Biomedical subjects

T Ottenhoff

Publications and source records attributed to T Ottenhoff.

13 recordsLinked to original sources

PubMed2001

DNA immunization of HLA transgenic mice with a plasmid expressing mycobacterial heat shock protein 65 results in HLA class I- and II-restricted T cell responses that can be augmented by cytokines.

Infection with Mycobacterium tuberculosis (MTB) remains a major cause of morbidity and mortality world-wide. An effective vaccination strategy is the immunization with plasmid DNA (pDNA), expressing an antigen (Ag) from a pathogen in vivo, which results in specific immune response against the encoded protein as well as the pathogen itself or cells infected with it. To test the ability to induce HLA-restricted T cell immune response against a mycobacterial antigen in humans by pDNA vaccination, we have used transgenic mice that express HLA class I (A*0201/Kb) or HLA class II (DRB1*0301) molecules. pDNA immunization with mycobacterial heat shock protein 65 (Mhsp65)-expressing plasmid (P3M.65) resulted in HLA-II-restricted, Ag-specific T cell-mediated immune responses characterized by proliferation and cytokine production. These T cell responses could be further augmented by the coinjection of P3M.65 and plasmid expressing murine GM-CSF. Furthermore, coimmunizing HLA-I transgenic mice with P3M.65 and a plasmid expressing murine IFN-gamma induced a specific cytotoxic T lymphocyte response restricted by HLA-A2. These results represent the first evidence of a concomitant in vivo induction of HLA class I- as well as class II-restricted T cell responses by pDNA immunization, which is induced or augmented by the codelivery of cytokine-expressing plasmids, supporting its potential use in clinical trials.

Animals↗

PubMed2001

Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance.

By applying density gradient electrophoresis (DGE) to human macrophages infected with Mycobacterium bovis BCG, we were able to separate three different bacterial fractions representing arrested phagosomes, phagolysosomes and mycobacterial clumps. After further purification of the phagosomal population, we found that isolated phagosomes containing live BCG were arrested in maturation as they exhibited only low amounts of the lysosomal glycoprotein LAMP-1 and processing of the lysosomal hydrolase cathepsin D was blocked. In addition, low amounts of MHC class I and class II molecules and the absence of HLA-DM suggest sequestration of mycobacterial phagosomes from antigen-processing pathways. We further investigated the involvement of the actin-binding protein coronin in intracellular survival of mycobacteria and showed that human coronin, as well as F-actin, were associated with early stages of mycobacterial phagocytosis but not with phagosome maintenance. Therefore, we conclude that the unique DGE migration pattern of arrested phagosomes is not as a result of retention of coronin, but that there are other proteins or lipids responsible for the block in maturation in human macrophages.

Bacterial Proteins↗

PubMed2000

Modulation of the major histocompatibility complex class II-associated peptide repertoire by human histocompatibility leukocyte antigen (HLA)-DO.

Antigen presentation by major histocompatibility complex class II molecules is essential for antibody production and T cell activation. For most class II alleles, peptide binding depends on the catalytic action of human histocompatibility leukocyte antigens (HLA)-DM. HLA-DO is selectively expressed in B cells and impedes the activity of DM, yet its physiological role remains unclear. Cell surface iodination assays and mass spectrometry of major histocompatibility complex class II-eluted peptides show that DO affects the antigenic peptide repertoire of class II. DO generates both quantitative and qualitative differences, and inhibits presentation of large-sized peptides. DO function was investigated under various pH conditions in in vitro peptide exchange assays and in antigen presentation assays using DO(-) and DO(+) transfectant cell lines as antigen-presenting cells, in which effective acidification of the endocytic pathway was prevented with bafilomycin A(1), an inhibitor of vacuolar ATPases. DO effectively inhibits antigen presentation of peptides that are loaded onto class II in endosomal compartments that are not very acidic. Thus, DO appears to be a unique, cell type-specific modulator mastering the class II-mediated immune response induced by B cells. DO may serve to increase the threshold for nonspecific B cell activation, restricting class II-peptide binding to late endosomal compartments, thereby affecting the peptide repertoire.

Antigen Presentation↗

PubMed1998

Strongly increased efficiency of altered peptide ligands by mannosylation.

Altered peptide ligands (APL) have the potential to modulate pathogenic T cell reactivity. High concentrations of APL, however, are required to achieve efficient blocking of the T cell response. We have therefore investigated whether improved delivery of APL to professional antigen-presenting cells (APC) can lead to more efficient application of such peptides. For this purpose APL were bis-mannosylated in order to facilitate their uptake by mannose receptor-positive dendritic cells (DC) in vitro. We present evidence that a 100- to 1000-fold lower concentration of bis-mannosylated APL was sufficient for complete blocking of the proliferative T cell response against the agonist peptide compared to the non-mannosylated APL. Moreover, bis-mannosylated APL were similarly effective in the inhibition of the T cell response against whole protein antigens. In contrast, unrelated, bis-mannosylated class II binding peptides were ineffective, indicating that the increased efficiency of the mannosylated APL was not due to competition for binding to class II molecules. Furthermore, a strong increase in the efficiency of presentation of APL was also observed when macrophages and peripheral blood mononuclear cells were used as APC. Thus, bis-mannosylation of APL greatly increases their potency to inhibit proliferative T cell responses. Moreover, it is likely that the use of bis-mannosylated APL will result in preferential presentation by mannose receptor-positive, professional APC. These results may be of relevance for more effective use of APL for immunoregulation in vivo.

Adjuvants, Immunologic↗

PubMed1997

Salivary anti-hsp65 antibodies as a diagnostic marker for gingivitis and a possible link to atherosclerosis.

Levels of specific salivary IgA antibodies against mycobacterial heat shock protein (hsp) 65 are significantly increased in patients with gingivitis when compared to clinically healthy subjects. The process of identifying the hsp65 epitopes recognized by the salivary antibodies, binding to overlapping 15-mer-hsp65 peptides, was assessed. Time-resolved fluorescence immunoassays using 15-mer overlapping peptides spanning the whole hsp65 molecule revealed six distinct sequences recognized by anti-hsp65 IgA antibodies. Due to the high degree of sequence homology between mycobacterial hsp65, cognates of the hsp60 family of oral bacterial flora and human hsp60, these six epitopes may serve as cross-reactive autoantigens in certain circumstances in vivo and could incite an autoimmune response that contributes to the initiation of gingivitis.

Adult↗

PubMed1997

Epitope specificity of anti-heat shock protein 65/60 serum antibodies in atherosclerosis.

Levels of specific antibodies (Ab) against mycobacterial and human heat shock protein (hsp) 65/60 are increased in the sera of patients with atherosclerotic lesions and have been demonstrated to be capable of mediating endothelial cytotoxicity. To clarify the antigen epitopes recognized by these serum Abs, Ab binding to hsp65 deletion mutants (Dms), as well as to overlapping 15-mer and 8-mer hsp65 peptides, was assessed. Western blotting of hsp65 Dms indicated the presence of at least one epitope between amino acid (aa) residues 171 and 276, recognized by both high-titer sera and affinity-purified anti-hsp65/60 Ab. Fluorescence immunoassays using 53 15-mer peptides and Pin ELISA using 526 7-mer peptides demonstrated three distinct, conserved sequences with high affinity to high-titer sera and purified anti-hsp65/60 Ab. Two N-terminal sequences, aa 97-109 and aa 179-187, and one C-terminal sequence, aa 504-512, were identified. These three epitopes recognized by anti-hsp65/60 Ab may serve as autoantigens in certain circumstances in vivo. This phenomenon could contribute to the initiation of atherosclerosis by an autoimmune reaction.

Amino Acid Sequence↗

PubMed1995

Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae.

The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox-active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N-terminal part of the protein corresponds to TR and the C-terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.

Amino Acid Sequence↗

PubMed1995

Molecular characterization and T-cell-stimulatory capacity of Mycobacterium leprae antigen T5.

A Mycobacterium leprae lambda gt11 clone designated T5 has previously been selected with sera from tuberculoid leprosy patients. Sequence analysis of this clone revealed the presence of two overlapping open reading frames (ORFs) present on the two cDNA strands. The first ORF codes for the serologically recognized antigen, which was fused with the lacZ gene in the lambda gt11 clone. The second ORF, present on the complementary strand, displays strong sequence homology with the aspartyl-tRNA synthetase genes of Escherichia coli and Thermus thermophilus. Here we show that the purified T5-derived product, overexpressed in E. coli, is recognized by T cells of the majority of the leprosy patients tested, including lepromatous leprosy patients who do not respond to whole M. leprae bacilli.

Amino Acid Sequence↗

PubMed1993

A novel, antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders.

It is now well established that cultured human melanocytes are capable of expressing immunologically important cell surface molecules and that they can produce cytokines. Not all cells with the ability to express MHC class II molecules are capable of effective Ag presentation. However, the dendritic nature of melanocytes, their strategic position within the skin, and their phagocytic capacity seem to suggest a role for these cells in processing and presenting Ag. This study demonstrates that cultured normal human skin melanocytes can present peptide Ag, and process and present the mycobacterial HSP65 kDa protein and whole Mycobacterium leprae sonicate to CD4+ cytotoxic proliferative T cell clones in an Ag-specific and HLA-class II-restricted manner. T cell stimulation was dependent on costimulatory signals, i.e., LFA-3/CD2 and LFA-1/ICAM-1. Besides eliciting a T cell proliferative response, our studies further demonstrate that melanocytes can function as target cells for T cell-mediated cytotoxicity. The described Ag-processing and -presenting functions of melanocytes, taken together with in vivo behavior of melanocytes in hypopigmentation, provide new clues for the etiopathogenesis of melanin pigmentary disorders.

Antigen Presentation↗

PubMed1990

Serum tumor necrosis factor levels and disease dissemination in leprosy and leishmaniasis.

It has been suggested that tumor necrosis factor alpha (TNF alpha) may serve as an important antigen-independent host defense mechanism against parasitic organisms. Sera from 66 patients with leishmaniasis and 68 patients with leprosy, all from Ethiopia, were tested for TNF alpha using an enzyme-linked immunoassay. Sera from patients with the multi-parasitic/bacillary type of disease (visceral or diffuse cutaneous leishmaniasis and lepromatous leprosy), known to be associated with absent or low specific T cell response, contained significantly higher TNF alpha titers than those of patients with pauci-parasitic/bacillary disease (localized cutaneous leishmaniasis and nonlepromatous leprosy). High titers of TNF alpha in the absence of a functioning T cell response do not appear to confer resistance against Leishmania aethiopica and Mycobacterium leprae.

Adolescent↗

PubMed1987

Human anti-idiotypic T lymphocyte clones are activated by autologous anti-rabies virus antibodies presented in association with HLA-DQ molecules.

The regulatory function of antigen-specific T cells in human antibody responses to protein and carbohydrate determinants of many viral and bacterial antigens has extensively been studied in systems involving in vitro triggering of B cells by antigens or polyclonal activators. Although amply documented in experimental murine models, the existence of T helper cells with receptor specificity for idiotypic determinants of B cell immunoglobulins has not been demonstrated in a human system. We are interested in T helper cell recognition of idiotypic determinants of virus-specific antibody, secreted by human B cells in response to viral antigens, and in the role which such idiotype-specific T helper cells play, alone or in concert with virus-specific T helper cells, to regulate the antibody response. Understanding of the function of different T helper cell subsets in an anti-viral antibody response and especially of the mechanisms of idiotype recognition by T cells is important for the development, and future application in man of idiotype vaccines, the potential of which has been indicated for different pathogens in several animal species. It was realized that for the efficient characterization of each of the T helper cell subsets, the availability of cloned populations of T cells would be inevitable. Furthermore, we argued that if, as predicted by Jerne, idiotype recognizing T helper cells are involved in physiological idiotype regulation in the course of an immune response--e.g., following encounter with virus--cloned populations of T cells should best be obtained by immunization protocols closely mimicking the physiological situation. In a previous report we described the induction of a secondary antibody response in human peripheral blood mononuclear cells (PBMC) in vitro by rabies virus antigen. This response was shown to be T helper cell dependent, and rabies virus-specific T helper cell clones have recently been obtained in our laboratory. The present study describes the generation of cloned lines of anti-idiotypic T4+ cells from rabies virus immune PBMC restimulated with rabies virus antigen in vitro. The cloned T cell lines were found to respond to circulating autologous antibody exhibiting specificity to rabies virus, but not to rabies virus antigen. The clonal proliferation, induced by this "auto-anticlonotypic" antibody, was found to be preceded by modulation of the T3/Ti molecular complex and required presentation of the antibodies by antigen presenting cells in association with HLA-DQ molecules. This observation of MHC-restricted idiotype recognition by human T cells, may have important consequences for the understanding of the regulation of th

Antibodies, Anti-Idiotypic↗

PubMed1986

Quantitative and qualitative differences in HLA-DR molecules correlated with antigen-presentation capacity.

The monoclonal antibodies 7.3.19.1 (anti-DRw52-like) and B8.11.2 (anti-DR framework) were used for the isolation and characterization of HLA class II molecules expressed by HLA-DR3 and DR5 homozygous B cell lines. Sequential immunoprecipitation studies demonstrated that from these cells class II molecules can be isolated which are characterized by the presence or absence of DR framework (DR) and DRw52-like (DRw62) determinants: (DR+, DRw52+), (DR+, DRw52-) and (DR-, DRw52+). The DR3 donor cells appeared to express only the (DR+, DRw52+) and (DR-, DRw52+) class II molecules whereas DR5-positive cells express only the (DR+, DRw52+) and (DR+, DRw52-) class II molecules. Besides qualitative differences some of the above-mentioned molecules appeared to differ in their levels of expression. To investigate whether this might have functional implications, cells with the HLA-DR3 and -5 haplotypes were used to present antigen purified protein derivative of tuberculin (PPD) to PPD-specific T cell lines and the blocking capacity of the two monoclonal antibodies 7.3.19.1 and B8.11.2 was determined. A remarkable correlation was observed between the type of class II molecule blocked by these monoclonal antibodies and its quantitative expression. However, (DR-, DRw52+) molecules, clearly expressed by DR3 cells, were not involved in the presentation of PPD. This indicates that not only quantitative but also qualitative aspects may play a role in the selection of the type of class II molecule that will be involved in antigen presentation.

Antibodies, Monoclonal↗

PubMed1986

T90/44 (9.3 antigen). A cell surface molecule with a function in human T cell activation.

T90/44 is a cell surface antigen which is present on human T cells of the helper and cytotoxic subsets and which binds the 9.3 monoclonal antibody (9.3 mAb). It is expressed in the form of 90-kDa disulfide-bonded dimers of a 44-kDa polypeptide and of free 44-kDa subunits. The function of T90/44 was investigated in a series of T cell function assays. 9.3 mAb was found to inhibit the activation of class II-restricted cloned T helper cells derived from leprosy patients and reactive with M. leprae antigens. The inhibition was first found at 1-10 ng/ml 9.3 mAb and regularly increased with the antibody concentration. The extent of the inhibition varied among different T cell clones in proportion to the respective different levels of T90/44 expression at their cell surface. The proliferative responses of peripheral blood lymphocytes (PBL) to purified protein derivative of M. tuberculosis (PPD) and tetanus toxoid were enhanced by the 9.3 mAb resulting in up to 20-30-fold increase of [3H]-thymidine incorporation. After phytohemagglutinin-induced activation of PBL, the number of T90/44 molecules per cell expressed at the cell surface rose from day 0 to day 7 by a factor of about 10. High concentrations of 9.3 mAb (5-10 micrograms/ml) at low cell densities and in the presence of monocytes in culture media supplemented by fetal calf serum were directly mitogenic for resting lymphocytes. The cytolytic effector functions of class I-restricted cytotoxic T lymphocytes (CTL) were not modulated by 9.3 mAb. The mixed lymphocyte reactions of three class I-restricted CTL to their specific target cells were found not to be significantly influenced by 9.3 mAb. In conclusion it is proposed that an antigen-independent T cell activation pathway can be entered at T90/44.

Antibodies, Monoclonal↗