Effector functions of heparin-binding hemagglutinin-specific CD8+ T lymphocytes in latent human tuberculosis

BACKGROUND: Most individuals infected with Mycobacterium tuberculosis do not develop tuberculosis (TB) and can be regarded as being protected by an appropriate immune response to the infection. The characterization of the immune responses of individuals with latent TB may thus be helpful in the definition of correlates of protection and the development of new vaccine strategies. The highly protective antigen heparin-binding hemagglutinin (HBHA) induces strong interferon (IFN)- gamma responses during latent, but not active, TB. Because of the recently recognized importance of CD8(+) T lymphocytes in anti-TB immunity, we characterized the CD8(+) T lymphocyte responses to HBHA in subjects with latent TB. RESULTS: HBHA-specific CD8(+) T lymphocytes expressed memory cell markers and synthesized HBHA-specific IFN- gamma . They also restricted mycobacterial growth and expressed cytotoxicity by a granule-dependent mechanism. This activity was associated with the intracellular expression of HBHA-induced perforin. Surprisingly, the perforin-producing CD8(+) T lymphocytes were distinct from the IFN- gamma -producing CD8(+) T lymphocytes. CONCLUSION: During latent TB, the HBHA-specific CD8(+) T lymphocyte population expresses all 3 effector functions associated with CD8(+) T lymphocyte-mediated protective immune mechanisms, which supports the notion that HBHA may be protective in humans and suggests that markers of HBHA-specific CD8(+) T lymphocyte responses may be useful in the monitoring of protection.     

Heparin-binding hemagglutinin, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis

Interactions of Mycobacterium tuberculosis with macrophages have long been recognized to be crucial to the pathogenesis of tuberculosis. The role of non-phagocytic cells is less well known. We have discovered a M. tuberculosis surface protein that interacts specifically with non-phagocytic cells, expresses hemagglutination activity and binds to sulfated glycoconjugates. It is therefore called heparin-binding hemagglutinin (HBHA). HBHA-deficient M. tuberculosis mutant strains are significantly impaired in their ability to disseminate from the lungs to other tissues, suggesting that the interaction with non-phagocytic cells, such as pulmonary epithelial cells, may play an important role in the extrapulmonary dissemination of the tubercle bacillus, one of the key steps that may lead to latency. Latently infected human individuals mount a strong T cell response to HBHA, whereas patients with active disease do not, suggesting that HBHA is a good marker for the immunodiagnosis of latent tuberculosis, and that HBHA-specific Th1 responses may contribute to protective immunity against active tuberculosis. Strong HBHA-mediated immuno-protection was shown in mouse challenge models. HBHA is a methylated protein and its antigenicity in latently infected subjects, as well as its protective immunogenicity strongly depends on the methylation pattern of HBHA. In both mice and man, the HBHA-specific IFN-gamma was produced by both the CD4(+) and the CD8(+) T cells. Furthermore, the HBHA-specific CD8(+) T cells expressed bactericidal and cytotoxic activities to mycobacteria-infected macrophages. This latter activity is most likely perforin mediated. Together, these observations strongly support the potential of methylated HBHA as an important component in future, acellular vaccines against tuberculosis.     

Regulatory T cells depress immune responses to protective antigens in active tuberculosis

RATIONALE: Tuberculosis (TB) remains a leading cause of death, and the role of T-cell responses to control Mycobacterium tuberculosis infections is well recognized. Patients with latent TB infection develop strong IFN-gamma responses to the protective antigen heparin-binding hemagglutinin (HBHA), whereas patients with active TB do not. OBJECTIVES: We investigated the mechanism of this difference and evaluated the possible involvement of regulatory T (Treg) cells and/or cytokines in the low HBHA T-cell responses of patients with active TB. METHODS: The impact of anti-transforming growth factor (TGF)-beta and anti-IL-10 antibodies and of Treg cell depletion on the HBHA-induced IFN-gamma secretion was analyzed, and the Treg cell phenotype was characterized by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Although the addition of anti-TGF-beta or anti-IL-10 antibodies had no effect on the HBHA-induced IFN-gamma secretion in patients with active TB, depletion of CD4(+)CD25(high)FOXP3(+) T lymphocytes resulted in the induction by HBHA of IFN-gamma concentrations that reached levels similar to those obtained for latent TB infection. No effect was noted on the early-secreted antigen target-6 or candidin T-cell responses. CONCLUSIONS: Specific CD4(+)CD25(high)FOXP3(+) T cells depress the T-cell-mediated immune responses to the protective mycobacterial antigen HBHA during active TB in humans.     

Increasing CD4+ T cells specific for tuberculosis correlate with improved clinical immunity after highly active antiretroviral therapy

Treatment advances have led to dramatic clinical improvements for patients with HIV-1 infection. These clinical improvements reflect treatment-related improvements in immune function, which are most striking in individuals who develop exaggerated immune inflammatory responses to occult opportunistic infections. The mechanisms accounting for these exaggerated immune responses are unknown. To gain insight into these mechanisms, we intensively studied a subject untreated for disseminated tuberculosis and HIV-1 coinfection who then began treatment for both diseases. We examined the changing frequencies of Mycobacterium tuberculosis (MTB)-specific CD4(+) T cells that produced interferon gamma (IFN-gamma) after short-term stimulation with MTB antigen, and we compared these frequencies with those in HIV-1-seronegative subjects with and without prior exposure to MTB antigens. For the HIV-1/MTB-coinfected subject, the proportion of peripheral blood CD4(+) T cells expressing MTB-specific IFN-gamma was 8.6% at 11 days, 11% at 33 days, and 33% at 95 days after starting treatment for HIV-1. CD4(+) IFN-gamma(+) T cells had a CD45RA(-)CD62L(-) (effector memory) phenotype and most coexpressed interleukin 2. Median frequencies of CD4(+) IFN-gamma(+) T cells from six subjects without and nine subjects with prior exposure to MTB antigens were 0.06 and 0.46%, respectively. We conclude that individuals starting treatment for disseminated tuberculosis and HIV-1 coinfection can accumulate remarkably large numbers of MTB-specific CD4(+) T cells in the peripheral blood. The rapid expansion of antigen-specific effector CD4(+) T cells is one mechanism to explain immediate improvements in clinical immunity after HIV-1 treatment. This mechanism provides a theoretical framework to understand the unusual inflammatory responses recently reported to occur after starting HIV-1 treatment.     

分枝杆菌肝素结合血凝素研究进展

结核病是由分枝杆菌感染引起的世界范围内流行的人畜共患病。肝素结合血凝素(heparin-binding adhesin,HBHA)是分枝杆菌表达的一种蛋白质,致病性分枝杆菌的HBHA蛋白具有黏附上皮细胞、扩散感染范围等功能。其发挥黏附功能的C端结构域能够刺激机体产生有效的Th1细胞免疫反应;HBHA特异的黏膜免疫反应能够提供一定的保护效果。这提示HBHA具有制作为疫苗的潜力。而且,HBHA蛋白在活动性结核病患者和潜伏感染者中引发的免疫反应存在差异,这使其可以成为新的诊断抗原。此外,在细胞水平上,HBHA可以利用补体系统进入巨噬细胞,促使巨噬细胞向M1极化,同时能够逃避自噬并诱发线粒体损伤和内质网应激介导功能,这为结核病的致病机制提供了更多的解释。HBHA与宿主之间相互作用认识的积累,可以为HBHA的应用性研究提供依据。     

Cellular responses to MPT-51, GlcB and ESAT-6 among MDR-TB and active tuberculosis patients in Brazil

Multi-drug resistant pulmonary tuberculosis (MDR-TB) may result from either insufficiency of the host cellular immune response or mycobacterial mechanisms of resistance. Mycobacterium tuberculosis-specific CD8+ and CD4+ T lymphocytes from MDR-TB patients are poorly studied. The aim of this study was to evaluate CD4+IFN-gamma+, CD4+IL-10+, CD8(+)IFN-gamma+ and CD8+IL-10+ cell populations by flow cytometry in non-resistant TB and multi-drug resistant tuberculosis (MDR-TB) patients from mid-central Brazil after stimulation with MPT-51, GlcB and ESAT-6 recombinant antigens from M. tuberculosis in comparison to tuberculin skin test negative (TST) healthy individuals. Non-resistant TB patients present specific cellular responses (CD4 and CD8, both IFN-gamma and IL-10) to GlcB, MPT-51 and ESAT-6; while MDR-TB patients present only CD8+IFN-gamma+ responses to ESAT-6 and CD8+IL-10+ responses to GlcB and ESAT-6. The results show that MDR-TB patients present impaired specific CD4 IFN-gamma and IL-10 responses and increased/normal specific CD8 IFN-gamma and IL-10 responses. This suggests an important role for CD8 function in these patients.     

Long-lived immune response to early secretory antigenic target 6 in individuals who had recovered from tuberculosis.

We sought to understand the persistence and relevance of the long-lived immune response to early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis in humans. ESAT-6 is recognized by memory cells involved in protection of animals against tuberculosis (TB). Recent reports also showed that ESAT-6 response can be recovered in patients with TB and in those soon after anti-TB therapy. We chose 18 individuals who had recovered from pulmonary TB (some in remission for >5 years), and 14 bacille Calmette-Guérin-vaccinated healthy individuals for this study. The results showed that peripheral blood mononuclear cells of 10 (55.6%) of 18 patients with TB remission responded to ESAT-6 with stimulation indices >3.0, whereas none of the healthy controls responded. Functional analysis showed that 13 (72.2%) of 18 patients with TB remission produced significant amounts of IFN-gamma in response to ESAT-6, whereas only 1 (7.1%) of the 14 healthy control subjects did so. It appears that responses to ESAT-6 can persist in individuals who had recovered from pulmonary TB.     

结核分枝杆菌黏附素HBHA在结核病免疫学诊断中的应用

目的 探讨结核分枝杆菌肝素结合血凝黏附素(Heparin-binding haemagglutinin adhesin,HBHA)在结核病免疫诊断方面的应用价值.方法 将在苏通培养基中培养至稳定期的卡介苗菌株(BCG)菌体超声裂解,离心后取上清并通过CL-6B层析柱,利用含0～500 mmol/L氯化钠的磷酸盐缓冲溶液(PBS)进行梯度洗脱后获得天然HBHA.以BCG基因组为模板PCR扩增结核分枝杆菌hbha基因片段,质粒pET-32a为载体,大肠杆菌作为宿主菌制备原核表达的HBHA重组蛋白.以纯化的天然HBHA蛋白和原核表达的HBHA蛋白为包被抗原,分别对肺结核组、肺外结核组、PPD阳性和PPD阴性健康对照组各47例血清通过ELISA方法检测抗HBHA的抗体水平.应用SPSS 11.5统计软件对各研究组数据进行方差齐性检验,然后进行t检验,并计算各研究组的敏感度和特异度.结果 含有375 mmol/L氯化钠的PBS洗脱液可以洗脱获得较纯的天然HBHA蛋白.利用重组HBHA所带的组氨酸标签进行特异分离、纯化.ELISA检测结果表明天然HBHA蛋白和重组HBHA蛋白、PPD阳性和PPD阴性健康对照组之间血清抗体水平差异不明显.肺结核组与肺外结核组的血清抗体ELISA检测的吸光度值在散点图中分布有明显差异,肺结核组吸光度值集中分布在0.30～0.40之间,肺外结核组吸光度值分布在0.35～0.45之间,经统计学检验结果差异具有统计学意义(t=12.224,P＜0.05).结核组(肺结核和肺外结核)与对照组(PPD阴性,PPD阳性)吸光度值分布具有显著不同,对照组全部小于0.30,结核组吸光度值则集中分布于0.30～0.45之间.经统计学检验(t=25.909,P＜0.05)血清抗体水平具有显著性差异.结论 结核分枝杆菌黏附素HBHA在结核病免疫学诊断的应用中具有较好的特异度和敏感度,有望用于结核病、尤其是肺外结核病的实验室辅助诊断.     

Detection of polyfunctional Mycobacterium tuberculosis-specific T cells and association with viral load in HIV-1-infected persons

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) epidemic is associated with a significant increase in the incidence of tuberculosis (TB); however, little is known about the quality of Mycobacterium tuberculosis (MTB)-specific cellular immune responses in coinfected individuals. METHODS: A total of 137 HIV-1-positive individuals in Durban, South Africa, were screened with the use of overlapping peptides spanning Ag85A, culture filtrate protein 10 (CFP-10), early secretory antigen target 6 (ESAT-6), and TB10.4, in an interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay. Intracellular cytokine staining for MTB-specific production of IFN-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2 was performed, as was ex vivo phenotyping of memory markers on MTB-specific T cells. RESULTS: A total of 41% of subjects responded to ESAT-6 and/or CFP-10, indicating the presence of latent MTB infection. The proportion of MTB-specific IFN-gamma(+)/TNF-alpha(+) CD4(+) cells was significantly higher than the proportion of IFN-gamma(+)/IL-2(+) CD4(+) cells (P = .0220), and the proportion of MTB-specific IL-2-secreting CD4 cells was inversely correlated with the HIV-1 load (P = .0098). MTB-specific CD8 T cells were predominately IFN-gamma(+)/TNF-alpha(+)/IL-2(-). Ex vivo memory phenotyping of MTB-specific CD4 and CD8 T cells indicated an early to intermediate differentiated phenotype for the population of effector memory cells. CONCLUSIONS: Polyfunctional MTB-specific CD4 and CD8 T cell responses are maintained in the peripheral blood of HIV-1-positive individuals, in the absence of active disease, and the functional capacity of these responses is affected by HIV-1 disease status.     

Human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy maintain activated CD8+ T cell subsets as a strong adaptive immune response to cytomegalovirus.

CD8(+) T lymphocyte function specific for human cytomegalovirus (CMV) was evaluated in 14 patients infected with human immunodeficiency virus (HIV) receiving highly active antiretroviral therapy (HAART) and 26 CMV-seropositive donors without HIV infection. Fifty-seven percent of the HIV-infected group had CMV-specific cytolytic activity in freshly isolated peripheral blood mononuclear cells (PBMC) against targets expressing CMV pp65. Both interferon (IFN)-gamma secretion by CD8(+) T cells and the frequency of human leukocyte antigen (HLA)-tetramer-positive T cells in HLA-A*0201-positive HIV-infected subjects correlated with CMV-specific cytolysis. In contrast, PBMC from healthy CMV-seropositive donors did not have either measurable CMV-specific cytolysis or secretion of IFN-gamma without in vitro stimulation. The T helper response to CMV antigens was vigorous in healthy CMV-seropositive donors but low in the cohort of HIV-infected patients. Potent CD8(+) cytotoxic T lymphocyte responses to CMV in HIV-infected patients receiving HAART is the converse of what is found in healthy CMV-seropositive subjects and may be the predominant adaptive immune response against CMV in HIV-infected patients.     

Purified protein derivative-activated type 1 cytokine-producing CD4+ T lymphocytes in the lung: a characteristic feature of active pulmonary and nonpulmonary tuberculosis

Because tuberculosis (TB) is primarily a pulmonary disease, we examined the cytokine responses of CD4(+) T lymphocytes in bronchoalveolar lavage (BAL) fluid after incubation with purified protein derivative (PPD) in human immunodeficiency virus-negative patients with TB and control subjects with nontuberculous respiratory disease. Parallel blood and BAL fluid samples from each subject were incubated with or without PPD, and the proportions of CD4(+) T lymphocytes producing interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha were measured by flow cytometry. The proportions of PPD-activated IFN-gamma- and TNF-alpha-producing CD4(+) cells were low among control subjects (median, 0.33% and 0.78%, respectively). By contrast, among patients with TB, strong IFN-gamma and TNF-alpha responses were demonstrated (median, 24.0% and 32.4%, respectively), regardless of whether the TB was pulmonary or nonpulmonary. Measurement of type 1 cytokine production by CD4(+) T lymphocytes in response to PPD in BAL fluid is a promising new diagnostic test for active TB in immunocompetent individuals.     

Apoptosis and T cell hyporesponsiveness in pulmonary tuberculosis

Mycobacterium tuberculosis (MTB)-induced T cell responses are depressed in peripheral blood mononuclear cells of persons with newly diagnosed pulmonary tuberculosis (TB), and levels of interferon (IFN)-gamma remain low even after completion of antituberculous therapy. Loss of MTB-reactive T cells through apoptotic mechanisms could account for this prolonged T cell hyporesponsiveness. T cell apoptosis was studied in TB patients and healthy control subjects. Both spontaneous and MTB-induced apoptosis (in CD4 and non-CD4 T cells) from TB patients was increased when compared with healthy control subjects, whereas coculture with control antigen (candida) had no effect on T cell apoptosis in either group of study subjects. An inverse correlation existed between increased MTB-induced T cell apoptosis and IFN-gamma and interleukin (IL)-2 immunoreactivities. Successful antituberculous chemotherapy resulted in a 50% reduction in both spontaneous and MTB-induced apoptosis, which coincided with 3- and 8-fold increases in levels of MTB-stimulated IL-2 and IFN-gamma, respectively. These data indicate that apoptotic pathways are operant during active MTB infection and may contribute to deletion of MTB-reactive T cells and the immunopathogenesis of this disease.     

HIV-1-related pleural tuberculosis: elevated production of IFN-gamma, but failure of immunity to Mycobacterium tuberculosis

BACKGROUND: Pleural tuberculosis can resolve spontaneously, suggesting that the inflammatory process may represent a protective immune response. However, pleural tuberculosis is strongly associated with HIV infection. It has been suggested that cell-mediated immune responses may be reduced, and direct bacterial invasion may have a role in pathogenesis, in HIV-positive cases. To test this hypothesis, we compared production of the pro-inflammatory cytokines, interferon (IFN)-gamma and tumour necrosis factor(TNF)-alpha, production of the immunosuppressive cytokine, interleukin (IL)-10, and mycobacterial culture positivity, in HIV-negative and HIV-positive patients with pleural tuberculosis. METHODS: Cytokine levels were measured in serum and pleural fluid, and in supernatants of blood and pleural fluid stimulated in vitro using mycobacterial antigens. Intracellular IFN-gamma and TNF-alpha production was measured after stimulation with phorbol myristate acetate and ionomycin in vitro. RESULTS: IFN-gamma was strikingly elevated in serum and pleural fluid in HIV-positive, compared to HIV-negative subjects (P < or = 0.02). TNF-alpha was elevated, but this was not statistically significant. IL-10 levels were higher in serum (P < 0.001), but similar in pleural fluid. IFN-gamma responses to soluble mycobacterial antigen in vitro were reduced in peripheral blood (P = 0.006), but not pleural fluid, of HIV-positive subjects. Intracellular cytokine staining suggested that CD8+ T cells were a major source of IFN-gamma in HIV-positive subjects. The proportion of subjects with a positive culture for Mycobacterium tuberculosis from pleural fluid was higher in the HIV-positive group. CONCLUSIONS: HIV-positive patients with pleural tuberculosis show elevated production of IFN-gamma, for which CD8+ T cells may be a major source. Mycobacterium tuberculosis can proliferate despite high levels of pro-inflammatory cytokines.     

Assessment of immune function by lymphoproliferation underestimates lymphocyte functional capacity in HIV patients treated with highly active antiretroviral therapy

The objective of this study was to evaluate T cell responses in HIV-infected patients after highly active antiretroviral therapy (HAART), using four assays of immune function, and to determine which best reflects the presence of CD4(+) T cells able to respond to CMV antigen. Peripheral blood mononuclear cells from 41 HIVinfected patients and 31 healthy HIV-seronegative controls were cultured with mitogen (PMA/Ca(2+) ionophore) or antigen (CMV). Production of interferon gamma (IFN-gamma) determined by ELISpot assay was compared with lymphoproliferation, IFN-gamma production assessed by ELISA, and CD69 expression and intracellular IFN-gamma assessed by flow cytometry. Cells from patients whose CD4(+) T cells counts increased 4-fold or to >200 cells/microl after HAART responded as well as control cells when assessed by IFN-gamma production and CD69 expression after mitogenic stimulation, but lymphoproliferation responses were depressed by about 52%. Patients who did not meet these criteria for immune reconstitution had lymphoproliferative responses up to 30-fold lower than control subjects, while intracellular IFN-gamma and CD69 expression and ELISpot counts were less than 3-fold lower. Responses to CMV antigen could not be detected by flow cytometry, but were readily detected by ELISpot in CMV-seropositive patients whose CD4(+) T cell counts had increased after HAART. This included patients with low responses assessed by lymphoproliferation. Moreover, ELISpot responses measured with fresh and frozen cells were comparable, while lymphoproliferation assays required fresh cells. In conclusion, the ELISpot assay is a sensitive and efficient technique for detecting CMV-specific IFN-gamma responses in samples that display poor responses when assessed by lymphoproliferation assays.     

Vdelta2+ gammadelta T cell function in Mycobacterium tuberculosis- and HIV-1-positive patients in the United States and Uganda: application of a whole-blood assay

BACKGROUND: Vgamma9(+)Vdelta2(+) gammadelta T cells (Vdelta 2(+) T cells) are activated by Mycobacterium tuberculosis and secrete interferon (IFN)-gamma. Vdelta 2(+) T cells recognize phosphoantigens, such as bromohydrin pyrophosphate (BrHPP), and link innate and adaptive immunity. METHODS: A whole-blood assay was developed that used IFN-gamma secretion in response to BrHPP as a measurement of Vdelta2(+) T cell function. RESULTS: Peak IFN-gamma levels were detected after stimulating whole blood with BrHPP for 7-9 days. IFN- gamma production in whole blood in response to BrHPP paralleled IFN-gamma production and Vdelta2(+) T cell expansion of peripheral-blood mononuclear cells. The assay was used to evaluate Vdelta2(+) T cell function in subjects in the United States (n = 24) and Uganda (n = 178) who were or were not infected with M. tuberculosis and/or human immunodeficiency virus (HIV) type 1. When 50 micromol/L BrHPP was used, 100% of healthy subjects produced IFN-gamma. The Vdelta2(+) T cell response was independent of the tuberculin skin test response. In Uganda, Vdelta2(+) T cell responses were decreased in patients with tuberculosis (n = 73) compared with responses in household contacts (n = 105). HIV-1-positive household contacts had lower responses than did HIV-1-negative household contacts. HIV-1-positive patients with tuberculosis had the lowest V delta 2(+) T cell responses. CONCLUSIONS: Tuberculosis and HIV-1 infection are associated with decreased Velta2(+) T cell function. Decreased Vdelta2(+) T cell function may contribute to increased risk for tuberculosis in HIV-1-positive patients.     

Impaired phenotype and function of monocyte derived dendritic cells in pulmonary tuberculosis

Pulmonary tuberculosis (PTB) is often associated with impaired immunological functions. Blood monocytes, which can differentiate into dendritic cells upon cytokine stimulation, play a central role in adequate immune reactivity. Here, we investigated the morphologic, phenotypic and functional characteristics of in vitro-generated monocyte derived dendritic cells (MoDC) from PTB patients in comparison with healthy subjects. Phenotypic analysis revealed a defective differentiation of MoDC in PTB patients as assessed by a strong down regulation of CD1a, MHC II, CD80 and CD83 expression and impaired allostimulatory function under the influence of IL-4 and GM-CSF. In contrast, the expression of CD86 was not affected and remained same as in healthy subjects. Furthermore, the maturation status of lipopolysaccharide (LPS) stimulated MoDC was not optimal in PTB. However, the MoDC of PTB patients produced significantly higher levels of TNF-alpha and IL-6 but lower levels of IL-12 compared to healthy subjects. These findings suggest that there is a fundamental defect in the differentiation and maturation of dendritic cells during PTB that may compromise the antigen presentation and subsequent immune functions.     

结核分枝杆菌肝素结合血凝黏附素与结核病

结核分枝杆菌肝素结合血凝黏附素是一种表面蛋白,有3个功能结构域,其C末端在翻译后进行甲基化修饰,与其免疫特性相关.可结合硫酸化糖,可通过补体C3介导与巨噬细胞的结合,与上皮细胞结合在肺外结核发病中有重要作用.通过对其细胞免疫及体液免疫作用的研究发现,其在诊断中有较好的作用,在免疫预防及免疫治疗中有广阔的应用前景.     

Increased levels of circulating interleukin-18 in patients with advanced tuberculosis

Interleukin-18 (IL-18) has recently been identified as an interferon- gamma-inducing factor and it plays an important role in the Th1 response. We measured serum levels of IL-18 and interferon-gamma (IFN-gamma) in 43 patients with pulmonary tuberculosis and 25 healthy control subjects. Significantly increased levels of circulating IL-18 and IFN-gamma were found in pulmonary tuberculosis as compared with those in healthy control subjects. Circulating IL-18 and IFN-gamma correlated with the extent of disease in pulmonary tuberculosis. We found significantly increased levels of circulating IL-18 and IFN-gamma in the patients with high-grade fever. Circulating IL-18 significantly correlated with circulating IFN-gamma. IL-18 may play an important role in immune response to human infection with Mycobacterium tuberculosis.     

Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis

Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live bacterial vaccine. However, limited information is available correlating route and dose of vaccination and induction of specific T cell responses with protection against tuberculosis. We compared efficacy of oral and systemic vaccination and correlated vaccine-induced T cell responses with protection in experimental tuberculosis of mice. After oral and systemic vaccination, we observed profound differences in persistence and dissemination of BCG and frequencies and location of specific IFN-gamma-secreting CD4(+) and CD8(+) T cells. Yet, both vaccination routes caused comparable levels of protection against aerosol challenge with Mycobacterium tuberculosis. Protection correlated best with rapid accumulation of specific CD8(+) T cells in infected tissues of challenged mice. In contrast, specific IFN-gamma production by CD4(+) T cells reflected the load of M. tuberculosis rather than the strength of protection. Our data question the measurement of IFN-gamma secretion by CD4(+) T cells and emphasize the need for new biomarkers for evaluation of tuberculosis vaccine efficacies.     

Influence of HLA-DRB1 alleles on Th1 and Th2 cytokine response to Mycobacterium tuberculosis antigens in pulmonary tuberculosis

The influence of human leukocyte antigens (HLA) on the immune response is well established. We investigated the regulatory role of HLA-DRB1 alleles on cytokine response to live M. tuberculosis and its culture filtrate antigen (CFA) in normal healthy subjects (NHS) and pulmonary tuberculosis (PTB) patients. Th1 (IFN-gamma and IL-12p40), Th2 (IL-4 and IL-5), pro-inflammatory (IL-6 and IL-8) and anti-inflammatory (TGF-beta and IL-10) cytokines were measured by ELISA in 72-h-old peripheral blood mononuclear cell culture supernatants from 58 NHS and 48 PTB patients. HLA-DRB1 genotyping was carried out by polymerase chain reaction and dot-blot hybridization with biotinylated sequence-specific oligonucleotide probes and detection by chemiluminescence. In response to live M. tuberculosis and CFA, significantly increased levels of IL-6, IL-8 and TGF-beta and decreased IFN-gamma, IL-12p40 and IL-10 were seen in PTB patients compared to NHS. We observed a significantly increased IFN-gamma response in HLA-DRB1*03-positive NHS (p=0.03) and decreased IFN-gamma response in HLA-DRB1*15-positive patients (p=0.04) than respective allele-negative individuals. An increased level of IL-12p40 in DRB1*10 (p=0.02) and IL-10 in DRB1*12- (p=0.03) positive NHS and an increased level of IL-6 in DRB1*04- (p=0.02) positive patients were observed. The study suggests that HLA-DRB1 alleles differentially modulate the various cytokine responses to M. tuberculosis antigens, which may influence the cellular and humoral immune responses to M. tuberculosis infection in a susceptible host.