Rare tuberculosis in recipients of allogeneic hematopoietic stem cell transplantation successfully treated with contezolid-a typical case report and literature review.

Background: Tuberculosis (TB) is a rare but potentially devastating complication in hematopoietic stem cell transplantation (HSCT) recipients. Myelosuppression-related antibiotics should be used cautiously in patients with hematological malignancies, especially those undergoing bone marrow transplantation and receiving bone marrow suppression therapy. Although linezolid has become the recommended drug for severe TB, its hematological toxicity is still an obstacle to its clinical application. Contezolid is a new representative of oxazolidinones in clinical development, showing superior anti-infection efficacy, but there have been no reports on the treatment of post-HSCT TB. Case presentation: We reported a patient with acute lymphoblastic leukemia suffered from pulmonary TB infection after HSCT. During anti-TB treatment, the patient had a poor response to linezolid-containing regimen, and developed side effects such as gingival bleeding and thrombocytopenia, so the administration was switched to contezolid. After 15 days of continuous treatment, the patient's platelet increased to 58×109/L, and he was discharged in stable condition. During subsequent anti-TB treatment with contezolid for more than 7 months, the platelets remained stable, and no hematological adverse reactions and no symptoms of peripheral neuropathy were observed. Moreover, repeat imaging showed that the bilateral lung lesions were significantly reduced, indicating a good outcome for the patient. Conclusion: This was the first successful case of post-HSCT TB patients treated with contezolid-containing antibiotic management strategies, which exhibited remarkable efficacy and good safety in this deadly disease.

Tissue-Resident Memory-Like CD8+ T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion.

Tissue-resident memory T (TRM) cells are well known to play critical roles in peripheral tissues during virus infection and tumor immunology. Our previous studies indicated that CD69+CD4+ and CD69+CD8+ T cells in tuberculous pleural effusion (TPE) were antigen-specific memory T cells. However, the phenotypical and functional characteristics of CD8+ TRM cells in tuberculosis remain unknown. We found that CD103+CD8+ T cells were the predominant subset of CD103+ lymphocytes in TPE; both CD103 and CD69 expressed on memory CD8+ T cells from TPE were significantly increased compared with those from paired peripheral blood. Phenotypically, CD103+CD69+ and CD103+CD69-CD8+ T cells expressed higher levels of CD45RO than CD103-CD69+CD8+ T cells did; CD103+CD69-CD8+ T cells highly expressed CD27, CD127, and CD62L and some chemokine receptors. We further compared the functional differences among the four distinct CD45RO+CD8+ T subsets identified by CD103 and CD69 expression. In consist with our published results, CD69+CD8+ T cells, but not CD103+CD8+, produced high levels of IFN-γ after treatment with BCG in the presence of BFA. Nevertheless, CD103-CD69+ and CD103+CD69+ memory CD8+ T cells expressed higher levels of Granzyme B, while CD103+CD69- memory CD8+ T cells were characterized as a possibly immunosuppressive subset by highly expressing CTLA-4, CD25, and FoxP3. Furthermore, TGF-ß extremely increased CD103 expression but not CD69 in vitro. Together, CD103+CD8+ T cells form the predominant subset of CD103+ lymphocytes in TPE; CD103 and CD69 expression defines distinct CD8+ TRM-like subsets exhibiting phenotypical and functional heterogeneity. Our findings provide an important theoretical basis to optimize and evaluate new tuberculosis vaccines.

[Plasma levels of interferon-inducible protein 10 in patients with active pulmonary tuberculosis with different affected areas].

OBJECTIVE: To explore the value of interferon-inducible protein 10 (IP-10) in the auxiliary diagnosis of tuberculosis and the judgment of the severity of disease. METHODS: From February, 2013 to February, 2017, a total of 193 patients with TB admitted in our hospital and 84 healthy control subjects were recruited consecutively. The peripheral blood plasma levels of interferon-γ (IFN-γ) and IP-10 were detected using liquid phase chip (Luminex) technique. According to the number of lung fields affected by TB, the patients were divided into group A (with lesions in 1-2 lung fields), group B (3-4 lung fields) and group C (5-6 lung fields), The expressions of IFN-γ and IP-10 in 3 groups were compared. RESULTS: The plasma levels of IP-10 were significantly higher in TB patients than in the control subjects (P < 0.05), but IFN-γ levels were comparable between the two groups (P > 0.05). Among the TB patients, plasma IP-10 levels was the highest in group C (P < 0.05), and IFN-γ levels did not differ significantly among the 3 groups (P > 0.05). CONCLUSIONS: Plasma IP-10 has a certain reference value in the auxiliary diagnosis of active tuberculosis and the judgment of the severity of the disease.

Mycobacterium tuberculosis Rv3615c is a highly immunodominant antigen and specifically induces potent Th1-type immune responses in tuberculosis pleurisy.

T-cell responses have been demonstrated to be essential for preventing Mycobacterium tuberculosis infection. The Th1-cytokines produced by T cells, such as INF-γ, IL-2, and TNF-α, not only limit the invasion of M. tuberculosis but also eliminate the pathogen at the site of infection. Bacillus Calmette-Guérin (BCG) is known to induce Th1-type responses but the protection is inadequate. Identification of immunogenic components, in addition to those expressed in BCG, and induction of a broad spectrum of Th1-type responses provide options for generating sufficient adaptive immunity. Here, we studied human pulmonary T-cell responses induced by the M. tuberculosis-specific antigen Rv3615c, a protein with a similar size and sequence homology to ESAT-6 and CFP-10, which induced dominant CD4+ T-cell responses in human tuberculosis (TB) models. We characterized T-cell responses including cytokine profiling, kinetics of activation, expansion, differentiation, TCR usage, and signaling of activation induced by Rv3615c compared with other M. tuberculosis-specific antigens. The expanded CD4+ T cells induced by Rv3615c predominately produced Th1, but less Th2 and Th17, cytokines and displayed effector/memory phenotypes (CD45RO+CD27-CD127-CCR7-). The magnitude of expansion and cytokine production was comparable to those induced by well-characterized the 6 kDa early secreted antigenic target (ESAT-6), the 10 kDa culture filtrate protein (CFP-10) and BCG. Rv3615c contained multiple epitopes Rv3615c1-15, Rv3615c6-20, Rv3615c66-80, Rv3615c71-85 and Rv3615c76-90 that activated CD4+ T cells. The Rv3615c-specific CD4+ T cells shared biased of T-cell receptor variable region of ß chain (TCR Vß) 1, 2, 4, 5.1, 7.1, 7.2 and/or 22 chains to promote their differentiation and proliferation respectively, by triggering a signaling cascade. Our data suggest that Rv3615c is a major target of Th1-type responses and can be a highly immunodominant antigen specific for M. tuberculosis infection.

Memory-Like Antigen-Specific Human NK Cells from TB Pleural Fluids Produced IL-22 in Response to IL-15 or Mycobacterium tuberculosis Antigens.

Our previous result indicated that memory-like human natural killer (NK) cells from TB pleural fluid cells (PFCs) produced large amounts of IFN-γ in response to Bacille Calmette Guerin (BCG). Furthermore, recent studies have shown that human lymphoid tissues harbored a unique NK cell subset that specialized in production of interleukin (IL)-22, a proinflammatory cytokine that mediates host defense against pathogens. Yet little information was available with regard to the properties of IL-22 production by memory-like human NK cells. In the present study, we found that cytokines IL-15 induced and IL-12 enhanced the levels of IL-22 by NK cells from TB PFCs. In addition, IL-22 but not IL-17 was produced by NK cells from PFCs in response to BCG and M.tb-related Ags. More importantly, the subset of specific IL-22-producing NK cells were distinct from IFN-γ-producing NK cells in PFCs. CD45RO+ or CD45RO- NK cells were sorted, co-cultured with autologous monocytes and stimulated with BCG for the production of IL-22. The result demonstrated that CD45RO+ but not CD45RO- NK cells produced significantly higher level of IL-22. Anti-IL-12Rß1 mAbs (2B10) partially inhibit the expression of IL-22 by NK cells under the culture with BCG. Consistently, BCG specific IL-22-producing NK cells from PFCs expressed CD45ROhighNKG2Dhighgranzyme Bhigh. In conclusion, our data demonstrated that memory-like antigen-specific CD45RO+ NK cells might participate in the recall immune response for M. tb infection via producing IL-22, which display a critical role to fight against M. tb.

Mycobacterium tuberculosis-Specific IL-21+IFN-γ+CD4+ T Cells Are Regulated by IL-12.

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+CD62Ll(ow)CCR7(low)CD40L(high)ICOS(high) 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.

Antigen-specific human NKT cells from tuberculosis patients produce IL-21 to help B cells for the production of immunoglobulins.

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(+)TCRvß11(+) 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 CD45RO(high)CD62L(low)CCR7(low). 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(+)TCRvß11(+) NKT cells. Sorted CD3(+)TCRvß11(+) NKT cells from PFMCs produced IFN-γ and IL-21 after stimulation, which expressed CD40L. Importantly, CD3(+)TCRvß11(+) NKT cells provided help to B cells for the production of IgG and IgA. Taken together, our data demonstrate that CD3(+)TCRvß11(+) 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.

[Distribution and drug-resistance of bacteria in the lower respiratory tract in patients with tuberculosis and severe pneumonia receiving invasive mechanical ventilation].

OBJECTIVE: To investigate the distribution and drug-resistance of bacteria in the lower respiratory tract in patients with tuberculosis and severe pneumonia receiving invasive mechanical ventilation. METHODS: The clinical data, lower respiratory tract infection pathogens and bacterial drug sensitivity were analyzed in 208 patients receiving invasive mechanical ventilation for tuberculosis and severe pneumonia. RESULTS: A total of 355 pathogenic microbial strains were obtained from the patients, among which 281 (79.2%) strains were Gram-negative bacteria, 62 (17.5%) were fungi, and 12 (3.4%) were Gram-positive bacteria. Mixed infections were found in 68 cases (19.2%). The sensitivity rates of meropenem, imipenem and amikacin were over 60% for Gram-negative bacteria, and those of teicoplanin, vancomycin, and fusidic acid were 100% for Gram-positive bacteria. CONCLUSION: The main pathogenic bacteria are Gram-negative bacteria, fungi and Gram-positive bacteria in the lower respiratory tract of patients with tuberculosis and severe pneumonia receiving mechanical ventilation. Meropenem, imipenem and amikacin are effective antibiotics for lower respiratory tract infections, and multi-drug resistance is frequent in these patients, which urges appropriate use of the antibiotics.

Mycobacterium tuberculosis-specific memory NKT cells in patients with tuberculous pleurisy.

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(+)TCRvß11(+) NKT cells at the local site of M. tuberculosis infection remains poorly defined. In the present study, we found that the numbers of CD3(+)TCRvß11(+) NKT cells in pleural fluid mononuclear cells (PFMCs) were significantly lower than those in peripheral blood mononuclear cells (PBMCs). However, CD3(+)TCRvß11(+) 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(+)TCRvß11(+) NKT cells from PFMCs produced high levels of IFN-γ. Sorted CD3(+)TCRvß11(+) 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(+)TCRvß11(+) 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(+)TCRvß11(+) NKT cells participated in the local immune responses against M. tuberculosis through the production of IFN-γ and the secretion of cytolytic molecules.

Tuberculosis antigen-induced expression of IFN-α in tuberculosis patients inhibits production of IL-1ß.

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-1ß in vivo. In addition, exogenous IFN-α significantly inhibited production of IL-1ß in PFMCs after stimulation with Bacillus Calmette-Guérin (BCG). To further evaluate the effect of endogenous IFN-α on BCG-induced IL-1ß 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-1ß. Notably, we showed that IFN-α inhibited production of IL-1ß through 2 distinct mechanisms: IFN-α signaling, via the STAT1 transcription factor, suppressed caspase-1-dependent IL-1ß maturation, and IFN-α induced the production of IL-10 in a STAT1-dependent manner in which IL-10 reduced the abundance of IL-1ß. In contrast, we found that IFN-α enhanced the production of IFN-γ, and IFN-γ also suppressed IL-1ß production in the PFMCs during BCG stimulation. Our findings demonstrate that IFN-α employs distinct pathways for regulating IL-1ß 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-1ß.

Mycobacterium tuberculosis-specific polyfunctional cytotoxic CD8+ T cells express CD69.

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.

Pleural fluid mononuclear cells (PFMCs) from tuberculous pleurisy can migrate in vitro in response to CXCL10.

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.

Human CD8+ T cells from TB pleurisy respond to four immunodominant epitopes in Mtb CFP10 restricted by HLA-B alleles.

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 ß chain (Vß) 12, 9, 7.2 or Vß4 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.

Human memory-like NK cells migrating to tuberculous pleural fluid via IP-10/CXCR3 and SDF-1/CXCR4 axis produce IFN-γ in response to Bacille Calmette Guerin.

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-12Rß1 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.

Distinct polyfunctional CD4+ T cell responses to BCG, ESAT-6 and CFP-10 in tuberculous pleurisy.

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.

Identification of M. tuberculosis-specific Th1 cells expressing CD69 generated in vivo in pleural fluid cells from patients with tuberculous pleurisy.

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-12Rß2. 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.

B lymphocytes that migrate to tuberculous pleural fluid via the SDF-1/CXCR4 axis actively respond to antigens specific for Mycobacterium tuberculosis.

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.

Human natural killer cells expressing the memory-associated marker CD45RO from tuberculous pleurisy respond more strongly and rapidly than CD45RO- natural killer cells following stimulation with interleukin-12.

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 ß1 and ß2 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.

Identification of Mycobacterium tuberculosis-specific Th1, Th17 and Th22 cells using the expression of CD40L in tuberculous pleurisy.

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.

Mycobacterium tuberculosis culture filtrate protein 10-specific effector/memory CD4⁺ and CD8⁺ T cells in tubercular pleural fluid, with biased usage of T cell receptor Vß chains.

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 Vß 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 Vß 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 Vß9, Vß12, or Vß7.2. 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.