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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. [ABSTRACT FROM AUTHOR]

Background: The search for immune correlates of protection against Mycobacterium tuberculosis (MTB) infection in humans is limited by the focus on peripheral blood measures. Bronchoalveolar lavage (BAL) can safely be done and provides insight into cellular function in the lung where infection is first established. In this study, blood and lung samples were assayed to determine if heavily MTB exposed persons who resist development of latent MTB infection (RSTR) vs those who develop latent MTB infection (LTBI), differ in the make-up of resident BAL innate and adaptive immune cells., Methods: Bronchoscopy was performed on 21 healthy long-term Ugandan RSTR and 25 LTBI participants. Immune cell distributions in BAL and peripheral blood were compared by differential cell counting and flow cytometry., Results: The bronchoscopy procedure was well tolerated with few adverse reactions. Differential macrophage and lymphocyte frequencies in BAL differed between RSTR and LTBI. When corrected for age, this difference lost statistical significance. BAL CD4+ and CD8+ T cells were almost entirely composed of effector memory T cells in contrast to PBMC, and did not differ between RSTR and LTBI. BAL NKT, γδ T cells and NK cells also did not differ between RTSR and LTBI participants. There was a marginally significant increase (p = 0.034) in CD8 T effector memory cells re-expressing CD45RA (TEMRA) in PBMC of LTBI vs RSTR participants., Conclusion: This observational case-control study comparing unstimulated BAL from RSTR vs LTBI, did not find evidence of large differences in the distribution of baseline BAL immune cells. PBMC TEMRA cell percentage was higher in LTBI relative to RSTR suggesting a role in the maintenance of latent MTB infection. Functional immune studies are required to determine if and how RSTR and LTBI BAL immune cells differ in response to MTB., Competing Interests: The authors have declared that no competing interests exist.

Despite widespread use of the Bacillus Calmette-Guerin vaccine, tuberculosis, caused by infection with Mycobacterium tuberculosis , remains a leading cause of morbidity and mortality worldwide. As CD8 + T cells are critical to tuberculosis host defense and a phase 2b vaccine trial of modified vaccinia Ankara expressing Ag85a that failed to demonstrate efficacy, also failed to induce a CD8 + T cell response, an effective tuberculosis vaccine may need to induce CD8 + T cells. However, little is known about CD8, as compared to CD4, antigens in tuberculosis. Herein, we report the results of the first ever HLA allele independent genome-wide CD8 antigen discovery program. Using CD8 + T cells derived from humans with latent tuberculosis infection or tuberculosis and an interferon-γ ELISPOT assay, we screened a synthetic peptide library representing 10% of the Mycobacterium tuberculosis proteome, selected to be enriched for Mycobacterium tuberculosis antigens. We defined a set of immunodominant CD8 antigens including part or all of 74 Mycobacterium tuberculosis proteins, only 16 of which are previously known CD8 antigens. Immunogenicity was associated with the degree of expression of mRNA and protein. Immunodominant antigens were enriched in cell wall proteins with preferential recognition of Esx protein family members, and within proteins comprising the Mycobacterium tuberculosis secretome. A validation study of immunodominant antigens demonstrated that these antigens were strongly recognized in Mycobacterium tuberculosis -infected individuals from a tuberculosis endemic region in Africa. The tuberculosis vaccine field will likely benefit from this greatly increased known repertoire of CD8 immunodominant antigens and definition of properties of Mycobacterium tuberculosis proteins important for CD8 antigenicity., Competing Interests: COMPETING INTERESTS Aeras TB Vaccine Foundation, a non-profit organization, has licensed technology from OHSU of which Drs. David and Deborah Lewinsohn are inventors and which is used in this research. In addition, OHSU and Drs. David Lewinsohn, Deborah Lewinsohn, and Melissa Nyendak have a financial interest in, and Ms. Cansler, Ms. Null, and Ms. Swarbrick are employees of, ViTi, a company that may have a commercial interest in the results of this research and technology. These potential individual and institutional conflicts of interest have been reviewed and managed by OHSU.

Nitazoxanide (NTZ) and its metabolite tizoxanide (TIZ) were studied as antimycobacterial agents in vitro (in mycobacterial growth indicator tube [MGIT] cultures) and in a whole blood bactericidal assay. Both NTZ and TIZ show high protein binding. In MGIT cultures (albumin concentration = 78 μM), inhibition of Mycobacterium tuberculosis growth occurred at total drug concentrations of ≥16 μg/ml, whereas in whole blood cultures (albumin concentration = 350 μM), ≥128 μg/ml was required. Free drug fractions at these two conditions were estimated to be 69% and 2%, respectively. Co-incubation of NTZ and TIZ in human plasma for 72 h nearly completely eliminated their ability to inhibit mycobacterial growth in MGIT. Interactions with plasma proteins may limit the potential of NTZ and TIZ as drugs for human tuberculosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Rationale: Healthy household contacts (HHC) of individuals with Tuberculosis (TB) with Tuberculin Skin Test (TST) conversions are considered to harbor latent Mycobacterium tuberculosis (Mtb), and at risk for TB. The immunologic, clinical, and public health implications of TST reversions that occur following Isoniazid preventive therapy (IPT) remain controversial., Objectives: To measure frequency of TST reversion following IPT, and variation in interferon-gamma (IFN-γ) responses to Mtb, in healthy Ugandan TB HHC with primary Mtb infection evidenced by TST conversion., Methods: Prospective cohort study of healthy, HIV-uninfected, TST-negative TB HHC with TST conversions. Repeat TST was performed 12 months following conversion (3 months following completion of 9 month IPT course) to assess for stable conversion vs. reversion. Whole blood IFN-γ responses to Mtb antigen 85B (MtbA85B) and whole Mtb bacilli (wMtb) were measured in a subset (n = 27 and n = 42, respectively) at enrollment and TST conversion, prior to initiation of IPT., Results: Of 122 subjects, TST reversion was noted in 25 (20.5%). There were no significant differences in demographic, clinical, or exposure variables between reverters and stable converters. At conversion, reverters had significantly smaller TST compared to stable converters (13.7 mm vs 16.4 mm, respectively; p = 0.003). At enrollment, there were no significant differences in IFN-γ responses to MtbA85B or wMTB between groups. At conversion, stable converters demonstrated significant increases in IFN-γ responses to Ag85B and wMtb compared to enrollment (p = 0.001, p<0.001, respectively), while there were no significant changes among reverters., Conclusions: TST reversion following IPT is common following primary Mtb infection and associated with unique patterns of Mtb-induced IFN-γ production. We have demonstrated that immune responses to primary Mtb infection are heterogeneous, and submit that prospective longitudinal studies of cell mediated immune responses to Mtb infection be prioritized to identify immune phenotypes protective against development of TB disease.

Contacts of active pulmonary tuberculosis (TB) patients are at risk for Mycobacterium tuberculosis (MTB) infection. Because most infections are controlled, studies during MTB infection provide insight into protective immunity. We compared immune responses of adult household contacts that did and did not convert the tuberculin skin test (TST). Innate and adaptive immune responses were measured by whole blood assay. Responses of TST converters (TSTC) were compared with persistently TST negative contacts (PTST-) and contacts who were TST+ at baseline (TST+). TLR-2, TLR-4, and IFN-γR responses to IFN-γ did not differ between the groups, nor did γδ T cell responses. T cell responses to MTB antigens differed markedly among TSTC, PTST-, and TST+ contacts. Thus, no differences in innate responses were found among the three household contact groups. However, adaptive T cell responses to MTB antigens did differ before and during MTB infection among PTST-, TSTC, and TST+ contacts.

Background: Human immunodeficiency virus (HIV)-tuberculosis coinfection is associated with heightened immune activation, viral replication, and T cell dysfunction. We compared changes in T cell activation and function between patients receiving concurrent treatment for HIV-tuberculosis coinfection and those receiving treatment for tuberculosis alone., Methods: HIV-infected adults with tuberculosis and CD4(+) T cell counts >350 cells/mm(3) were randomized to receive tuberculosis treatment alone (control arm; n = 36) or 6 months of antiretroviral therapy (ART) concurrent with tuberculosis treatment (intervention arm; n = 38). HIV viral load, T cell subsets, T cell activation, and cytokine production were measured at enrollment and every 3 months for 12 months., Results: Differences in absolute CD4(+) and CD8(+) T cell counts were not observed between arms. Viral load was reduced while participants received ART; control patients maintained viral load at baseline levels. Both arms had significant reductions in T cell expression of CD38 and HLA-DR. Interferon-γ production in response to mitogen increased significantly in the intervention arm., Conclusions: In HIV-infected adults with tuberculosis and CD4(+) T cell counts >350 cells/mm(3), both tuberculosis treatment and concurrent HIV-tuberculosis treatment reduce T cell activation and stabilize T cell counts. Concurrent ART with tuberculosis treatment does not provide additional, sustained reductions in T cell activation among individuals with preserved immunologic function.

Background: Both HIV and TB cause a state of heightened immune activation. Immune activation in HIV is associated with progression to AIDS. Prior studies, focusing on persons with advanced HIV, have shown no decline in markers of cellular activation in response to TB therapy alone., Methodology: This prospective cohort study, composed of participants within a larger phase 3 open-label randomized controlled clinical trial, measured the impact of TB treatment on immune activation in persons with non-advanced HIV infection (CD4>350 cells/mm3) and pulmonary TB. HIV load, CD4 count, and markers of immune activation (CD38 and HLA-DR on CD4 and CD8 T cells) were measured prior to starting, during, and for 6 months after completion of standard 6 month anti-tuberculosis (TB) therapy in 38 HIV infected Ugandans with smear and culture confirmed pulmonary TB., Results: Expression of CD38, and co-expression of CD38 and HLA-DR, on CD8 cells declined significantly within 3 months of starting standard TB therapy in the absence of anti-retroviral therapy, and remained suppressed for 6 months after completion of therapy. In contrast, HIV load and CD4 count remained unchanged throughout the study period., Conclusion: TB therapy leads to measurable decreases in immune activation in persons with HIV/TB co-infection and CD4 counts>350 cells/mm3.

To characterize changes in serum cytokine levels in human immunodeficiency virus type 1 (HIV-1)-infected persons with Mycobacterium avium complex (MAC) bacteremia, the levels of IL-1alpha (interleukin-1alpha), IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), soluble type II TNF receptor (sTNF-RII), and transforming growth factor beta (TGF-beta) 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-alpha, sTNF-RII, and TGF-beta levels were noted at baseline for all subjects, a result consistent with advanced HIV-1 disease. IL-1alpha 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-alpha in serum, which were evident for IL-6 by 4 to 6 weeks and for TNF-alpha by 8 to 16 weeks. Thus, antibiotic treatment for MAC results in decreased levels of IL-6 and TNF-alpha in serum in HIV-1-infected persons who are not on HAART.

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.

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 from > or = 1 to < 1. 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 with > or = 500 CD4 cells/mm3, Vdelta2 T cell responses to bacterial antigens remain intact.

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.

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.

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.

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.