Benjamin Wizel

The NKp46 Receptor Contributes to NK Cell Lysis of Mononuclear Phagocytes Infected with an Intracellular Bacterium

We used human tuberculosis as a model to investigate the role of NK cytotoxic mechanisms in the immune response to intracellular infection. Freshly isolated NK cells and NK cell lines from healthy donors lysed Mycobacterium tuberculosis-infected monocytes to a greater extent than uninfected monocytes. Lysis of infected monocytes was associated with increased expression of mRNA for the NKp46 receptor, but not the NKp44 receptor. Antisera to NKp46 markedly inhibited lysis of infected monocytes. NK cell-mediated lysis was not due to reduced expression of MHC class I molecules on the surface of infected monocytes or to enhanced production of IL-18 or IFN-γ. NK cell lytic activity against M. tuberculosis-infected monocytes and NKp46 mRNA expression were reduced in tuberculosis patients with ineffective immunity to M. tuberculosis compared with findings in healthy donors. These observations suggest that 1) the NKp46 receptor participates in NK cell-mediated lysis of cells infected with an intracellular pathogen, and 2) the reduced functional capacity of NK cells is associated with severe manifestations of infectious disease.

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.

Chlamydia pneumoniae Inclusion Membrane Protein Cpn0585 Interacts with Multiple Rab GTPases

ABSTRACT Chlamydiae are intracellular bacteria that develop within a membrane-bound vacuole called an inclusion. To ensure that the inclusion is a safe niche for chlamydial replication, chlamydiae exploit a number of host cell processes, including membrane-trafficking pathways. Recently, several Rab GTPases were found to associate with the inclusions of various chlamydial species. Here we report that Cpn0585, a Chlamydia pneumoniae inclusion membrane protein (Inc), interacts with multiple Rab GTPases. The results from yeast two-hybrid experiments revealed that an amino-terminally truncated form of Cpn0585 (Cpn0585102-651) interacts with Rab1, Rab10, and Rab11 but not with Rab4 or Rab6. Cpn0585-Rab GTPase interactions are direct and GTP dependent as shown in glutathione S-transferase pull-down assays using native and recombinant Cpn0585. In C. pneumoniae-infected HEp-2 cells transfected with enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, the colocalization with Cpn0585 at the inclusion membrane was partial for EGFP-Rab1 and EGFP-Rab10, but extensive for wild-type EGFP-Rab11A and the constitutively active GTPase-deficient EGFP-Rab11AQ70L. Moreover, Cpn0585 colocalized with EGFP-Rab11AQ70L as early as 2 h postinfection. Upon delivery into live C. pneumoniae-infected cells, Cpn0585628-651-specific antibodies bound to the inclusion membrane, demonstrating that the Rab GTPase-interacting domain of Cpn0585 faces the host cell cytosol. Finally, ectopic expression of Cpn0585102-651 partially inhibited the development of C. pneumoniae inclusions in EGFP. but not in EGFP-Rab11AQ70L-expressing HEp-2 cells. Collectively, these data suggest that Cpn0585 is involved in the recruitment of Rab GTPases to the inclusion membrane and that interfering with this function may adversely impact the fitness of the C. pneumoniae inclusion for chlamydial replication.

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.

Multiple Chlamydia pneumoniae Antigens Prime CD8+ Tc1 Responses That Inhibit Intracellular Growth of This Vacuolar Pathogen

CD8+ T cells play an essential role in immunity to Chlamydia pneumoniae (Cpn). However, the target Ags recognized by Cpn-specific CD8+ T cells have not been identified, and the mechanisms by which this T cell subset contributes to protection remain unknown. In this work we demonstrate that Cpn infection primes a pathogen-specific CD8+ T cell response in mice. Eighteen H-2b binding peptides representing sequences from 12 Cpn Ags sensitized target cells for MHC class I-restricted lysis by CD8+ CTL generated from the spleens and lungs of infected mice. Peptide-specific IFN-γ-secreting CD8+ T cells were present in local and systemic compartments after primary infection, and these cells expanded after pathogen re-exposure. CD8+ T cell lines to the 18 Cpn epitope-bearing peptides were cytotoxic, displayed a memory phenotype, and secreted IFN-γ and TNF-α, but not IL-4. These CTL lines lysed Cpn-infected macrophages, and the lytic activity was inhibited by brefeldin A, indicating endogenous processing of CTL Ags. Finally, Cpn peptide-specific CD8+ CTL suppressed chlamydial growth in vitro by direct lysis of infected cells and by secretion of IFN-γ and other soluble factors. These studies provide information on the mechanisms by which CD8+ CTL protect against Cpn, furnish the tools to investigate their possible role in immunopathology, and lay the foundation for future work to develop vaccines against acute and chronic Cpn infections.

Mosquito immune responses and malaria transmission: lessons from insect model systems and implications for vertebrate innate immunity and vaccine development

The introduction of novel biochemical, genetic, molecular and cell biology tools to the study of insect immunity has generated an information explosion in recent years. Due to the biodiversity of insects, complementary model systems have been developed. The conceptual framework built based on these systems is used to discuss our current understanding of mosquito immune responses and their implications for malaria transmission. The areas of insect and vertebrate innate immunity are merging as new information confirms the remarkable extent of the evolutionary conservation, at a molecular level, in the signaling pathways mediating these responses in such distant species. Our current understanding of the molecular language that allows the vertebrate innate immune system to identify parasites, such as malaria, and direct the acquired immune system to mount a protective immune response is very limited. Insect vectors of parasitic diseases, such as mosquitoes, could represent excellent models to understand the molecular responses of epithelial cells to parasite invasion. This information could broaden our understanding of vertebrate responses to parasitic infection and could have extensive implications for anti-malarial vaccine development.

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.

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.

A CD8+ T Cell Heptaepitope Minigene Vaccine Induces Protective Immunity against Chlamydia pneumoniae

An intact T cell compartment and IFN-γ signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8+ T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8+ Tc1 response against various Cpn Ags, and that CD8+ CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2b-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-γ-producing CD8+ CTL against all seven CTL epitopes. CD8+ T cell lines generated to minigene-encoded CTL epitopes secreted IFN-γ and TNF-α and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with β2-microglobulin−/− mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8+ T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4+ T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8+ T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.

Reduced Expression of Nuclear Cyclic Adenosine 5′-Monophospate Response Element-Binding Proteins and IFN-γ Promoter Function in Disease Due to an Intracellular Pathogen

Mycobacterium tuberculosis-induced IFN-γ protein and mRNA expression have been shown to be reduced in tuberculosis patients, compared with healthy tuberculin reactors. To determine whether this decrease was associated with reduced activity of the IFN-γ promoter, we first studied binding of nuclear proteins to the radiolabeled proximal IFN-γ promoter (−71 to −40 bp), using EMSAs with nuclear extracts of freshly isolated peripheral blood T cells. Nuclear extracts of T cells from most tuberculosis patients showed markedly reduced expression of proteins that bind to the proximal IFN-γ promoter, compared with findings in nuclear extracts of T cells from healthy tuberculin reactors. These DNA-binding complexes contained CREB proteins, based on competitive EMSAs, supershift assays, and Western blotting with an anti-CREB Ab. Transient transfection of PBLs with a luciferase reporter construct under the control of the IFN-γ promoter revealed reduced IFN-γ promoter activity in tuberculosis patients. Transient transfection of Jurkat cells with a dominant-negative CREB repressor plasmid reduced IFN-γ promoter activity. These data suggest that reduced expression of CREB nuclear proteins in tuberculosis patients results in decreased IFN-γ promoter activity and reduced IFN-γ production.