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Clifford V Harding

Publications and source records attributed to Clifford V Harding.

At least 37 records · Page 2Linked to original sources
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

Mycobacterium tuberculosis LprG (Rv1411c): a novel TLR-2 ligand that inhibits human macrophage class II MHC antigen processing.

MHC class II (MHC-II)-restricted CD4(+) T cells are essential for control of Mycobacterium tuberculosis infection. This report describes the identification and purification of LprG (Rv1411c) as an inhibitor of primary human macrophage MHC-II Ag processing. LprG is a 24-kDa lipoprotein found in the M. tuberculosis cell wall. Prolonged exposure (>16 h) of human macrophages to LprG resulted in marked inhibition of MHC-II Ag processing. Inhibition of MHC-II Ag processing was dependent on TLR-2. Short-term exposure (<6 h) to LprG stimulated TLR-2-dependent TNF-alpha production. Thus, LprG can exploit TLR-2 signaling to inhibit MHC-II Ag processing in human macrophages. Inhibition of MHC-II Ag processing by mycobacterial lipoproteins may allow M. tuberculosis, within infected macrophages, to avoid recognition by 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

In vitro human memory CD8 T cell expansion in response to cytomegalovirus requires CD4+ T cell help.

Requirements for human memory CD8(+) T cell expansion are incompletely understood. We found that human cytomegalovirus (HCMV) induced expansion of memory CD8(+) T cells in vitro without requiring intracellular viral peptide synthesis. Peptide-major histocompatibility complex class I tetramer binding confirmed expansion of cells with HCMV-peptide specificity. Expansion of memory CD8(+) T cells was completely dependent on the presence and function of CD4(+) T cells, whose "help" also could be induced by exposure to irrelevant antigen. Recombinant interleukin (IL)-2 or IL-15 could substitute for help provided by CD4(+) T cells, whereas CD8(+) T cell expansion was blocked by anti-IL-2 but not anti-IL-15 antibody. Human memory CD8(+) T cells expand dramatically in vitro in response to cross-presentation of HCMV antigens, and, in contrast to observations made in murine systems, this proliferation was critically dependent on CD4(+) T cells that provide essential IL-2. Thus, in humans, cross-presentation and expansion of memory CD8(+) T cells may be compromised in disease states that result in deficits in CD4(+) T cell numbers or function, such as may be seen in human immunodeficiency virus type 1 infection.

CD4-Positive T-Lymphocytes↗
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↗
PubMed2004

Inhibition of major histocompatibility complex II expression and antigen processing in murine alveolar macrophages by Mycobacterium bovis BCG and the 19-kilodalton mycobacterial lipoprotein.

Alveolar macrophages constitute a primary defense against Mycobacterium tuberculosis, but they are unable to control M. tuberculosis without acquired T-cell immunity. This study determined the antigen-presenting cell function of murine alveolar macrophages and the ability of the model mycobacterium, Mycobacterium bovis BCG, to modulate it. The majority (80 to 85%) of alveolar macrophages expressed both CD80 (B7.1) and CD11c, and 20 to 30% coexpressed major histocompatibility complex II (MHC-II). Gamma interferon (IFN-gamma) enhanced MHC-II but not B7.1 expression. Naive or IFN-gamma-treated alveolar macrophages did not express CD86 (B7.2), CD11b, Mac-3, CD40, or F4/80. M. bovis BCG and the 19-kDa mycobacterial lipoprotein inhibited IFN-gamma-regulated MHC-II expression on alveolar macrophages, and inhibition was dependent on Toll-like receptor 2. The inhibition of MHC-II expression by the 19-kDa lipoprotein was associated with decreased presentation of soluble antigen to T cells. Thus, susceptibility to tuberculosis may result from the ability of mycobacteria to interfere with MHC-II expression and antigen presentation by alveolar macrophages.

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

CpG DNA induces a class II transactivator-independent increase in class II MHC by stabilizing class II MHC mRNA in B lymphocytes.

Microbial products, such as CpG DNA and LPS, enhance class II MHC (MHC-II) expression and Ag presentation by dendritic cells, but this effect does not occur with macrophages and is largely unexplored in B cells. Although MHC-II expression is influenced by transcriptional regulation, which is governed by class II transactivator (CIITA) in all cells, microbial products enhance MHC-II expression by dendritic cells in part by increasing MHC-II protein stability. In this study, we show that the CpG-induced increase in MHC-II expression by B lymphocytes is not due to protein stabilization or changes in CIITA expression or activity, but instead is due to increased stability of MHC-II mRNA. This CIITA-independent mechanism adds a new layer of complexity to regulation of MHC-II and may increase T cell help for B cell Ab responses to microbial or vaccine Ags.

Adjuvants, Immunologic↗
PubMed2003

Alternate class I MHC antigen processing is inhibited by Toll-like receptor signaling pathogen-associated molecular patterns: Mycobacterium tuberculosis 19-kDa lipoprotein, CpG DNA, and lipopolysaccharide.

Pathogen-associated molecular patterns (PAMPs) signal through Toll-like receptors (TLRs) to activate immune responses, but prolonged exposure to PAMPs from Mycobacterium tuberculosis (MTB) and other pathogens inhibits class II MHC (MHC-II) expression and Ag processing, which may allow MTB to evade CD4(+) T cell immunity. Alternate class I MHC (MHC-I) processing allows macrophages to present Ags from MTB and other bacteria to CD8(+) T cells, but the effect of PAMPs on this processing pathway is unknown. In our studies, MTB and TLR-signaling PAMPs, MTB 19-kDa lipoprotein, CpG DNA, and LPS, inhibited alternate MHC-I processing of latex-conjugated Ag by IFN-gamma-activated macrophages. Inhibition was dependent on TLR-2 for MTB 19-kDa lipoprotein (but not whole MTB or the other PAMPs); inhibition was dependent on myeloid differentiation factor 88 for MTB and all of the individual PAMPs. Inhibition of MHC-II and alternate MHC-I processing was delayed, appearing after 16 h of PAMP exposure, as would occur in chronically infected macrophages. Despite inhibition of alternate MHC-I Ag processing, there was no inhibition of MHC-I expression, MHC-I-restricted presentation of exogenous peptide or conventional MHC-I processing of cytosolic Ag. MTB 19-kDa lipoprotein and other PAMPs inhibited phagosome maturation and phagosome Ag degradation in a myeloid differentiation factor 88-dependent manner; this may limit availability of peptides to bind MHC-I. By inhibiting both MHC-II and alternate MHC-I Ag processing, pathogens that establish prolonged infection of macrophages (>16 h), e.g., MTB, may immunologically silence macrophages and evade surveillance by both CD4(+) and CD8(+) T cells, promoting chronic infection.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Inhibition of IFN-gamma-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion.

Mycobacterium tuberculosis (MTB) persists inside macrophages despite vigorous immune responses. MTB and MTB 19-kDa lipoprotein inhibit class II MHC (MHC-II) expression and Ag processing by a Toll-like receptor 2-dependent mechanism that is shown in this study to involve a defect in IFN-gamma induction of class II transactivator (CIITA). Exposure of macrophages to MTB or MTB 19-kDa lipoprotein inhibited IFN-gamma-induced MHC-II expression, but not IL-4-induced MHC-II expression, by preventing induction of mRNA for CIITA (total, type I, and type IV), IFN regulatory factor-1, and MHC-II. MTB 19-kDa lipoprotein induced mRNA for suppressor of cytokine signaling (SOCS)1 but did not inhibit IFN-gamma-induced Stat1 phosphorylation. Furthermore, the lipoprotein inhibited MHC-II Ag processing in SOCS1(-/-) macrophages. MTB 19-kDa lipoprotein did not inhibit translocation of phosphorylated Stat1 to the nucleus or Stat1 binding to and transactivation of IFN-gamma-sensitive promoter constructs. Thus, MTB 19-kDa lipoprotein inhibited IFN-gamma signaling independent of SOCS1 and without interfering with the activation of Stat1. Inhibition of IFN-gamma-induced CIITA by MTB 19-kDa lipoprotein may allow MTB to evade detection by CD4(+) T cells.

Active Transport, Cell Nucleus↗
PubMed2003

Tapasin-/- and TAP1-/- macrophages are deficient in vacuolar alternate class I MHC (MHC-I) processing due to decreased MHC-I stability at phagolysosomal pH.

Alternate class I MHC (MHC-I) Ag processing via cytosolic or vacuolar pathways leads to cross-presentation of exogenous Ag to CD8 T cells. Vacuolar alternate MHC-I processing involves phagolysosomal Ag proteolysis and peptide binding to MHC-I in post-Golgi compartments. We report the first study of alternate MHC-I Ag processing in tapasin(-/-) cells and experiments with tapasin(-/-) and TAP1(-/-) macrophages that characterize alternate MHC-I processing. Tapasin promotes retention of MHC-I in the endoplasmic reticulum (ER) for loading with high affinity peptides, whereas tapasin(-/-) cells allow poorly loaded MHC-I molecules to exit the ER. Hypothetically, we considered that a large proportion of post-Golgi MHC-I on tapasin(-/-) cells might be peptide-receptive, enhancing alternate MHC-I processing. In contrast, alternate MHC-I processing was diminished in both tapasin(-/-) and TAP1(-/-) macrophages. Nonetheless, these cells efficiently presented exogenous peptide, suggesting a loss of MHC-I stability or function specific to vacuolar processing compartments. Tapasin(-/-) and TAP1(-/-) macrophages had decreased MHC-I stability and increased susceptibility of MHC-I to inactivation by acidic conditions (correlating with vacuolar pH). Incubation of tapasin(-/-) or TAP1(-/-) cells at 26 degrees C decreased susceptibility of MHC-I to acid pH and reversed the deficiency in alternate MHC-I processing. Thus, tapasin and TAP are required for MHC-I to bind ER-derived stabilizing peptides to achieve the stability needed for alternate MHC-I processing via peptide exchange in acidic vacuolar processing compartments. Acidic pH destabilizes MHC-I, but also promotes peptide exchange, thereby enhancing alternate MHC-I Ag processing. These results are consistent with alternate MHC-I Ag processing mechanisms that involve binding of peptides to MHC-I within acidic vacuolar compartments.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Localization of peptide/MHC class II complexes in macrophages following antigen processing of viable Streptococcus pyogenes.

The subcellular localization of peptide/MHC complexes was investigated during processing of the surface M5 protein from Streptococcus pyogenes. Bone marrow-derived macrophages were pulsed with viable S. pyogenes for 20 min followed by various periods of chase. T hybridoma cells detected complexes of one epitope, M5(17-31) with E(d) on the surface of macrophages within 30 min of chase. In contrast, complexes with another epitope, M5(308-319) with A(d) peaked later. Intracellular localization of peptide/MHC-II complexes was studied by subcellular fractionation and detection of complexes in fractions by T hybridoma cells. M5(17-31)/E(d) complexes were detected in light membrane fractions containing plasma membrane and early endosomes by 10-30 min. M5(308-319)/A(d) complexes were detected in these light membranes after 3 h of chase. Thus, the time course of M5(308-319)/A(d) presentation was delayed relative to M5(17-31)/E(d). However, neither type of complex was detected at any time in fractions containing phagosomes. Both species of peptide/MHC complexes localized to endocytic compartments, indicating a role for endosomes in presentation of antigens from phagocytosed bacteria.

Animals↗
PubMed2003

CCR5 promoter polymorphism determines macrophage CCR5 density and magnitude of HIV-1 propagation in vitro.

The common CCR5 promoter polymorphism at position -2459 (A/G) has been associated with differences in the rate of progression to AIDS, where HIV-1-infected individuals with the CCR5 -2459 G/G genotype exhibit slower disease progression than those with the A/A genotype. Mechanisms underlying the relationship between these polymorphisms and disease progression are not known. Here through in vitro infection of peripheral blood mononuclear cells obtained from healthy Caucasian blood donors with macrophage-tropic HIV-1 isolates we observed low, medium, and high viral propagation in association with G/G, A/G, and A/A promoter genotypes, respectively. Flow cytometric analysis of unstimulated CD14+ monocytes from these same donors revealed a similar hierarchy of CCR5 receptor density in association with promoter genotypes. Finally, PBMC from persons with the G/G promoter polymorphism produced higher levels of beta-chemokines after in vitro stimulation. Thus, the CCR5 -2459 (A/G) promoter polymorphism determines CCR5 expression and predicts the magnitude of HIV-1 propagation in vitro. These findings may provide important insight regarding the regulation of mechanisms that influence the rate of HIV-1 propagation and progression to AIDS.

Cell Division↗
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

Regulation of class II MHC expression in APCs: roles of types I, III, and IV class II transactivator.

Class II transactivator (CIITA) is necessary for expression of class II MHC (MHC-II) molecules. In mice, CIITA expression is regulated by three promoters (pI, pIII, and pIV), producing types I, III, and IV CIITA. The relative roles of different CIITA types remain unclear. Unstimulated bone marrow-derived macrophages expressed low levels of CIITA mRNA; type I CIITA was nine times more abundant than type IV (type III CIITA was barely detected). Exposure to IFN-gamma (6 h) dramatically increased types I and IV CIITA mRNA to similar absolute levels. Type IV CIITA declined over time, but type I was stable for over 72 h. Thus, the dominant form of CIITA evolved with time during activation by IFN-gamma, and type I CIITA explained prolonged expression of MHC-II by macrophages. mRNA half-life was shorter for type I than type IV CIITA, suggesting that sustained transcription contributed to stable expression of type I CIITA induced by IFN-gamma. Splenic B cells expressed mRNA for type III CIITA but very little for types I or IV. Treatment with IL-4 increased surface expression of MHC-II protein, but mRNA for MHC-II and CIITA (total, I, III, and IV) remained unchanged, suggesting posttranslational regulation. Splenic dendritic cells expressed type I CIITA but little type III or IV; CpG DNA induced their maturation and decreased types I and III CIITA, consistent with decreased MHC-II protein synthesis. CIITA types differ in regulation in various APCs under different stimuli, and the predominant type of CIITA varies at different stages of APC activation.

Animals↗
PubMed2002

Differences in antigen processing with haplotype-mismatched MHC class II heterodimers: Aalpha(d)Abeta(b) heterodimers participate in early endosomal processing.

MHC class II heterozygotes form haplotype-mismatched heterodimers (combining alpha and beta chains of different alleles). Transfected L-cells expressing Aalpha(d)Abeta(d) or Aalpha(d)Abeta(b) presented exogenous OVA(323-339) peptide to T cells with similar high efficiency, while Aalpha(b)Abeta(b) was less efficient and Aalpha(b)Abeta(d) was ineffective. In contrast, Aalpha(d)Abeta(b) greatly exceeded Aalpha(d)Abeta(d) in processing of intact OVA for presentation of OVA(323-339); Aalpha(b)Abeta(b) was even less efficient and Aalpha(b)Abeta(d) was ineffective. Of macrophages from C57BL/6 (H-2(b)), DBA/2 (H-2(d)) and B6D2F1 (H-2(bxd))mice, B6D2F1 macrophages had highest I-A expression and efficiency for OVA processing or presentation of exogenous OVA(323-339) peptide. Blocking antibodies specific for I-A chains showed that OVA processing by B6D2F1 macrophages primarily involved haplotype-mismatched Aalpha(d)Abeta(b) heterodimers, whereas Aalpha(d)Abeta(d) and Aalpha(b)Abeta(b) contributed more to presentation of exogenous OVA(323-339) peptide. OVA(323-339):I-A complexes were formed from OVA within 10 min with B6D2F1 macrophages but not until 20 min with C57BL/6 or DBA/2 macrophages, and OVA processing was more resistant to inhibition of late endocytic function by hypothermia (18 degrees C) in B6D2F1 than C57BL/6 or DBA/2 macrophages. These results indicate that Aalpha(d)Abeta(b) haplotype-mismatched heterodimers may contribute to antigen processing in early endocytic compartments.

Amino Acid Sequence↗