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BACKGROUND: Approximately 5% of patients with drug-susceptible tuberculosis have a relapse after 6 months of first-line therapy, as do approximately 20% of patients after 4 months of short-course therapy. We postulated that by analyzing pretreatment isolates of Mycobacterium tuberculosis obtained from patients who subsequently had a relapse or were cured, we could determine any correlations between the minimum inhibitory concentration (MIC) of a drug below the standard resistance breakpoint and the relapse risk after treatment. METHODS: Using data from the Tuberculosis Trials Consortium Study 22 (development cohort), we assessed relapse and cure isolates to determine the MIC values of isoniazid and rifampin that were below the standard resistance breakpoint (0.1 μg per milliliter for isoniazid and 1.0 μg per milliliter for rifampin). We combined this analysis with clinical, radiologic, and laboratory data to generate predictive relapse models, which we validated by analyzing data from the DMID 01–009 study (validation cohort). RESULTS: In the development cohort, the mean (±SD) MIC of isoniazid below the breakpoint was 0.0334±0.0085 μg per milliliter in the relapse group and 0.0286±0.0092 μg per milliliter in the cure group, which represented a higher value in the relapse group by a factor of 1.17 (P=0.02). The corresponding MIC values of rifampin were 0.0695±0.0276 and 0.0453±0.0223 μg per milliliter, respectively, which represented a higher value in the relapse group by a factor of 1.53 (P

Background Human genetic susceptibility for tuberculosis (TB) has been demonstrated by several studies, but few have examined multiple innate and adaptive immunity genes comprehensively, age-specific effects, and/or resistance to Mycobacterium tuberculosis (Mtb) infection (RSTR). We hypothesized that RSTR, defined by a persistently negative tuberculin skin test, may have different genetic influences than Mtb disease. Methods We examined 29 candidate genes in pathways that mediate immune responses to Mtb in subjects in a household contact study in Kampala, Uganda. We genotyped 546 haplotype-tagging single nucleotide polymorphisms (SNPs) in 835 individuals from 481 families; 28.7% had TB, 10.5% were RSTR, and the remaining 60.8% had latent Mtb infection. Results Among our most significant findings were SNPs in TICAM2 (p=3.6×10−6) and IL1B (p=4.3×10−5) associated with TB. Multiple SNPs in IL4 and TOLLIP were associated with TB (p

SUMMARY SETTING: Patients with smear-positive, newly diagnosed pulmonary tuberculosis (TB) presenting to the out- patient TB clinic in Kampala, Uganda. OBJECTIVE: To compare colony-forming unit (cfu) counting and time to positive (TTP) in Mycobacteria Growth Indicator Tube (MGIT) culture as measures of early bactericidal activity (EBA). DESIGN: Patients were enrolled in an EBA feasibility study of standard TB chemotherapy. Sixteen-hour over- night sputum collections were obtained before and on days 2, 4, 7, 10, 12 and 14 of treatment for quantitative culture on selective Middlebrook 7H11 agar media and TTP in the MGIT liquid culture system. RESULTS: Log cfu and TTP were correlated over all time points (rs = −0.71, P < 0.001). Within-subject (day to day) variation as a percentage of total variation was very similar between the two measures: 25.7% for cfu and 25% for TTP. Mean EBA 0-14, 0-2 and 2-14 mea- sured by TTP were similar to those previously reported. CONCLUSION: TTP measured by an automated, stan- dardized, commercially available culture system corre- lates with cfu determinations. EBA measured by TTP provides similar information to cfu counting, and is re- producible across sites and in different patient popula- tions. These findings support replacing cfu counting with TTP as the primary measurement in EBA studies.

(See the editorial commentary by Von Reyn, on pages 817–9.) The epidemics of tuberculosis and human immunodeficiency virus type 1 (HIV) infection converged with the greatest intensity in sub-Saharan Africa. Both the incidence and the prevalence of tuberculosis increased as the HIV infection epidemic swept through the continent. In Africa today, tuberculosis may affect up to 30% of coinfected persons [1] and is the leading cause of death. Even with effective antituberculosis treatment, case fatality rates for tuberculosis may be as high as 20%, especially in the face of severe immunosuppression [2, 3]. Moreover, tuberculosis may accelerate the clinical course of HIV infection and lead to poor outcomes [4–7]. Newly revised guidelines from the World Health Organization (WHO) recommend the use of antiretroviral therapy for all HIV-seropositive persons with tuberculosis disease, regardless of their CD4+ T-cell count [8]. Recent evidence indicates that antiretroviral therapy should be started early in the course of tuberculosis treatment because it improves survival [9, 10]. Although there is abundant information on the benefit of antiretroviral therapy in HIV-infected tuberculosis patients with advanced immunosuppression [11–13], there are fewer data on the optimal management of tuberculosis among patients with CD4+ T-cell counts of >350 cells/μL. The present study was designed in the early years of the antiretroviral therapy rollout in Africa to determine whether a 6-month course of antiretroviral therapy given concurrently with tuberculosis treatment would improve the clinical, immunologic, and virologic outcomes among patients with CD4+ T-cell counts of >350 cells/μL. The rationale of the study was that a punctuated course of antiretroviral therapy in patients with high CD4+ T-cell counts would suppress viral replication during therapy for tuberculosis, block the effects of immune activation on T cells harboring HIV, slow the pace of HIV disease progression, and improve clinical outcomes. Although treatment interruption is no longer a viable option given recent advances in the field of HIV infection management [14], the present study provides evidence of clinical benefit when starting antiretroviral therapy in tuberculosis patients with preserved immunity.

Although neutrophils have been identified as sources of inflammatory cytokines and chemokines, little is known about their immunologic function during mycobacterial infection in the lungs. In this study, we examined the growth ofMycobacterium bovisBCG in the lungs under experimental conditions that altered neutrophil recruitment to the lungs. Depletion and recruitment of neutrophils was associated with respective increases and decreases inM. bovisBCG growth. Thus, neutrophils may enhance mycobacteriocidal immunity in the lungs.

Testing new drugs is critical to improving the treatment of tuberculosis. Quantitative cultures of Mycobacterium tuberculosis on solid media have been used in Phase 1 and 2 trials, but are time and resource intensive. Time to detection (TTD) of growth of M. tuberculosis in automated liquid culture systems is an alternative. TTD has been shown to correlate with CFU in quantitative cultures, and is faster and simpler to perform. We compared TTD in the BACTEC 460 liquid culture system with CFU in a clinical trial that included 110 subjects. Comparing all sputum cultures collected between baseline and 2 months we found a strong negative correlation between log(10) CFU and TTD (rho = -0.91). In addition, when TTD at baseline was compared with 1 and 2 month sputum culture positivity, subjects whose cultures were negative after 1 and 2 months had a significantly longer median baseline TTD compared with subjects whose cultures were positive at 1 and 2 months (5 vs. 3 days and 3 vs. 2 days, respectively). TTD compares closely with CFU and represents a faster, simpler alternative to quantitative cultures.

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor β [TGF-β]) cytokines. IL-10 and TGF-β are produced by M. tuberculosis -infected macrophages. The effect of IL-10 and TGF-β on M. tuberculosis -reactive human CD4 + and γδ T cells, the two major human T-cell subsets activated by M. tuberculosis , was investigated. Both IL-10 and TGF-β inhibited proliferation and gamma interferon production by CD4 + and γδ T cells. IL-10 was a more potent inhibitor than TGF-β for both T-cell subsets. Combinations of IL-10 and TGF-β did not result in additive or synergistic inhibition. IL-10 inhibited γδ and CD4 + T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4 + T cells and, to a lesser extent, for γδ T cells. TGF-β inhibited both CD4 + and γδ T cells directly and had little effect on APC function for γδ and CD4 + T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis -infected monocytes to a greater extent than TGF-β. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-β both inhibited CD4 + and γδ T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis .

Mycobacterium tuberculosis readily activates both CD4 + and Vδ2 + γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis -infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and γδ T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2 + γδ T cells. For γδ T cells, recognition of M. tuberculosis -infected monocytes was dependent on Vδ2 + T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4 + T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vδ2 + γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4 + T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4 + and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4 + T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4 + and γδ T cells.

Background Nutritional changes during and after tuberculosis treatment have not been well described. We therefore determined the effect of wasting on rate of mean change in lean tissue and fat mass as measured by bioelectrical impedance analysis (BIA), and mean change in body mass index (BMI) during and after tuberculosis treatment. Methods In a prospective cohort study of 717 adult patients, BMI and height-normalized indices of lean tissue (LMI) and fat mass (FMI) as measured by BIA were assessed at baseline, 3, 12, and 24 months. Results Men with wasting at baseline regained LMI at a greater rate than FMI (4.55 kg/m2 (95% confidence interval (CI): 1.26, 7.83 versus 3.16 (95% CI: 0.80, 5.52)) per month, respectively during initial tuberculosis therapy. In contrast, women with wasting regained FMI at greater rate than LMI (3.55 kg/m2 (95% CI: 0.40, 6.70) versus 2.07 (95% CI: -0.74, 4.88)), respectively. Men with wasting regained BMI at a rate of 6.45 kg/m2 (95% CI: 3.02, 9.87) in the first three months whereas women, had a rate of 3.30 kg/m2 (95% CI: -0.11, 6.72). There were minimal changes in body composition after month 3 and during months 12 to 24. Conclusion Wasted tuberculosis patients regain weight with treatment but the type of gain differs by gender and patients may remain underweight after the initial phase of treatment.

OBJECTIVE To determine the proportion of recurrent tuberculosis (TB) due to relapse with the patient's initial strain or reinfection with a new strain of Mycobacterium tuberculosis 1-2 years after anti-tuberculosis treatment in Uganda, a sub-Saharan TB-endemic country. DESIGN Records of patients with culture-confirmed TB who completed treatment at an urban Ugandan clinic were reviewed. Restriction fragment length polymorphism (RFLP) patterns were used to determine relapse or reinfection. Associations between human immunodeficiency virus (HIV) positivity and type of TB recurrence were determined. RESULTS Of 1701 patients cured of their initial TB episode with a median follow-up of 1.24 years, 171 (10%) had TB recurrence (8.4 per 100 person-years). Rate and risk factors for recurrence were similar to other studies from sub-Saharan Africa. Insertion sequence (IS) 6110-based RFLP of paired isolates from 98 recurrences identified 80 relapses and 18 reinfections. Relapses among HIV-positive and -negative patients were respectively 79% and 85% of recurrences. CONCLUSIONS Relapse was more common and presented earlier than reinfection in both HIV-positive and -negative TB patients 1-2 years after completing treatment. These findings impact both the choice of retreatment drug regimen, as relapsing patients are at higher risk for acquired drug resistance, and clinical trials of new TB regimens with relapse as clinical endpoint.

The newly identified cytokine, IL-15 enhanced antigen-induced proliferation of PBMC obtained from HIV-1-seropositive subjects. When compared to IL-2 which enhanced both spontaneous and antigen-induced lymphocyte proliferative responses, IL-15 rarely increased spontaneous lymphocyte proliferation. Additionally, in cultures of lymphocytes obtained from 15 HIV-1-infected patients with < 300 circulating CD4- lymphocytes/microliter IL-15 induced significant HIV-1 expression (46, 21, and 71 pg/ml) in only 3 of 15 experiments and IL-2 induced significant HIV-1 expression (range 16- > 5000 pg/ml) in 11 of 15 experiments (P < 0.01, Fischer's exact test). Simultaneous assays of cytokine-induced spontaneous lymphocyte proliferation and HIV-1 expression revealed similar dose-response relationships for induction of HIV-1 and lymphocyte proliferation by IL-2. Thus, IL-15 helps to correct the impaired proliferative response of CD4+ lymphocytes from HIV-1-infected persons without the mitogenic effect of IL-2 that also may induce HIV-1 expression.

Mycobacterium tuberculosis and its antigens are potent inducers of cytokine expression by mononuclear phagocytes. In this study, the ability of live M. tuberculosis to stimulate interleukin-12 (IL-12) expression by human monocytes was examined. Monocytes were purified from peripheral blood mononuclear cells by adherence and either infected with M. tuberculosis or exposed to soluble protein antigens of M. tuberculosis (purified protein derivative [PPD]). Live M. tuberculosis (10(6) to 10(7) CFU/ml) was a potent stimulus for interleukin-12 (IL-12). By using reverse transcription-PCR, p40 mRNA was detected at 3 h, peaked at 6 to 12 h, and decayed to baseline levels at 18 to 24 h following infection. Bioactive IL-12 (p70) was measured by the phytohemagglutinin blast proliferation assay and confirmed the p40 mRNA results. In contrast, soluble PPD at concentrations known to readily induce IL-1 and tumor necrosis factor alpha expression by monocytes (10 to 100 microg/ml) was a poor stimulus for IL-12 p40 mRNA expression. The different efficiencies of M. tuberculosis bacilli and PPD for IL-12 expression by monocytes was in part due to a requirement for phagocytosis. Induction of IL-12 in response to M. tuberculosis was reduced by cytochalasin D. Furthermore, phagocytosis of dead M. tuberculosis or inert 2-micron-diameter polystyrene beads by monocytes induced IL-12 p40 mRNA. In contrast, 0.5-micron-diameter beads, which can enter cells through pinocytosis, did not stimulate IL-12 expression. Functionally, IL-12 readily enhanced PPD-stimulated IFN-gamma production and CD4+ T-cell-mediated cytotoxicity by peripheral blood mononuclear cells from healthy tuberculin-positive donors but induced less enhancement when live M. tuberculosis was the antigen. These results suggest that IL-12 is upregulated as part of the early cytokine response of mononuclear phagocytes to M. tuberculosis and that the cellular events associated with phagocytosis are themselves a potent signal for IL-12 production. IL-12 released by infected macrophages in turn can further upregulate M. tuberculosis-specific CD4+ T-cell effector function.

BALB/c mice inoculated with live Brugia malayi microfilariae (mf), or immunized with a soluble filarial extract (B. malayi Ag (BmA)), develop a pronounced Th2-like response over time. In contrast, single or repeated immunizations with a soluble Mycobacterium tuberculosis Ag preparation (purified protein derivative, PPD) stimulates a Th1, but not Th2 response (IFN-gamma > IL-4, IL-5). To determine if the Th1 response to PPD can be modulated by the ongoing helminth-induced Th2 activity, mice were: 1) immunized simultaneously with BmA and PPD; 2) immunized first with BmA, then with PPD; or 3) inoculated with live mf and immunized with PPD at various times thereafter. Simultaneous immunization with both Ag had no effect on the Th response induced by PPD, i.e., it was strictly Th1. In contrast, establishment of a Th2 response by either inoculation of live mf or immunization with BmA before administration of PPD skewed the PPD-specific Th response such that IL-4 and IL-5 were produced in addition to IFN-gamma. IL-4 and IL-5 levels produced in response to PPD under these conditions were further elevated in vitro in the presence of neutralizing IFN-gamma. Finally, in vivo neutralization of IL-4 diminished induction of Th2 responses to PPD. These results demonstrate that ongoing Th2 responses to helminth Ag modulate the Th response to mycobacterial Ag by an IL-4 dependent mechanism.

The T cell-mediated acquired immune response to infection with Mycobacterium tuberculosis, both in humans and in experimental models in the mouse, is a complex event believed to involve a variety of T cell subsets that manifest themselves in numerous functions, including protection, delayed-type hypersensitivity, cytolysis, and the establishment of a state of memory immunity. These functions in turn involve the secretion of an array of cytokines, several of which direct cells of the monocyte/macrophage axis to contain and destroy the invading bacilli. This article reviews the development of these ideas, both from clinical experience and from basic research in animal models. In addition, the newly emerging hypothesis that the secreted or export proteins of M. tuberculosis are the key protective antigens leading to the initial expression of acquired specific resistance to this organism is examined.

The role of specific Th subsets in the regulation of acquired resistance to the filarial parasite Brugia malayi is not known. We examined pathologic and cytokine responses in filarial antigen-sensitized BALB/c mice inoculated intraperitoneally with live microfilariae. Animals immunized three times with soluble microfilarial antigen demonstrated accelerated clearance of live parasites (12 +/- 5% of parasites recovered from the peritoneal cavity 4 days after inoculation vs 57 +/- 6% in controls, P0.001). Elimination of microfilariae by immunized mice was associated with local eosinophilia (1.5 x 10(7) eosinophils/ml peritoneal wash fluid compared with 2 x 10(5) eosinophils/ml in unimmunized animals), development of local eosinophil-containing granulomas, and elevated serum IgE levels (7.0 +/- 1.4 vs 2.1 +/- 0.9 micrograms/ml in controls, P0.01). CD4+ cells from the site of parasite challenge produced Th2-associated cytokines exclusively (IL-4 and IL-5, not IFN-gamma and IL-2) in response to Brugia antigen, whereas spleen and lymph node cells produced both Th1- and Th2-associated cytokines. Mice immunized a single time with microfilarial antigen did not clear parasites in this time, and peritoneal exudate cells from these animals produced IFN-gamma but not IL-5. These results indicate that acquired resistance to B. malayi microfilariae in mice is associated with induction of a Th2 and not a Th1 response at the site of parasite elimination.

We have developed a novel method for screening a Mycobacterium bovis (BCG) cosmid library in Mycobacterium smegmatis for the detection of immunostimulatory T-cell antigens (Ag). Distinctive protein banding patterns were demonstrated in culture filtrates of three of 30 recombinant M. smegmatis clones: pBCCS13 (41 and 73 kDa); pBCCS221 (30, 50 and 68 kDa); pBCCS223 (100 kDa). Western immunoblots indicated that monoclonal antibodies (mAb) directed to the previously characterized 19-, 30-, 38-, 65- and 71-kDa mycobacterial Ag were not reactive with the distinctive recombinant proteins. Furthermore, T-cell Western blots demonstrated that fractions containing the distinctive proteins were immunostimulatory. A given tuberculin-positive donor expressed unique patterns of blastogenic reactivity to protein fractions isolated from each of the three recombinant clones. Restriction enzyme digests of the three recombinant BCG inserts revealed distinctive DNA-banding patterns. The immunostimulatory Ag, therefore, are most likely encoded within different regions of the BCG genome, as contained within three distinct inserts. T-cell Western blots further indicated a heterogeneity in the repertoire of BCG-responsive T cells since tuberculin-positive donors varied in the pattern of reactivity to protein fractions isolated from the same recombinant filtrate. Most likely, immunity to M. tuberculosis results from activation of a heterogeneous array of T cells targeted to multiple immunostimulatory Ag. The method we describe should greatly enhance our ability to define the full spectrum of T-cell Ag encoded by mycobacteria, particularly those which are secreted proteins.

gamma delta T cells, both human and murine, have been found to be highly responsive to mycobacterial antigens. However, the role and function of gamma delta T cells in the immune response to Mycobacterium tuberculosis remain largely unknown. In earlier studies, we demonstrated that monocytes infected with live M. tuberculosis were particularly effective inducers of human peripheral blood gamma delta T cells. The present studies were performed to further characterize the interaction between human mononuclear phagocytes, gamma delta T cells, and live M. tuberculosis, in comparison with CD4+ T cells. First, we found that resting gamma delta T cells expanded in vitro by live M. tuberculosis were specific for M. tuberculosis, and that heat killing and washing the mycobacteria removed the antigen(s) for gamma delta T cells. In contrast, the heat-killed mycobacteria retained significant antigenicity for CD4+ T cells. Second, live M. tuberculosis-expanded gamma delta T cells from healthy tuberculin-positive donors did not respond significantly to the antigens in M. tuberculosis culture filtrate, including the 65- and 71-kDa mycobacterial heat shock proteins. Third, the activation of gamma delta T cells by live mycobacteria was dependent on antigen-presenting cells, and mononuclear phagocytes were found to be very efficient antigen-presenting cells both for resting peripheral blood gamma delta T cells and for activated expanded gamma delta T cells. The mononuclear phagocyte carried the necessary costimulatory factors necessary for gamma delta T-cell proliferation. Fourth, the antigen repertoire and HLA requirements for CD4+ memory T cells and those for gamma delta T cells appear to be quite distinct from each other. CD4+ T cells recognized both soluble protein antigens and whole organisms in a class II major histocompatibility complex-restricted manner, whereas gamma delta T cells appeared to recognize only constituents associated with the whole organism and were not restricted by class I or class II major histocompatibility complex molecules. Finally, the assay system described to expand and purify responding CD4+ and gamma delta T cells after stimulation with live M. tuberculosis represented a simple approach to the direct comparison of these two T-cell populations in the interaction with mononuclear phagocytes infected with M. tuberculosis. Such studies provide insight not only into the relative roles of human CD4+ and gamma delta T cells in the human immune response to intracellular bacterial pathogens such as M. tuberculosis but also into the basic biologic role of human gamma delta T cells in antimicrobial immunity.

mRNA is a marker of cell viability. Quantifying Mycobacterium tuberculosis mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of M. tuberculosis viability.

CD4+ T cells regulate the protective immune response which follows exposure to Mycobacterium tuberculosis by activating macrophages through the cytokines the CD4+ T cells secrete. In addition CD4+ T cells have been shown to be directly cytotoxic for antigen-pulsed mononuclear phagocytes (monocytes-macrophages). To explore the functional interaction between mycobacterial antigen-specific CD4+ T cells and mononuclear phagocytes further, CD4+ T-cell clones were derived from healthy purified protein derivative-positive individuals. Five T-cell clones were selected for detailed analysis. None responded to the purified recombinant or native mycobacterial antigens of 14, 19, 65, 71, and 30 (alpha-antigen/Ag6) kDa. However, the T-cell clones demonstrated heterogeneity in antigen recognition as measured by their Western blot (immunoblot) responses. Some T-cell clones made only interleukin 2, while others made only interleukin 4; all produced gamma interferon, although in differing amounts. Four of five T-cells clones were cytotoxic for purified protein derivative-pulsed monocytes at 1:1 and 10:1 effector-target cell ratios. When monocytes infected with live M. tuberculosis were used as targets, comparable levels of cytotoxicity were observed. The cytotoxicity was major histocompatibility complex class II restricted and inhibited by antibodies to ICAM-1 and LFA-1 and not by antibodies to tumor necrosis factor alpha, lymphotoxin, and gamma interferon. Cytotoxicity by CD4+ T cells for monocytes pulsed with mycobacterial antigens or infected with live M. tuberculosis is a common property of these cells and appears to be independent of the repertoire of lymphokines produced and not limited to recognition of defined mycobacterial heat shock proteins. Lysis of heavily infected mononuclear phagocytes may be one manner in which CD4+ T cells regulate host immune response to M. tuberculosis.

Susceptibility or resistance to infection with Leishmania major correlates with the ability of mice to produce characteristic panels of lymphokines in response to the parasite. To investigate the role of antigen-presenting cells in this phenomenon, we developed a model system which used congenic (H-2d) susceptible and resistant mice. L. major-specific T cells were isolated from infected BALB/c and B10.D2 mice, and the cells were restimulated in vitro on syngenic or congenic antigen-presenting cells. BALB/c L. major-reactive T cells restimulated with either antigen-presenting cell produced high levels of interleukin-4 and low levels of gamma interferon. In contrast, T cells from B10.D2 mice produced gamma interferon. Radiation-induced chimeras reconstituted with BALB/c bone marrow also produced more interleukin-4 in response to L. major than did chimeras reconstituted with B10.D2 bone marrow. To test whether this pattern of cytokine secretion was unique to infection with L. major, we infected the mice with a second intracellular pathogen, Mycobacterium bovis BCG. Mycobacterium-specific T cells from both BALB/c and B10.D2 mice produced interleukin-2 and no interleukin-4. Finally, when BALB/c mice were vaccinated with avirulent L. major, the induced resistance correlated with reduced production of interleukin-4 but no increase in gamma interferon production. Instead, T cells from the vaccinated mice produced high levels of tumor necrosis factor. This suggests that tumor necrosis factor, in addition to gamma interferon, may be involved in resistance to L. major and that interleukin-4 may inhibit the leishmanicidal activity of tumor necrosis factor and/or gamma interferon.

Murine CD4+ T cell clones have been classified into at least two subsets, Th1 and Th2, on the basis of their distinct lymphokine secretion profiles and functions. In the present study, we compared the functional responses of Th1 and Th2 clones to Ag presentation by splenic B cells and peritoneal macrophages. Th2 clones secreted IL-4 in response to Ag presented by resting B cells, but their optimal proliferation required the addition of IL-1 or a source of IL-1. The degree of IL-1 dependence varied among the four Th2 clones examined. In contrast, Th1 clones secreted IL-2 and proliferated in response to Ag presented by both B cells and macrophages, without any requirement for exogenous IL-1. Furthermore, the proliferation of Th2 clones in response to Ag presented by splenocytes or macrophages was inhibited by an IL-1R antagonist. These results indicate that IL-1 is an important costimulator for the expansion of the Th2 subset of CD4+ T cells. The different requirements for the proliferation of Th1 and Th2 cells may be responsible for the preferential expansion of one or the other subset under different conditions of immunization.

To test the hypothesis that resting and previously activated B lymphocytes differ in their proliferative and differentiative responses to various Th cell-derived stimuli, we have examined the interactions of purified small (resting) and large (activated) murine B cells with rabbit Ig-specific Th1 and Th2 clones in the presence of the Ag analogue, rabbit anti-mouse Ig antibody. Small numbers of Th2 cells induce strong Ag-dependent proliferation of and Ig secretion by both resting and activated B lymphocytes. In contrast, Th1 clones stimulate lower responses of activated B cells and fail to stimulate small resting B cells. An interaction with Th1 clones does make small B cells responsive to the Th2-derived cytokine, IL-4, indicating that Th1 clones are capable of delivering some but not all the stimuli necessary for the induction of humoral immunity. Finally, in order to compare the responses of small and large B cells to cognate interactions and secreted cytokines, we used an autoreactive I-Ak-specific Th2 line. This line induces proliferation of and Ig secretion by I-Ak expressing but not H-2d resting and activated B cells as a result of cognate interactions. However, when the H-2d B cells are bystanders in the presence of cytokine secretion by this Th2 line, or are directly exposed to Th2-derived cytokines, both small and large B cells are induced to proliferate but only the large B cells secrete antibody. These results indicate that the magnitude and nature of antibody responses depend on three principal factors: the cytokines produced by Th cells, the state of activation of the responding B lymphocytes, and whether the B cells are recipients of cognate help or are bystanders at the site of T cell stimulation. Our findings also confirm the view that cognate T-B interactions are most efficient for initiating B cell responses and may allow B cells to subsequently respond to a variety of T cell-derived cytokines.

Voluntary counseling and testing (VCT) for the human immunodeficiency virus (HIV) is recommended for persons treated for tuberculosis (TB). Opportunities to diagnose HIV may be missed by limiting HIV testing to only persons diagnosed with TB. Among TB suspects in Uganda, we determined HIV prevalence, risk behaviors, and willingness to refer family for VCT.Consenting adult patients presenting for evaluation at a referral TB clinic received same-day VCT. TB diagnosis data were abstracted from clinical records.Among 665 eligible patients, 565 (85%) consented to VCT. Among these, 238 (42%) were HIV-positive. Of the HIV-infected patients, 37% had received a non-TB diagnosis. HIV seroprevalence was higher in patients with a non-TB diagnosis (49%) than those diagnosed with TB (39%) (P = 0.02). Fewer than 6% of HIV-infected patients reported always using condoms with sexual partners. The majority of patients (86%) reported being 'very willing' to refer family members for VCT.Over 35% of HIV-infected cases in our population would have been undetected if HIV testing was limited to cases with diagnosed TB. The high HIV seroprevalence in both TB and non-TB cases merits HIV testing for all patients evaluated at TB clinics. HIV-infected TB suspects reporting high-risk behavior are at risk for HIV transmission, and should receive risk-reduction counseling.

To evaluate the early bactericidal activity (EBA) of the new fluoroquinolones levofloxacin, gatifloxacin and moxifloxacin in patients with pulmonary tuberculosis (PTB).Randomized, open-label trial. Forty adults with newly diagnosed smear-positive PTB (10 per arm) were assigned to receive isoniazid (INH) 300 mg, levofloxacin 1000 mg, gatifloxacin 400 mg, or moxifloxacin 400 mg daily for 7 days. Sputum for quantitative culture was collected for 2 days before and daily during 7 days of monotherapy. Bactericidal activity was estimated by measuring the decline in bacilli during the first 2 days (EBA 0-2) and last 5 days of monotherapy (extended EBA, EBA 2-7). Laboratory staff were blinded to treatment assignment.The EBA 0-2 of INH (0.67 log10 cfu/ml/day) was greater than that of moxifloxacin and gatifloxacin (0.33 and 0.35 log10 cfu/ml/day, respectively), but not of levofloxacin 1000 mg daily (0.45 log10 cfu/ml/day) (P = 0.14). Bactericidal activity between days 2 and 7 was similar for all three fluoroquinolones. In a pooled comparison, the EBA 2-7 of the fluoroquinolones was greater than for INH.Moxifloxacin, gatifloxacin, and high-dose levofloxacin have excellent EBA, only slightly less than for INH, and greater extended EBA. These drugs warrant further study in the treatment of drug-susceptible TB.

Summary Active tuberculosis (TB) is associated with prolonged suppression of Mycobacterium tuberculosis (MTB)-specific immune responses, but mechanisms involved are understood incompletely. We investigated a potential role for CD4+CD25+ regulatory T cells in depressed anti-MTB immunity by evaluating serially CD4 cell phenotype and interferon (IFN)-γ production by mononuclear cells from patients with TB. At diagnosis, frequencies of CD4+CD25+ T cells were increased in blood from TB patients compared to healthy purified protein derivative (PPD)-positive controls (with a history of prior TB exposure), and remained elevated at completion of therapy (6 months). By contrast, expression of another activation marker, CD69, by CD4 T cells was increased at diagnosis, but declined rapidly to control levels with treatment. Among CD4+CD25+ T cells from TB patients at diagnosis those expressing high levels of CD25, probably representing regulatory T cells, were increased 2·9-fold when compared to control subjects, while MTB-stimulated IFN-γ levels in whole blood supernatants were depressed. A role for CD4+CD25+ T cells in depressed IFN-γ production during TB was substantiated in depletion experiments, where CD25+-depleted CD4 T cells produced increased amounts of IFN-γ upon MTB stimulation compared to unseparated T cells. At follow-up, IFN-γ production improved most significantly in blood from TB patients with high baseline frequencies of CD4+CD25+ T cells (more than threefold higher than controls for both total and CD25hi+ CD4 T cells), who also had a significant drop in frequencies of both total and ‘regulatory’ CD4+CD25+ T cells in response to treatment. Expansion of CD4+CD25+ regulatory T cells during active TB may play a role in depressed T cell IFN-γ production.

A hallmark of M. tuberculosis infection is the ability of most (90-95%) healthy adults to control infection through acquired immunity, in which antigen specific T cells and macrophages arrest growth of M. tuberculosis bacilli and maintain control over persistent bacilli. In addition to CD4+ T cells, other T cell subsets such as, gammadelta, CD8+ and CD1-restricted T cells have roles in the immune response to M. tuberculosis. A diverse T cell response allows the host to recognize a wider range of mycobacterial antigens presented by different families of antigen-presenting molecules, and thus greater ability to detect the pathogen. Macrophages are key antigen presenting cells for T cells, and M. tuberculosis survives and persists in this central immune cell. This is likely an important factor in generating this T cell diversity. Furthermore, the slow growth and chronic nature of M. tuberculosis infection results in prolonged exposure to antigens, and hence further T cell sensitization. The effector mechanisms used by T cells to control M. tuberculosis are poorly understood. To survive in macrophages, M. tuberculosis has evolved mechanisms to block immune responses. These include modulation of phagosomes, neutralization of macrophage effector molecules, stimulating the secretion of inhibitory cytokines, and interfering with processing of antigens for T cells. The relative importance of these blocking mechanisms likely depends on the stage of M. tuberculosis infection: primary infection, persistence, reactivation or active tuberculosis. The balance of the host-pathogen interaction in M. tuberculosis infection is determined by the interaction of T cells and infected macrophages. The outcome of this interaction results either in control of M. tuberculosis infection or active disease. A better understanding of this interaction will result in improved approaches to treatment and prevention of tuberculosis.

The Th-2 response to microfilariae of Brugia malayi in BALB/c mice is associated with peritoneal eosinophilia. These eosinophils had an activated hypodense phenotype in vivo and expressed high levels of the class II major histocompatibility complex (MHC). Interleukin 4 up-regulated the class II MHC, whereas gamma interferon inhibited class II MHC expression on eosinophils.

Certain microbial substances, e.g., LPS, can activate neutrophils or prime them to enhance their response to other activating agents, e.g., fMLP. We investigated the role of the Mycobacterium tuberculosis (MTB) 19-kDa lipoprotein in activation of human neutrophils. MTB 19-kDa lipoprotein initiated phenotypic changes characteristic of neutrophil activation, including down-regulation of CD62 ligand (L-selectin) and up-regulation of CD35 (CR1) and CD11b/CD18 (CR3, Mac-1). In addition, exposure of neutrophils to MTB 19-kDa lipoprotein enhanced the subsequent oxidative burst in response to fMLP as assessed by oxidation of dihydrorhodamine 123 (determined by flow cytometry). LPS also produced these effects with similar kinetics, but an oligodeoxynucleotide containing a CpG motif failed to induce any priming or activation response. Although the effects of LPS required the presence of serum, neutrophil activation by MTB 19-kDa lipoprotein occurred independently of serum factors, suggesting the involvement of different receptors and signaling mechanisms for LPS and MTB 19-kDa lipoprotein. Thus, MTB 19-kDa lipoprotein serves as a pathogen-associated molecular pattern that promotes neutrophil priming and activation.

Despite the continued importance of tuberculosis as a world-wide threat to public health, little is known about the mechanisms used by human lymphocytes to contain and kill the intracellular pathogenMycobacterium tuberculosis. We previously described an in vitro model of infection of human monocytes (MN) with virulentM. tuberculosisstrain H37Rv in which the ability of peripheral blood lymphocytes to limit intracellular growth of the organism could be measured. In the current study, we determined that lymphocyte-mediated killing of intracellularM. tuberculosisoccurs within the first 24 h of coculture with infected MN. Natural killer (NK) cells isolated from both purified protein derivative (PPD)-positive and PPD-negative subjects were capable of mediating this early killing of intracellular H37Rv. NK cell-mediated killing of intracellularM. tuberculosiswas not associated with the production of gamma interferon. Transferred supernatants of cocultured NK cells andM. tuberculosis-infected MN could not mediate the killing of intracellularM. tuberculosis, and Transwell studies indicated that direct cell-to-cell contact was required for NK cells to mediate the killing of the organism. Killing was not dependent upon exocytosis of NK cell cytotoxic granules. NK cells induced apoptosis of mycobacterium-infected MN, but neither killing of intracellularM. tuberculosisby NK cells nor NK cell-induced apoptosis of infected MN was inhibited by blocking the interaction of FasL and Fas. Thus, human NK cells may mediate killing of intracellularM. tuberculosisvia alternative apoptotic pathways.

To characterize changes in serum cytokine levels in human immunodeficiency virus type 1 (HIV-1)-infected persons withMycobacterium aviumcomplex (MAC) bacteremia, the levels of IL-1α (interleukin-1α), IL-6, IL-10, tumor necrosis factor alpha (TNF-α), soluble type II TNF receptor (sTNF-RII), and transforming growth factorβ(TGF-β) in serum were measured in two cohorts of HIV-1-infected persons with MAC bacteremia. The first cohort was part of a MAC prophylaxis study. Patients with bacteremia were matched with controls without bacteremia. Elevated IL-6, IL-10, TNF-α, sTNF-RII, and TGF-β levels were noted at baseline for all subjects, a result consistent with advanced HIV-1 disease. IL-1α was not detected. No differences in cytokine levels in serum were noted at baseline and at the time of bacteremia between patients with MAC and controls. In the second cohort, subjects had serum samples collected at the time of MAC bacteremia and thereafter while on macrolide therapy. Serum samples at time of bacteremia were collected from HIV-1-infected persons at a time when neither highly active antiretroviral therapy (HAART) nor MAC prophylaxis was used routinely. MAC treatment resulted in decreased levels of IL-6 and TNF-α in serum, which were evident for IL-6 by 4 to 6 weeks and for TNF-α by 8 to 16 weeks. Thus, antibiotic treatment for MAC results in decreased levels of IL-6 and TNF-α in serum in HIV-1-infected persons who are not on HAART.

Latency-associated peptide of transforming growth factor β (TGF-β) (LAP) was used to determine whether in vivo modulation of TGF-β bioactivity enhanced pulmonary immunity toMycobacterium bovisBCG infection in C57BL/6 mice. LAP decreased BCG growth in the lung and enhanced antigen-specific T-cell proliferation and gamma interferon mRNA expression. Thus, susceptibility of the lung to primary BCG infection may be partially mediated by the immunosuppressive effects of TGF-β.

Since the first descriptions of mycobacterial reactivity for gammadelta T cells in 1989, studies of gammadelta T-cell responses to M. tuberculosis in humans and animal models have increased our understanding of the complex role(s) of this T-cell subset not only in the immune response to M. tuberculosis, but also to microbial pathogens in general. Although CD4+ T cells remain the dominant and critical T-cell subset in protection against M. tuberculosis, gammadelta T cells appear to have an important complementary role, which may be primarily expressed in and around maturing granulomas. This is a difficult area to study in humans. Gammadelta T cells are potent sources of IFN-gamma and competent cytotoxic effector cells, but differ from CD4+ T cells in the antigens they recognize and the manner in which M. tuberculosis-infected macrophages process and present antigens to these two subsets. One of the most fascinating features of Vgamma9/Vgamma2+ gammadelta T cells is their responsiveness to non-peptidic molecules. Solving the mechanism(s) of antigen recognition and presentation of these molecules to gammadelta T cells should help determine whether gammadelta T cells are responding to universal 'supernatigen'-like motifs expressed by a broad range of microbes or in fact discriminate among a diversity of peptidic and nonpeptidic microbial antigens. Enhanced understanding of the function of and antigen recognition by Vgamma9+/Vgamma2+ T cells is not only important for immunity to M. tuberculosis but also for T-cell responses to microbial pathogens in general.

Mycobacterium tuberculosis bacilli readily activate CD4(+) and gammadelta T cells. CD4(+) and gammadelta T cells were compared for their ability to regulate IFN-gamma, TNF-alpha, and IL-10 production, cytokines with significant roles in the immune response to M. tuberculosis. PBMC from healthy tuberculin positive donors were stimulated with live M. tuberculosis-H37Ra. CD4(+) and gammadelta T cells were purified by negative selection and tested in response to autologous monocytes infected with M. tuberculosis. Both subsets produced equal amounts of secreted IFN-gamma. However, the precursor frequency of IFN-gamma secreting gammadelta T cells was half that of CD4(+) T cells, indicating that gammadelta T cells were more efficient producers of IFN-gamma than CD4(+) T cells. TNF-alpha production was markedly enhanced by addition of CD4(+) and gammadelta T cells to M. tuberculosis infected monocytes, and TNF-alpha was produced by both T cells and monocytes. No differences in TNF-alpha enhancement were noted between CD4(+) and gammadelta T cells. IL-10 production by M. tuberculosis infected monocytes was not modulated by CD4(+) or gammadelta T cells. Thus CD4(+) and gammadelta T cells had similar roles in differential regulation of IFN-gamma, TNF-alpha, and IL-10 secretion in response to M. tuberculosis infected monocytes. However, the interaction between T cells and infected monocytes differed for each cytokine. IFN-gamma production was dependent on antigen presentation and costimulators provided by monocytes. TNF-alpha levels were increased by addition of TNF-alpha produced by T cells and IL-10 production by monocytes was not modulated by CD4(+) or gammadelta T cells.

Human immune responses to M. tuberculosis are characterized by activation of multiple T cell subsets including CD4+, CD8+, and gammadelta T cells, and the role of CD8+ alphabeta TCR+ T cells in this response is poorly understood. Stimulation of T cells from healthy tuberculin skin test-positive persons with live M. tuberculosis-H37Ra or soluble M. tuberculosis Ags readily up-regulated IL-2Ralpha (CD25) expression on CD8+ T cells. Purified resting and activated CD8+ T cells produced IFN-gamma and proliferated to both M. tuberculosis bacilli and soluble mycobacterial Ags with monocytes as APC. Precursor frequency of mycobacterial Ag-specific CD8+ T cells by IFN-gamma enzyme-linked immunospot was 5-10-fold lower than the precursor frequency of CD4+ T cells, and IFN-gamma secretion by CD8+ T cells was 50-100-fold lower. CD8+ T cells secreted approximately 10-fold less IFN-gamma per cell than CD4+ T cells in response to mycobacterial Ags. CD8+ T cell responses to M. tuberculosis bacilli were blocked by anti-MHC class I antibody and required Ag processing. Processing of M. tuberculosis bacilli by monocytes for presentation to MHC class I-restricted CD8+ T cells was insensitive to brefeldin A treatment, which blocks the conventional MHC class I Ag-processing pathway. These results represent the first demonstration that human cells can process pathogen Ags via an alternate Ag-processing pathway for MHC class I and suggest a mechanism for participation of IFN-gamma-secreting CD8+ T cells in the human immune responses to M. tuberculosis.

Regulation of interleukin (IL)-12 production by coexpression of tumor necrosis factor (TNF)-alpha, IL-10, and transforming growth factor (TGF)-beta in human monocytes infected with Mycobacterium tuberculosis H37Ra was analyzed. Also, since IL-12 induces interferon (IFN)-gamma, the effect of IFN-gamma on IL-12 expression was examined. IL-12 mRNA was measured by reverse transcriptase-polymerase chain reaction and IL-12 protein by ELISA. IL-12 p35 mRNA was constitutive and inducible. IL-12 p70 protein paralleled IL-12 p40 protein expression. TNF-alpha protein expression occurred earlier than IL-12 p40 protein but was not required for IL-12 induction. Addition or neutralization of TGF-beta did not significantly alter IL-12 induction. In contrast, recombinant IL-10 reduced IL-12 and neutralization of IL-10 minimally enhanced IL-12. A pronounced increase in IL-12 followed IFN-gamma pretreatment, which selectively up-regulated IL-12 p35 mRNA. Further understanding of operative cytokine networks during M. tuberculosis infection may improve strategies for vaccine development and immunotherapy.

Protective human immunity to Mycobacterium tuberculosis (M. tb) has proven difficult to characterize, in part because of technical obstacles to in vitro infection of human cells with virulent M. tb. We established a reproducible method of infecting human monocytes (MN) with the virulent M. tb strain H37Rv that did not reduce MN viability. TNF-alpha had no effect on replication of H37Rv within MN, and IFN-gamma mediated only a 1.9-fold reduction in bacterial growth. In contrast, nonadherent cells (NAC) from purified protein derivative (PPD)-positive and PPD-negative subjects reduced intracellular growth of H37Rv by 6- and 10.6-fold, respectively (p = 0.007 and p = 0.005). CD4+ T cells were essential to growth inhibition mediated by NAC of PPD-positive subjects, whereas containment of M. tb by NAC of PPD-negative subjects did not require CD4+ cells. CD8+ T cells did not contribute to protection mediated by NAC of either group. Supernatants of cocultured H37Rv-infected MN and NAC only partially reduced intracellular growth of M. tb despite containing nanogram concentrations of TNF-alpha and IFN-gamma. Neutralizing antibodies to TNF-alpha, IFN-gamma, and IL-12 failed to affect the NAC-mediated growth limitation. NAC treated with emetine retained approximately 40% of their capacity to contain intracellular H37Rv, however. These studies indicate that protective human recall responses to M. tb are mediated primarily by CD4+ T cells, whereas CD4-CD8- lymphocytes may contribute to innate immunity to M. tb. The ability of NAC to activate M. tb-infected MN is only partly attributable to soluble mediators and may also involve contact-mediated mechanisms.

T cell-mediated cytotoxicity against Mycobacterium tuberculosis (MTB)-infected macrophages may be a major mechanism of specific host defense, but little is known about such activities in the lung. Thus, the capacity of alveolar lymphocyte MTB-specific cell lines (AL) and alveolar macrophages (AM) from tuberculin skin test-positive healthy subjects to serve as CTL and target cells, respectively, in response to MTB (H37Ra) or purified protein derivative (PPD) was investigated. Mycobacterial Ag-pulsed AM were targets of blood CTL activity at E:T ratios ofor = 30:1 (51Cr release assay), but were significantly more resistant to cytotoxicity than autologous blood monocytes. PPD- plus IL-2-expanded AL and blood lymphocytes were cytotoxic for autologous mycobacterium-stimulated monocytes at E:T ratios ofor = 10:1. The CTL activity of lymphocytes expanded with PPD was predominantly class II MHC restricted, whereas the CTL activity of lymphocytes expanded with PPD plus IL-2 was both class I and class II MHC restricted. Both CD4+ and CD8+ T cells were enriched in BL and AL expanded with PPD and IL-2, and both subsets had mycobacterium-specific CTL activity. Such novel cytotoxic responses by CD4+ and CD8+ T cells may be a major mechanism of defense against MTB at the site of disease activity.

Vdelta2 gammadelta T cells are readily activated by microbial antigens. In persons infected with human immunodeficiency virus type 1 (HIV-1), the number of gammadelta T cells remains the same or increases in association with reversal of the Vdelta2/Vdelta1 ratio fromor = 1 to1. Vdelta2 T cell responses to microbial antigens were tested in 11 HIV-1-infected (or = 500 CD4 cells/mm3) and 7 uninfected persons. In persons with HIV-1 infection, Mycobacterium tuberculosis expanded Vdelta2 cells in 1 person as did Salmonella typhimurium in 4; however, Candida albicans antigens did not lead to more Vdelta2 cells. Vdelta2 responses to M. tuberculosis were enhanced by interleukin (IL)-2 in HIV-1-infected persons (from 1 subject to 7; P.01) and were associated with increased interferon-gamma production. Bacterial antigens and IL-2 increased HIV-1 replication; M. tuberculosis antigens induced the greatest increase. Thus, in HIV-1-infected persons withor = 500 CD4 cells/mm3, Vdelta2 T cell responses to bacterial antigens remain intact.

The 30-kDa secreted antigen of Mycobacterium tuberculosis was a strong inducer of tumor necrosis factor alpha in human monocytes. Our findings suggest that tumor necrosis factor alpha production may be up-regulated at both the posttranscriptional and transcriptional levels. Regulation at the posttranscriptional level probably reflects enhanced translational efficiency.

Alveolar macrophages form the first line of defense against inhaled droplets containing Mycobacterium tuberculosis by controlling mycobacterial growth and regulating T cell responses. CD4+ and gamma delta T cells, two major T cell subsets activated by M. tuberculosis, require accessory cells for activation. However, the ability of alveolar macrophages to function as accessory cells for T cell activation remains controversial. We sought to determine the ability of alveolar macrophages to serve as accessory cells for resting (HLA-DR-, IL-2R-) and activated (HLA-DR+, IL-2R+) gamma delta T cells in response to M. tuberculosis and its Ag, and to compare accessory cell function for gamma delta T cells of alveolar macrophages and blood monocytes obtained from the same donor. Alveolar macrophages were found to serve as accessory cells for both resting and activated gamma delta T cells in response to M. tuberculosis Ag. At high alveolar macrophage to T cell ratios (3:1), however, expansion of resting gamma delta T cells was inhibited by alveolar macrophages. The inhibition of resting gamma delta T cells by alveolar macrophages was dose-dependent, required their presence during the first 24 h, and was partially overcome by IL-2. Alveolar macrophages did not inhibit activated gamma delta T cells even at high accessory cell to T cell ratios, and alveolar macrophages functioned as well as monocytes as accessory cells. Monocytes were not inhibitory for either resting or activated gamma delta T cells. These findings support the following model. In the normal alveolus the alveolar macrophage to T cell ratio isor = 9:1, and therefore the threshold for resting gamma delta T cell activation is likely to be high. Once a nonspecific inflammatory response occurs, such as after invasion by M. tuberculosis, this ratio is altered, favoring gamma delta T cell activation by alveolar macrophages.

CD4+ and gamma delta T cells are activated readily by Mycobacterium tuberculosis. To examine their role in the human immune response to M. tuberculosis, CD4+ and gamma delta T cells from healthy tuberculin-positive donor were studied for patterns of Ag recognition, cytotoxicity, and cytokine production in response to M. tuberculosis-infected mononuclear phagocytes. Both T cell subsets responded to intact M. tuberculosis and its cytosolic Ags. However, CD4+ and gamma delta T cells differed in the range of cytosolic Ags recognized: reactivity to a wide m.w. range of Ags for CD4+ T cells, and a restricted pattern for gamma delta T cells, with dominance of Ags of 10 to 15 kDa. Both T cell subsets were equally cytotoxic for M. tuberculosis-infected monocytes. Furthermore, both CD4+ and gamma delta T cells produced large amounts of IFN-gamma: mean pg/ml of IFN-gamma in supernatants was 2458 +/- 213 for CD4+ and 2349 +/- 245 for gamma delta T cells. By filter-spot ELISA (ELISPOT), the frequency of IFN-gamma-secreting gamma delta T cells was one-half of that of CD4+ T cells in response to M. tuberculosis, suggesting that gamma delta T cells on a per cell basis were more efficient producers of IFN-gamma than CD4+ T cells. In contrast, CD4+ T cells produced more IL-2 than gamma delta T cells, which correlated with diminished T cell proliferation of gamma delta T cells compared with CD4+ T cells. These results indicate that CD4+ and gamma delta T cell subsets have similar effector functions (cytotoxicity, IFN-gamma production) in response to M. tuberculosis-infected macrophages, despite differences in the Ags recognized, IL-2 production, and efficiency of IFN-gamma production.

gamma delta T-cell receptor-bearing T cells (gamma delta T cells) are readily activated by intracellular bacterial pathogens such as Mycobacterium tuberculosis. The bacterial antigens responsible for gamma delta T-cell activation remain poorly characterized. We have found that heat treatment of live M. tuberculosis bacilli released into the supernatant an antigen which stimulated human gamma delta T cells. gamma delta T-cell activation was measured by determining the increase in percentage of gamma delta T cells by flow cytometry in peripheral blood mononuclear cells stimulated with antigen and by proliferation of gamma delta T-cell lines with monocytes as antigen-presenting cells. Supernatant from heat-treated M. tuberculosis was fractionated by fast-performance liquid chromatography (FPLC) on a Superose 12 column. Maximal gamma delta T-cell activation was measured for a fraction of 10 to 14 kDa. Separation of the supernatant by preparative isoelectric focusing demonstrated peak activity at a pI of < 4.0. On two-dimensional gel electrophoresis, the 10- to 14-kDa FPLC fraction contained at least seven distinct molecules, of which two had a pI of < 4.5. Protease treatment reduced the bioactivity of the 10- to 14-kDa FPLC fraction for both resting and activated gamma delta T cells. Murine antibodies raised to the 10- to 14-kDa fraction reacted by enzyme-linked immunosorbent assay with antigens of 10 to 14 kDa in lysate of M. tuberculosis. In addition, gamma delta T cells proliferated in response to an antigen of 10 to 14 kDa present in M. tuberculosis lysate. gamma delta T-cell-stimulating antigen was not found in culture filtrate of M. tuberculosis but was associated with the bacterial pellet and lysate of M. tuberculosis. These results provide a preliminary characterization of a 10- to 14-kDa, cell-associated, heat-stable, low-pI protein antigen of M. tuberculosis which is a major stimulus for human gamma delta T cells.

The purpose of this study was to examine the murine T-helper-cell (Th) cytokine response to the human filarial parasite Brugia malayi. In the first 14 days following intraperitoneal inoculation of live microfilariae into BALB/c mice, filarial antigen-driven splenic lymphoid cells produced gamma interferon (IFN-gamma) and little or no interleukin-5 (IL-5). After this time, IL-5 production increased (to 10 to 12 ng per 5 x 10(6) cells) coincident with a marked diminution in IFN-gamma generation. A single subcutaneous immunization with soluble microfilarial antigens also induced an IFN-gamma but no IL-5 response, whereas immunization three times elicited a predominant Th2-like reaction characterized by IL-4 and IL-5 production by CD4+ lymph node lymphocytes and a 10-fold increase in serum immunoglobulin E. The importance of IL-10 in establishing the balance between parasite-specific Th1 and Th2 responses was demonstrated by the ability of neutralizing monoclonal antibody to this cytokine to increase IFN-gamma production by splenic and lymph node cells from mice chronically exposed to live microfilariae or immunized multiple times with soluble filarial antigens.

Gamma delta (gamma delta) T cell receptor (TCR) expressing T cells comprise 3% of human peripheral blood lymphocytes, yet their role in the immune response remains largely unknown. There is evidence both in humans and in animal models that these cells participate in the immune response to mycobacterial antigens. In mice, exposure to mycobacterial antigens leads to the expansion of gamma delta T cells in draining lymph nodes and lungs. In humans, gamma delta T cell lines with reactivity to mycobacterial antigens have been derived from synovial fluid of a rheumatoid arthritis patient, skin lesions of leprosy patients, and peripheral blood of a healthy tuberculin reactor. Very little is known, however, about the factors which induce human gamma delta T cells to expand. In studies comparing the human T cell response to live and heat-killed Mycobacterium tuberculosis (MT), we have found that monocytes infected with live MT are very effective inducers of human gamma delta T cell expansion. After 7 d of exposure to live MT, gamma delta T cells were greatly increased in all healthy tuberculin reactors (PPD+) tested and frequently were the predominant T cell population. In contrast, heat-killed MT or purified protein products of MT induced a CD4+, alpha beta TCR+ T cell response with very little increase in gamma delta T cells. Furthermore, a similar selective induction of gamma delta T cells was observed when monocytes infected with live Salmonella were used to stimulate T cells. Heat-killed Salmonella, like heat-killed MT, induced a predominantly CD4+ alpha beta TCR+ T cell response. These findings suggest that human gamma delta T cells are a major reactive T cell population during the early stages of infection with living intracellular bacteria and are therefore likely to exert an important role in the initial interaction between host and parasite.

Little is known about the immunodominant or protective antigens of Mycobacterium tuberculosis in humans. Cell-mediated immunity is necessary for protection, and healthy tuberculin-positive individuals are relatively resistant to exogenous reinfection. We compared the targets of the cell-mediated immune response in healthy tuberculin-positive individuals to those of tuberculosis patients and tuberculin-negative persons. By using T-cell Western blotting (immunoblotting) of nitrocellulose-bound M. tuberculosis culture filtrate, peaks of T-cell blastogenic activity were identified in the healthy tuberculin reactors at 30, 37, 44, 57, 64, 71 and 88 kDa. Three of these fractions (30, 64, and 71 kDa) coincided with previously characterized proteins: antigen 6/alpha antigen, HSP60, and HSP70, respectively. The blastogenic responses to purified M. tuberculosis antigen 6/alpha antigen and BCG HSP60 were assessed. When cultured with purified antigen 6/alpha antigen, lymphocytes of healthy tuberculin reactors demonstrated greater [3H]thymidine incorporation than either healthy tuberculin-negative controls or tuberculous patients (8,113 +/- 1,939 delta cpm versus 645 +/- 425 delta cpm and 1,019 +/- 710 delta cpm, respectively; P less than 0.01). Healthy reactors also responded to HSP60, although to a lesser degree than antigen 6/alpha antigen (4,276 +/- 1,095 delta cpm; P less than 0.05). Partially purified HSP70 bound to nitrocellulose paper elicited a significant lymphocyte blastogenic response in two of six of the tuberculous patients but in none of the eight healthy tuberculin reactors. Lymphocytes of none of five tuberculin-negative controls responded to recombinant antigens at 14 or 19 kDa or to HSP70. Antibody reactivity generally was inversely correlated with blastogenic response: tuberculous sera had high titer antibody to M. tuberculosis culture filtrate in a range from 35 to 180 kDa. This is the first systematic evaluation of the human response to a panel of native and recombinant antigens in healthy tuberculin reactors and tuberculous patients. Antigens which stimulated prominent lymphocyte blastogenic responses were identified in seven fractions on T-cell Western blot analysis. Two of these may represent previously characterized proteins; the others may contain immunodominant proteins that will require further characterization.