scotland)
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Nov 2011 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Th1 cell-mediated immune responses at the site of active infection are important to restrict the growth of M. tuberculosis (MTB) and for the spontaneous resolution of patients with tuberculous pleurisy (TBP). In the present study, we found that without any stimulation, CD4(+) T cells in pleural fluid cells (PFCs) from patients with TBP expressed significantly higher levels of CD69 than PBMCs from patients with tuberculosis (TB) or healthy donors. CD4(+)CD69(+) T cells expressed T-bet and IL-12Rβ2. After stimulation with MTB-specific antigens, CD4(+)CD69(+) T cells expressed significantly higher levels of IFN-γ, IL-2 and TNF-α than CD4(+)CD69(-) T cells, demonstrating that CD4(+)CD69(+) T cells were MTB-specific Th1 cells. In addition, CD4(+)CD69(+) T cells were mostly polyfunctional Th1 cells that simultaneously produced IFN-γ, IL-2, TNF-α and displayed an effector or effector memory phenotype (CD45RA(-)CCR7(-)CD62L(-)CD27(-)). Moreover, the percentages of CD4(+)CD69(+) T cells were significantly and positively correlated with polyfunctional T cells. Interestingly, sorted CD4(+)CD69(+) but not CD4(+)CD69(-) fractions by flow cytometry produced IFN-γ, IL-2 and TNF-α that were significantly regulated by CD4(+)CD25(+) Treg cells. Taken together, based on the expression of CD69, we found a direct quantitative and qualitative method to detect and evaluate the in vivo generated MTB-specific polyfunctional CD4(+) T cells in PFCs from patients with TBP. This method can be used for the potential diagnosis and enrichment or isolation of MTB-specific Th1 cells in the investigations.
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are known as innate immune lymphocytes that respond rapidly when challenged by pathogens but little is known about adaptive immune features including memory related to NK cells from human beings. In the present study, we demonstrate for the first time that human NK cells expressing the memory-associated marker CD45RO were persistent in pleural fluid cells (PFCs) from tuberculous patients. CD45RO(+) NK cells produced significantly more interferon-γ and were more cytotoxic compared with CD45RO(-) NK cells from PFCs when stimulated with interleukin-12 (IL-12). Consistently, IL-12 enhanced the expression of granzyme B, CD69, CD25, NKG2D, IL-12 receptors β1 and β2 on CD45RO(+) NK cells from PFCs. Our experiments contribute to a better understanding of the NK cells from PFCs and indicate that human CD45RO(+) NK cells from PFCs expressing a 'memory-like' phenotype may have an important role in defending against infection by Mycobacterium tuberculosis.
[Show abstract][Hide abstract] ABSTRACT: T cell-mediated immunity is critical for the control of Mycobacterium tuberculosis infection. Identifying the precise immune mechanisms that lead to control of initial M. tuberculosis infection and preventing reactivation of latent infection are crucial for combating tuberculosis. However, a detailed understanding
of the role of T cells in the immune response to infection has been hindered. In addition, there are few flow cytometry studies
characterizing the Vβ repertoires of T cell receptors (TCRs) at local sites of M. tuberculosis infection in adult tuberculosis. In this study, we used culture filtrate protein 10 (CFP-10) from M. tuberculosis to characterize T cells at local sites of infection. We simultaneously analyzed the correlation of the production of cytokines
with TCR Vβ repertoires in CFP-10-specific CD4+ and CD8+ T cell subsets. For the first time, we demonstrate that CFP-10-specific CD4+ or CD8+ T cells from tubercular pleural fluid can produce high levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha
(TNF-α) and upregulate the expression of CD107a/b on the cell surface. The CFP-10-specific cells were effector/memory cells
with a CD45RO+ CD62L− CCR7− CD27− expression profile. In addition, we found CFP-10-specific CD4+ and CD8+ T cells in tubercular pleural fluid, with biased usage of TCR Vβ9, Vβ12, or Vβ7.2. Our findings of CFP-10-specific CD4+ and CD8+ T cells in tubercular pleural fluid are critical for understanding the mechanisms of the local cellular immune response and
developing more effective therapeutic interventions in cases of M. tuberculosis infection.
Preview · Article · May 2011 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4(+) T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4(+)CD40L(+) but not CD4(+)CD40L(-) T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4(+)CD40L(+) T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA(-)CD45RO(+)CCR7(-)CD62L(-)ICOS(-)). To determine the specificity of CD4(+)CD40L(+) T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4(+)CD40L(+) and CD4(+)CD40L(-) T cells by flow cytometry. We further demonstrated that sorted CD4(+)CD40L(+), but not CD4(+)CD40L(-) fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4(+) T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB.
[Show abstract][Hide abstract] ABSTRACT: The local milieu at the site of Mycobacterium tuberculosis infection that modulates T-cell functions is the main battleground for the host to build counter-M. tuberculosis immune responses. CD4+T cells are enriched predominantly in tuberculosis pleurisy and their roles are of considerable importance, but their nature and functional profiles linked with local condition remain elusive. Here we evaluated the functions of M. tuberculosis-specific CD4+T cells from the major three profiles: cytokines production, cell activation and division. Results showed that pleural fluid (PF) from tuberculosis patients in a dose dependent manner inhibited the production of IFN-γ, IL-2 and TNF-α by M. tuberculosis-specific peptides or BCG activated CD4+T cells from pleural fluid mononuclear cells (PFMCs). Surface staining for activation molecules indicated that PF could also blunt cell activation process. CFSE labeling showed that antigen-specific CD4+T cell division ceased following co-incubation with PF. Pre- or post-treatment with PF could disturb subsequent cell activities. The strong inhibitory effect mediated by PF on CD4+T cells was functional predominance. Moreover, application of inhibitors of IDO, adenosine, neutralizing Abs to IL-10 and TGF-β could partially reverse IFN-γ production. Our current research provided novel information that the functions of antigen-specific CD4+T cells coincubated with PF were apparently impaired, which were distinct from cells that cultured in fresh culture medium. We concluded that CD4+T cell mediated antigen-specific cellular immune response that occurred locally might be impaired by PF.
No preview · Article · Apr 2011 · Immunology letters
[Show abstract][Hide abstract] ABSTRACT: Immunosuppressive mediators in tuberculosis pleurisy (pleural fluid (PF)) are associated with the course of disease, but they remain poorly defined. To study the local immune status of patients with tuberculosis pleurisy, we examined the effect of PF on the functions of T cells and the differentiation of Th1 cells. PF could inhibit the ability of T cells to produce cytokines. However, tumor-necrosis factor (TNF)-α derived from non-T cells was not impaired. Further analysis indicated that cell activation and cell cycle progression were also suppressed. Moreover, PF could inhibit Th1 cell differentiation. Importantly, we found that inhibitors of indoleamine 2,3-dioxygenase (IDO) and adenosine and neutralizing antibodies against IL-10 and transforming growth factor (TGF)-β could reverse cytokine production, suggesting that IDO, adenosine, IL-10 and Transforming growth factor-β1 in PF might take part in impairing T-cell functions. Taken together, our data demonstrate for the first time that several immunopathological factors participate in the downregulation of T-cell functions in local PF.