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David H Canaday

Publications and source records attributed to David H Canaday.

15 recordsLinked to original sources
PubMed2006

Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 that regulates innate immunity and APC function.

TLR2 recognizes components of Mycobacterium tuberculosis (Mtb) and initiates responses by APCs that influence both innate and adaptive immunity. Mtb lipoproteins are an important class of TLR2 ligand, but only two, LpqH and LprG, have been characterized to date. In this study, we characterize a third Mtb lipoprotein, LprA, and determine its effects on host macrophages and dendritic cells. LprA is a cell wall-associated lipoprotein with no homologs outside the slow-growing mycobacteria. Using Mycobacterium smegmatis as an expression host, we purified 6x His-tagged LprA both with and without its acyl modifications. Acylated LprA had agonist activity for both human and murine TLR2 and induced expression of TNF-alpha, IL-10, and IL-12. LprA also induced dendritic cell maturation as shown by increased expression of CD40, CD80, and class II MHC (MHC-II). In macrophages, prolonged (24 h) incubation with LprA decreased IFN-gamma-induced MHC-II Ag processing and presentation, consistent with an observed decrease in MHC-II expression (macrophage viability was not affected and apoptosis was not induced by LprA). Reduced MHC-II Ag presentation may represent a negative feedback mechanism for control of inflammation that may be subverted by Mtb for immune evasion. Thus, Mtb LprA is a TLR2 agonist that induces cytokine responses and regulates APC function.

Acylation↗
PubMed2006

Role of phagosomes and major histocompatibility complex class II (MHC-II) compartment in MHC-II antigen processing of Mycobacterium tuberculosis in human macrophages.

Mycobacterium tuberculosis resides in phagosomes inside macrophages. In this study, we analyzed the kinetics and location of M. tuberculosis peptide-major histocompatibility complex class II (MHC-II) complexes in M. tuberculosis-infected human macrophages. M. tuberculosis peptide-MHC-II complexes were detected with polyclonal autologous M. tuberculosis-specific CD4+ T cells or F9A6 T hybridoma cells specific for M. tuberculosis antigen (Ag) 85B (96-111). Macrophages processed heat-killed M. tuberculosis more rapidly and efficiently than live M. tuberculosis. To determine where M. tuberculosis peptide-MHC-II complexes were formed intracellularly, macrophages incubated with heat-killed M. tuberculosis were homogenized, and subcellular compartments were separated on Percoll density gradients analyzed with T cells. In THP-1 cells, M. tuberculosis Ag 85B (96- 111)-DR1 complexes appeared initially in phagosomes, followed by MHC class II compartment (MIIC) and the plasma membrane fractions. In monocyte-derived macrophages, M. tuberculosis peptide-MHC-II complexes appeared only in MIIC fractions and subsequently on the plasma membrane. Although phagosomes from both cell types acquired lysosome-associated membrane protein 1 (LAMP-1) and MHC-II, THP-1 phagosomes that support formation of M. tuberculosis peptide-MHC-II complexes had increased levels of both LAMP-1 and MHC-II. Thus, M. tuberculosis phagosomes with high levels of MHC-II and LAMP-1 and MIIC both have the potential to form peptide-MHC-II complexes from M. tuberculosis antigens in human macrophages.

Cell Line↗
PubMed2005

Mouse endothelial cells cross-present lymphocyte-derived antigen on class I MHC via a TAP1- and proteasome-dependent pathway.

In vivo studies suggest that vascular endothelial cells (ECs) can acquire and cross-present exogenous Ag on MHC-I but the cellular mechanisms underlying this observation remain unknown. We tested whether primary female mouse aortic ECs could cross-present exogenous male Ag to the T cell hybridoma, MHH, specific for HYUty plus D(b). MHC-I-deficient male spleen cells provided a source of male Ag that could not directly stimulate the MHH cells. Addition of male but not female MHC-I-deficient spleen cells to wild-type syngeneic female EC induced MHH stimulation, demonstrating EC cross-presentation. Lactacystin treatment of the donor male MHC-I-deficient spleen cells, to inhibit proteasome function, markedly enhanced EC cross-presentation showing that the process is most efficient for intact proteins rather than degraded peptide fragments. Additional experiments revealed that this EC Ag-processing pathway is both proteasome and TAP1 dependent. These studies demonstrate that cultured murine aortic ECs can process and present MHC-I-restricted Ag derived from a separate, live cell, and they offer insight into the molecular requirements involved in this EC Ag presentation process. Through this pathway, ECs expressing cross-presented peptides can participate in the effector phase of T cell-mediated inflammatory responses such as autoimmunity, anti-tumor immunity, and transplant rejection.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Mycobacterium tuberculosis heat shock fusion protein enhances class I MHC cross-processing and -presentation by B lymphocytes.

Exogenous heat shock protein (HSP):peptide complexes are processed for cross-presentation of HSP-chaperoned peptides on class I MHC (MHC-I) molecules. Fusion proteins containing HSP and Ag sequences facilitate MHC-I cross-presentation of linked antigenic epitopes. Processing of HSP-associated Ag has been attributed to dendritic cells and macrophages. We now provide the first evidence to show processing of HSP-associated Ag for MHC-I cross-presentation by B lymphocytes. Fusion of OVA sequence (rOVA, containing OVA(230-359) sequence) to Mycobacterium tuberculosis HSP70 greatly enhanced rOVA processing and MHC-I cross-presentation of OVA(257-264):K(b) complexes by B cells. Enhanced processing was dependent on linkage of rOVA sequence to HSP70. M. tuberculosis HSP70-OVA fusion protein enhanced cross-processing by a CD91-dependent process that was independent of TLR4 and MyD88. The enhancement occurred through a post-Golgi, proteasome-independent mechanism. These results indicate that HSPs enhance delivery and cross-processing of HSP-linked Ag by B cells, which could provide a novel contribution to the generation of CD8(+) T cell responses. HSP fusion proteins have potential advantages for use in vaccines to enhance priming of CD8(+) T cell responses.

Adjuvants, Immunologic↗
PubMed2005

Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens.

Extracellular antigens are internalized and processed before binding MHC class II molecules within endosomal and lysosomal compartments of professional antigen presenting cells (APC) for subsequent presentation to T cells. Yet select cytoplasmic peptides derived from autoantigens also intersect and bind class II molecules via an unknown mechanism. In human B lymphoblasts, inhibition of the peptide transporter associated with antigen processing (TAP) failed to alter class II-restricted cytoplasmic epitope presentation. By contrast, decreased display of cytoplasmic epitopes via class II molecules was observed in cells with diminished expression of the lysosome-associated membrane protein-2 (Lamp-2). Overexpression of Lamp-2 isoform A (Lamp-2a), an established component of chaperone-mediated autophagy, enhanced cytoplasmic autoantigen presentation. Manipulating APC expression of heat shock cognate protein 70 (hsc70), a cofactor for Lamp-2a, also altered cytoplasmic class II peptide presentation. These results demonstrate a novel role for the lysosomal Lamp-2a-hsc70 complex in promoting immunological recognition and antigen presentation.

ATP-Binding Cassette Transporters↗
PubMed2004

Bacterial heat shock proteins enhance class II MHC antigen processing and presentation of chaperoned peptides to CD4+ T cells.

APCs process heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC molecules, but the ability of HSPs to contribute chaperoned peptides for class II MHC (MHC-II) Ag processing and presentation is unclear. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-II presentation, as detected by T hybridoma cells. Bacterial HSPs enhanced MHC-II presentation only if peptide was complexed to the HSP, suggesting that the key HSP function was enhanced delivery or processing of chaperoned peptide Ag rather than generalized enhancement of APC function. HSP-enhanced processing was intact in MyD88 knockout cells, which lack most TLR signaling, further suggesting the effect was not due to TLR-induced induction of accessory molecules. Bacterial HSPs enhanced uptake of peptide, which may contribute to increased MHC-II presentation. In addition, HSPs enhanced binding of peptide to MHC-II molecules at pH 5.0 (the pH of vacuolar compartments), but not at pH 7.4, indicating another mechanism for enhancement of MHC-II Ag processing. Bacterial HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD4+ T cells.

Amino Acid Sequence↗
PubMed2004

Bacterial heat shock proteins promote CD91-dependent class I MHC cross-presentation of chaperoned peptide to CD8+ T cells by cytosolic mechanisms in dendritic cells versus vacuolar mechanisms in macrophages.

APCs process mammalian heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC (MHC-I) molecules to CD8(+) T cells. HSPs are also expressed in prokaryotes and chaperone microbial peptides, but the ability of prokaryotic HSPs to contribute chaperoned peptides for Ag presentation is unknown. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-I presentation by both murine macrophages and dendritic cells. HSP-enhanced MHC-I peptide presentation occurred only if peptide was complexed to the prokaryotic HSP and was dependent on CD91, establishing CD91 as a receptor for prokaryotic as well as mammalian HSPs. Inhibition of cytosolic processing mechanisms (e.g., by transporter for Ag presentation deficiency or brefeldin A) blocked HSP-enhanced peptide presentation in dendritic cells but not macrophages. Thus, prokaryotic HSPs deliver chaperoned peptide for alternate MHC-I Ag processing and cross-presentation via cytosolic mechanisms in dendritic cells and vacuolar mechanisms in macrophages. Prokaryotic HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD8(+) T cells.

Adjuvants, Immunologic↗
PubMed2004

Prolonged toll-like receptor signaling by Mycobacterium tuberculosis and its 19-kilodalton lipoprotein inhibits gamma interferon-induced regulation of selected genes in macrophages.

Infection of macrophages with Mycobacterium tuberculosis or exposure to M. tuberculosis 19-kDa lipoprotein for >16 h inhibits gamma interferon (IFN-gamma)-induced major histocompatibility complex class II (MHC-II) expression by a mechanism involving Toll-like receptors (TLRs). M. tuberculosis was found to inhibit murine macrophage MHC-II antigen (Ag) processing activity induced by IFN-gamma but not by interleukin-4 (IL-4), suggesting inhibition of IFN-gamma-induced gene regulation. We designed an approach to test the ability of M. tuberculosis-infected cells to respond to IFN-gamma. To model chronic infection with M. tuberculosis with accompanying prolonged TLR signaling, macrophages were infected with M. tuberculosis or incubated with M. tuberculosis 19-kDa lipoprotein for 24 h prior to the addition of IFN-gamma. Microarray gene expression studies were then used to determine whether prolonged TLR signaling by M. tuberculosis broadly inhibits IFN-gamma regulation of macrophage gene expression. Of 347 IFN-gamma-induced genes, M. tuberculosis and 19-kDa lipoprotein inhibited induction of 42 and 36%, respectively. Key genes or gene products were also examined by quantitative reverse transcription-PCR and flow cytometry, confirming and extending the results obtained by microarray studies. M. tuberculosis inhibited IFN-gamma induction of genes involved in MHC-II Ag processing, Ag presentation, and recruitment of T cells. These effects were largely dependent on myeloid differentiation factor 88, implying a role for TLRs. Thus, prolonged TLR signaling by M. tuberculosis inhibits certain macrophage responses to IFN-gamma, particularly those related to MHC-II Ag presentation. This inhibition may promote M. tuberculosis evasion of T-cell responses and persistence of infection in tuberculosis.

Animals↗
PubMed2003

T-cell hybridomas from HLA-transgenic mice as tools for analysis of human antigen processing.

The study of antigen processing and presentation by human antigen presenting cells (APC) has been limited by difficulties of producing and maintaining human T-cell clones. Murine T-cell hybridomas have advantages for detecting specific peptide-MHC complexes on APC. Human antigen-specific immortalized T-cell lines have not been successfully produced. We report and validate the use of transgenic mice with human MHC genes for HLA-A2, DR1 and DR4 to produce murine T-cell hybridomas that are restricted to human HLA alleles and respond to human macrophages, dendritic cells (DC), and B-cell lines. Hybridomas restricted by human MHC-I and -II specific for influenza matrix protein, tetanus toxoid, diphtheria antigen CRM(197), and various M. tuberculosis antigens were produced. Epitope specificity was determined for several hybridomas. T hybridomas recognized peptide-MHC complexes on fixed APC for analysis of kinetics or susceptibility to inhibitors of antigen processing. T hybridomas restricted by human MHC represent convenient and powerful tools for the study of antigen processing by human APC.

Amino Acid Sequence↗
PubMed2003

Human immunity to M. tuberculosis: T cell subsets and antigen processing.

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.

Antigen Presentation↗
PubMed2003

Antigen processing of the heptavalent pneumococcal conjugate vaccine carrier protein CRM(197) differs depending on the serotype of the attached polysaccharide.

The pneumococcal (Pn) conjugate vaccine includes seven different polysaccharides (PS) conjugated to CRM(197). Utilizing antigen-processing cells and a CRM(197)-specific mouse T-cell hybridoma, we found that the serotype of conjugated PnPS dramatically affected antigen processing of CRM(197). Unconjugated CRM(197) and serotype conjugates 14 and 18C were processed more efficiently.

Amino Acid Sequence↗
PubMed2003

The Mycobacterium tuberculosis 19-kilodalton lipoprotein inhibits gamma interferon-regulated HLA-DR and Fc gamma R1 on human macrophages through Toll-like receptor 2.

Mycobacterium tuberculosis survives in macrophages in the face of acquired CD4(+) T-cell immunity, which controls but does not eliminate the organism. Gamma interferon (IFN-gamma) has a central role in host defenses against M. tuberculosis by activating macrophages and regulating major histocompatibility complex class II (MHC-II) antigen (Ag) processing. M. tuberculosis interferes with IFN-gamma receptor (IFN-gamma R) signaling in macrophages, but the molecules responsible for this inhibition are poorly defined. This study determined that the 19-kDa lipoprotein from M. tuberculosis inhibits IFN-gamma-regulated HLA-DR protein and mRNA expression in human macrophages. Inhibition of HLA-DR expression was associated with decreased processing and presentation of soluble protein Ags and M. tuberculosis bacilli to MHC-II-restricted T cells. Inhibition of HLA-DR required prolonged exposure to 19-kDa lipoprotein and was blocked with a monoclonal antibody specific for Toll-like receptor 2 (TLR-2). The 19-kDa lipoprotein also inhibited IFN-gamma-induced expression of Fc gamma RI. Thus, M. tuberculosis, through 19-kDa lipoprotein activation of TLR-2, inhibits IFN-gamma R signaling in human macrophages, resulting in decreased MHC-II Ag processing and recognition by MHC-II-restricted CD4 T cells. These findings provide a mechanism for M. tuberculosis persistence in macrophages.

Animals↗
PubMed2002

ATP and control of intracellular growth of mycobacteria by T cells.

Extracellular ATP at millimolar concentrations inhibits growth of mycobacteria in human macrophages. Whether T cells can produce sufficient ATP is unknown. CD4(+) and CD8(+) T cells did not release sufficient ATP through either degranulation or lysis of bystander cells to restrict growth of Mycobacterium bovis BCG in monocytes.

Adenosine Triphosphate↗
PubMed2002

Identification by mass spectrometry of CD8(+)-T-cell Mycobacterium tuberculosis epitopes within the Rv0341 gene product.

Identification of Mycobacterium tuberculosis proteins that can provide immunological protection against tuberculosis is essential for the development of a more effective vaccine. To identify new vaccine targets, we have used immunoaffinity chromatography to isolate class I HLA-A*0201-peptide complexes from M. tuberculosis-infected cells and sequenced the isolated peptides by mass spectrometry. From this material, we have identified three peptides derived from a single M. tuberculosis protein that is encoded by the M. tuberculosis Rv0341 gene. Although no known protein encoded by the Rv0341 gene has been described, it is predicted to give rise to a 479-amino-acid protein with a molecular mass of 43.9 kDa. The three peptides identified are all nested and were found to be antigenic, in that they were capable of inducing peptide-specific, CD8(+) T cells from healthy blood donors in vitro and capable of recognizing and lysing M. tuberculosis-infected dendritic cells. This methodology provides a powerful tool for the identification of M. tuberculosis proteins that can be evaluated as potential vaccine candidates.

Antigens, Bacterial↗