Homayoun Shams

NK Cells Regulate CD8+ T Cell Effector Function in Response to an Intracellular Pathogen

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-γ+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+IFN-γ+cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-γ, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-γ, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-γ. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-γ+ T cells by producing IFN-γ, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-γ and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.

Cytokine Profiles in Immunocompetent Persons Infected with Mycobacterium avium Complex

To evaluate the immunologic factors that contribute to protection against Mycobacterium avium complex (MAC), cytokine production by peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus-negative persons with pulmonary MAC (MAC patients) and healthy control subjects with a delayed hypersensitivity skin test response to M. avium sensitin (MAS-positive control subjects) was measured. In MAC patients, mycobacterium-stimulated PBMC produced higher concentrations of interleukin (IL)-10 but lower concentrations of interferon (IFN)-gamma, IL-12, and tumor necrosis factor (TNF)-alpha, compared with PBMC from MAS-positive control subjects. Immunolabeling for intracellular IL-10 revealed that this cytokine was produced by both monocytes and T cells. Alveolar macrophages produced TNF-alpha and IL-10 in response to MAC, which suggests that these cytokines are produced in the lungs of patients with pulmonary disease caused by this pathogen. Our findings suggest that IFN-gamma, TNF-alpha, and IL-12 contribute to protection against MAC, whereas IL-10 is immunosuppressive.

Exposure to Cigarette Smoke Inhibits the Pulmonary T-Cell Response to Influenza Virus and Mycobacterium tuberculosis

ABSTRACT Smoking is associated with increased susceptibility to tuberculosis and influenza. However, little information is available on the mechanisms underlying this increased susceptibility. Mice were left unexposed or were exposed to cigarette smoke and then infected with Mycobacterium tuberculosis by aerosol or influenza A by intranasal infection. Some mice were given a DNA vaccine encoding an immunogenic M. tuberculosis protein. Gamma interferon (IFN-γ) production by T cells from the lungs and spleens was measured. Cigarette smoke exposure inhibited the lung T-cell production of IFN-γ during stimulation in vitro with anti-CD3, after vaccination with a construct expressing an immunogenic mycobacterial protein, and during infection with M. tuberculosis and influenza A virus in vivo. Reduced IFN-γ production was mediated through the decreased phosphorylation of transcription factors that positively regulate IFN-γ expression. Cigarette smoke exposure increased the bacterial burden in mice infected with M. tuberculosis and increased weight loss and mortality in mice infected with influenza virus. This study provides the first demonstration that cigarette smoke exposure directly inhibits the pulmonary T-cell response to M. tuberculosis and influenza virus in a physiologically relevant animal model, increasing susceptibility to both pathogens.

GM-CSF in the Lung Protects against Lethal Influenza Infection

RATIONALE Alveolar macrophages contribute to host defenses against influenza in animal models. Enhancing alveolar macrophage function may contribute to protection against influenza. OBJECTIVES To determine if increased expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the lung increases resistance to influenza. METHODS Wild-type mice and transgenic mice that expressed GM-CSF in the lung were infected with influenza virus, and lung pathology, weight loss, and mortality were measured. We also administered GM-CSF to the lungs of wild-type mice that were infected with influenza virus. MEASUREMENTS AND MAIN RESULTS Wild-type mice all died after infection with different strains of influenza virus, but all transgenic mice expressing GM-CSF in the lungs survived. The latter also had greatly reduced weight loss and lung injury, and showed histologic evidence of a rapid host inflammatory response that controlled infection. The resistance of transgenic mice to influenza was abrogated by elimination of alveolar phagocytes, but not by depletion of T cells, B cells, or neutrophils. Transgenic mice had far more alveolar macrophages than did wild-type mice, and they were more resistant to influenza-induced apoptosis. Delivery of intranasal GM-CSF to wild-type mice also conferred resistance to influenza. CONCLUSIONS GM-CSF confers resistance to influenza by enhancing innate immune mechanisms that depend on alveolar macrophages. Pulmonary delivery of this cytokine has the potential to reduce the morbidity and mortality due to influenza virus.

Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+ T cells in the context of multiple HLA alleles

The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP1071–85, that elicited IFN-γ production and CTL activity by both CD4+ and CD8+ T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP1071–85 contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4+ cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8+ cells of A2+ donors. T6 elicited responses by CD4+ cells in the context of DRB1*04 and DQB1*03, and by CD8+ cells of B35+ donors. Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimal epitopes for both CD4+ and CD8+ cells. As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations. The capacity of CFP1071–85 to stimulate IFN-γ production and CTL activity by CD4+ and CD8+ cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine.

IS6110 functions as a mobile, monocyte‐activated promoter in Mycobacterium tuberculosis

The mobile insertion sequence, IS6110, is an important marker in tracking of Mycobacterium tuberculosis strains. Here, we demonstrate that IS6110 can upregulate downstream genes through an outward‐directed promoter in its 3′ end, thus adding to the significance of this element. Promoter activity was orientation dependent and was localized within a 110 bp fragment adjacent to the right terminal inverted repeat. Transcripts from this promoter, named OP6110, begin ≈ 85 bp upstream of the 3′ end of IS6110. Use of green fluorescent protein (GFP) expression constructs showed that OP6110 was upregulated in M. tuberculosis during growth in human monocytes and in late growth phases in broth. Analysis of natural insertion sites in M. tuberculosis showed that IS6110 upregulated expression of several downstream genes during growth in human monocytes, including Rv2280 in H37Rv and the PE‐PGRS gene, Rv1468c, in the clinical strain 210, which is a member of the Beijing family. Transcription between IS6110 and downstream genes was confirmed by reverse transcription polymerase chain reaction. The ability to activate genes during infection suggests that IS6110 has the potential to influence growth characteristics of different strains, and indicates another mechanism by which IS6110 can impact M. tuberculosis evolution.

ESAT-6 inhibits production of IFN-γ by Mycobacterium tuberculosis-responsive human T cells

The Mycobacterium tuberculosis early secreted Ag of 6 kDa (ESAT-6) is a potent Ag for human T cells and is a putative vaccine candidate. However, ESAT-6 also contributes to virulence in animal models, mediates cellular cytolysis, and inhibits IL-12 production by mononuclear phagocytes. We evaluated the effects of ESAT-6 and its molecular chaperone, culture filtrate protein of 10 kDa (CFP10), on the capacity of human T cells to produce IFN-γ and proliferate in response to TCR activation. Recombinant ESAT-6, but not CFP10, markedly inhibited IFN-γ production by T cells stimulated with M. tuberculosis or with the combination of anti-CD3 and anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T cell production of IL-17 and TNF-α but not IL-2. Preincubation of ESAT-6 with CFP10 under conditions that favor dimer formation did not affect inhibition of IFN-γ. ESAT-6 decreased IFN-γ transcription and reduced expression of the transcription factors, ATF-2 and c-Jun, which normally bind to the IFN-γ proximal promoter and stimulate mRNA expression. ESAT-6 inhibited T cell IFN-γ secretion through mechanisms that did not involve cellular cytotoxicity or apoptosis. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing activation of ZAP70. We conclude that ESAT-6 directly inhibits human T cell responses to mycobacterial Ags by affecting TCR signaling pathways downstream of ZAP70.

The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo

The ability of Mycobacterium tuberculosis to grow in macrophages is central to its pathogenicity. We found previously that the widespread 210 strain of M. tuberculosis grew more rapidly than other strains in human macrophages. Because principal sigma factors influence virulence in some bacteria, we analysed mRNA expression of the principal sigma factor, sigA, in M. tuberculosis isolates during growth in human macrophages. Isolates of the 210 strain had higher sigA mRNA levels and higher intracellular growth rates, compared with other clinical strains and the laboratory strain H37Rv. SigA was also upregulated in the 210 isolate TB294 during growth in macrophages, compared with growth in broth. In contrast, H37Rv sigA mRNA levels did not change under these conditions. Overexpression of sigA enhanced growth of recombinant M. tuberculosis in macrophages and in lungs of mice after aerosol infection, whereas recombinant strains expressing antisense transcripts to sigA showed decreased growth in both models. In the presence of superoxide, sense sigA transformants showed greater resistance than vector controls, and the antisense sigA transformant did not grow. We conclude that M. tuberculosis sigA modulates the expression of genes that contribute to virulence, enhancing growth in human macrophages and during the early phases of pulmonary infection in vivo. This effect may be mediated in part by increased resistance to reactive oxygen intermediates.

CREB, ATF, and AP-1 Transcription Factors Regulate IFN-γ Secretion by Human T Cells in Response to Mycobacterial Antigen

IFN-γ production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-γ, −73 to −48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-γ in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-γ upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-γ proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-γ and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-γ proximal promoter and that they up-regulate IFN-γ transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation.

Contribution of CD8(+) T cells to gamma interferon production in human tuberculosis.

ABSTRACT The proportions of peripheral blood mononuclear cells (PBMC), CD4+ T cells, and CD8+ T cells that produce gamma interferon (IFN-γ) in response to Mycobacterium tuberculosis were markedly reduced in tuberculosis patients, particularly in those with severe disease. Depletion of CD4+ but not CD8+ cells prior to stimulation of PBMC with M. tuberculosis abolished IFN-γ production. These results show that (i) IFN-γ production by CD8+ and CD4+ cells correlates with the clinical manifestations ofM. tuberculosis infection and (ii) IFN-γ production by CD8+ cells depends on CD4+ cells.