Keith A. Chervenak

Selective expansion of human gamma delta T cells by monocytes infected with live Mycobacterium 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.

Linkage and association analysis of candidate genes for TB and TNFα cytokine expression: evidence for association with IFNGR1, IL-10, and TNF receptor 1 genes

Tuberculosis (TB) is a growing public health threat globally and several studies suggest a role of host genetic susceptibility in increased TB risk. As part of a household contact study in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB by developing an intermediate phenotype model for TB susceptibility, analyzing levels of tumor necrosis factor-α (TNFα) in response to culture filtrate as the phenotype. In the present study, we analyzed candidate genes related to TNFα regulation and found that interleukin (IL)-10, interferon-gamma receptor 1 (IFNGR1), and TNFα receptor 1 (TNFR1) genes were linked and associated to both TB and TNFα. We also show that these associations are with progression to active disease and not susceptibility to latent infection. This is the first report of an association between TB and TNFR1 in a human population and our findings for IL-10 and IFNGR1 replicate previous findings. By observing pleiotropic effects on both phenotypes, we show construct validity of our intermediate phenotype model, which enables the characterization of the role of these genetic polymorphisms on TB pathogenesis. This study further illustrates the utility of such a model for disentangling complex traits.

Effect of Potent Antiretroviral Therapy on Immune Responses to Mycobacterium avium in Human Immunodeficiency Virus—Infected Subjects

To characterize the influence of highly active antiretroviral therapy (HAART) on cell-mediated immunity (CMI) to Mycobacterium avium complex (MAC), we measured immune responses to M. avium in human immunodeficiency virus (HIV)-infected individuals before and during HAART, in subjects with a history of disseminated MAC (DMAC), and in HIV-uninfected control subjects. Forty-seven percent of untreated HIV-infected patients and 78% of control subjects exhibited in vitro proliferative responses to M. avium (P=.03). Proliferative responses to M. avium increased after HAART for 3 months and were present in 77% of subjects after 6 months. Mean interferon-gamma production increased from 199 to 1156 pg/mL after HAART (P=.06). Proliferative responses to M. avium occurred in 76% of DMAC subjects receiving HAART. CD4 and CD8 but not gammadelta T cells expanded in response to M. avium. CMI to M. avium reconstitutes rapidly after HAART and appears sustained even with partial viral suppression.

Innate and Adaptive Immune Responses during Acute M. tuberculosis Infection in Adult Household Contacts in Kampala, Uganda

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.

Serum Interleukin-6 (IL-6), IL-10, Tumor Necrosis Factor (TNF) Alpha, Soluble Type II TNF Receptor, and Transforming Growth Factor Beta Levels in Human Immunodeficiency Virus Type 1-Infected Individuals with Mycobacterium avium Complex Disease

ABSTRACT To characterize changes in serum cytokine levels in human immunodeficiency virus type 1 (HIV-1)-infected persons withMycobacterium avium complex (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 factor β (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.

A Locus at 5q33.3 Confers Resistance to Tuberculosis in Highly Susceptible Individuals

Immunosuppression resulting from HIV infection increases the risk of progression to active tuberculosis (TB) both in individuals newly exposed to Mycobacterium tuberculosis (MTB) and in those with latent infections. We hypothesized that HIV-positive individuals who do not develop TB, despite living in areas where it is hyperendemic, provide a model of natural resistance. We performed a genome-wide association study of TB resistance by using 581 HIV-positive Ugandans and Tanzanians enrolled in prospective cohort studies of TB; 267 of these individuals developed active TB, and 314 did not. A common variant, rs4921437 at 5q33.3, was significantly associated with TB (odds ratio = 0.37, p = 2.11 × 10(-8)). This variant lies within a genomic region that includes IL12B and is embedded in an H3K27Ac histone mark. The locus also displays consistent patterns of linkage disequilibrium across African populations and has signals of strong selection in populations from equatorial Africa. Along with prior studies demonstrating that therapy with IL-12 (the cytokine encoded in part by IL12B, associated with longer survival following MTB infection in mice deficient in CD4 T cells), our results suggest that this pathway might be an excellent target for the development of new modalities for treating TB, especially for HIV-positive individuals. Our results also indicate that studying extreme disease resistance in the face of extensive exposure can increase the power to detect associations in complex infectious disease.

Polymorphisms in TICAM2 and IL1B are associated with TB

Human genetic susceptibility for tuberculosis (TB) has been demonstrated by several studies, but few have examined the multiple innate and adaptive immunity genes comprehensively, age-specific effects and/or resistance to Mycobacterium tuberculosis (Mtb) infection (resistors (RSTRs)). We hypothesized that RSTRs, defined by a persistently negative tuberculin skin test, may have different genetic influences than Mtb disease. 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 RSTRs, and the remaining 60.8% had latent Mtb infection. 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<0.05). Age–genotype interaction analysis revealed SNPs in IL18 and TLR6 that were suggestively associated with TB in children aged ⩽10 years (P=2.9 × 10−3). By contrast, RSTR was associated with SNPs in NOD2, SLC6A3 and TLR4 (nominal P<0.05); these genes were not associated with TB, suggesting distinct genetic influences. We report the first association between TICAM2 polymorphisms and TB and between IL18 and pediatric TB.

Vδ2+ γδ T Cell Function in Mycobacterium tuberculosis– and HIV-1–Positive Patients in the United States and Uganda: Application of a Whole-Blood Assay

γδ T cells represent 1%–5% of circulating human T cells [1, 2]. Vγ9+Vδ2+ γδ T cells (Vδ2+ T cells), the dominant circulating γδ T cell subset in adults, are activated in response to intracellular pathogens, such as Mycobacterium tuberculosis [3–7]. Vδ2+ T cells secrete interferon (IFN)–γ and have cytotoxic T lymphocyte and natural killer cell–like effector function. Vδ2+ T cells recognize small organic phosphate antigens (phosphoantigens) and other nonpeptidic molecules. The tuberculosis antigens found inside M. tuberculosis bacilli were among the first phosphoantigens to be purified [8, 9]. Others include isopentenyl pyrophosphate (IPP) [10, 11] and 3-formyl-1-butyl pyrophosphate [12]. The synthetic analogue bromohydrin pyrophosphate (BrHPP) activates Vδ2+ T cells at nanomolar concentrations [13, 14]. Phosphoantigens are produced by many pathogens and are recognized in a non–major histocompatibility complex–restricted manner. Thus, Vδ2+ T cells can provide a link between innate and adaptive immunity to a wide range of pathogens. In primates, Vδ2+ T cell responses increase during primary mycobacterial infection and increase further on challenge after vaccination with bacille Calmette-Guerin (BCG) [15, 16]. BCG vaccination of adults increases Vδ2+ T cell responses [17]. Vδ2+ T cells are activated in vitro by M. tuberculosis [18]. Studies investigating total γδ or Vδ2+ T cell function in peripheral blood and lungs of patients with tuberculosis, their household contacts, and healthy subjects have provided contradictory results. Some studies demonstrated increased numbers of γδ T cells in peripheral blood of patients with tuberculosis [19, 20], and others demonstrated decreased γδ T cell responses to M. tuberculosis antigens in patients with tuberculosis [21–27]. This variability was likely due to the different antigens used to activate γδ T cells, the stage of infection or disease, and the age of the subjects. Alterations in the γδ T cell repertoire and its function also have been described in HIV-1–positive patients and simian immunodeficiency virus–infected primates [28–32]. Vδ2+ T cell activation is dependent on interleukin (IL)–2 produced by CD4+ T cells, which is diminished in HIV-1 infection [33]. Vδ2+ T cells lyse HIV-1–infected cells and block entry of HIV-1 via β-chemokine secretion [34]. Therefore, decreased Vδ2+ T cell function may contribute not only to the loss of anti–HIV-1 immunity but also to diminished immune protection against M. tuberculosis. Most studies of γδ T cell function have applied multicolor flow cytometry to enumerate Vδ2+ T cells in mixed populations of peripheral-blood mononuclear cells (PBMCs) stimulated with crude microbial antigens and rely on IL-2 produced by CD4+ T cells for Vδ2+ T cell expansion. Flow-cytometric assays are not optimal for field studies in settings of endemicity for tuberculosis. A growing literature has validated the use of whole-blood assays to measure immune function in population-based studies of M. tuberculosis infection and disease [35–38]. We have developed a simple whole-blood assay that measures IFN-γ secretion in response to the phosphoantigen BrHPP in the presence of IL-2 to assess Vδ2+ T cell function independent of CD4+ T cell responses. This assay was used to assess Vδ2+ T cell responses in M. tuberculosis– and HIV-1–positive patients in the United States and Uganda.

Effects of Antiretroviral Therapy on Immune Function of HIV-infected Adults with Pulmonary Tuberculosis and CD4+ >350 Cells/mm3

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.

Genetic and Shared Environmental Influences on Interferon-γ Production in Response to Mycobacterium tuberculosis Antigens in a Ugandan Population

Interferon-γ (IFN-γ) is a key cytokine in the immune response to Mycobacterium tuberculosis (Mtb). Many studies established IFN-γ responses are influenced by host genetics, however differed widely by the study design and heritability estimation method. We estimated heritability of IFN-γ responses to Mtb culture filtrate (CF), ESAT-6, and Antigen 85B (Ag85B) in 1,104 Ugandans from a household contact study. Our method separately evaluates shared environmental and genetic variance, therefore heritability estimates were not upwardly biased, ranging from 11.6% for Ag85B to 22.9% for CF. Subset analyses of individuals with latent Mtb infection or without human immunodeficiency virus infection yielded higher heritability estimates, suggesting 10-30% of variation in IFN-γ is caused by a shared environment. Immunosuppression does not negate the role of genetics on IFN-γ response. These estimates are remarkably close to those reported for components of the innate immune response. These findings have implications for the interpretation of IFN-γ response assays and vaccine studies.