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Biomedical subjects

Daniel M Altmann

Publications and source records attributed to Daniel M Altmann.

At least 19 recordsLinked to original sources
PubMed2006

T cell responses to a non-glycosylated epitope predominate in type II collagen-immunised HLA-DRB1*0101 transgenic mice.

AIM: To study collagen-induced arthritis in human leucocyte antigen (HLA)-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules (MHC-II) and to determine T cell specificity against the arthritogenic CII(259-273) epitope of type II collagen either unmodified or post-translationally glycosylated at Lys(264). METHODS: Arthritis was induced by immunisation with human type II collagen in complete Freund's adjuvant and measured by footpad swelling, clinical score and histology. T cell responses were assessed by proliferation of spleen and lymph node cells and in antigen presentation assays, using T cell hybridomas specific for the glycosylated and non-glycosylated CII(259-273) epitope. RESULTS: The incidence of arthritis was 50% in DR1-transgenic mice lacking endogenous MHC-II molecules. Recall T cell responses in draining lymph nodes and spleen were consistently greater against the non-glycosylated epitope than to the glycosylated CII(259-273). Most of the T cell hybridomas generated from CII-immunised mice recognised the non-glycosylated CII epitope and this form of the epitope was also presented with 100-fold higher efficiency and 1 h faster kinetics by both macrophages and dendritic cells. CONCLUSION: This study shows that T cell responses to the non-glycosylated epitope of heterologous (human) CII are dominant in HLA-DR1 transgenic mice lacking MHC-II, which could contribute to the pathogenicity of autoimmune arthritis.

Animals↗
PubMed2006

The TCR Vbeta signature of bacterial superantigens spreads with stimulus strength.

Superantigens (Sags) induce large-scale stimulation of T lymphocytes by a mechanism distinct from conventional antigen presentation, involving direct MHC binding and stimulation of TCR families based on Vbeta gene usage. The specific Vbeta targets of a given Sag have, since the earliest studies in murine models, been considered a hallmark of that toxin. Bacterial Sags are implicated in the aetiology of a wide range of human diseases, although their role has been most clearly defined in toxic shock syndrome. While Sags have been defined by the Vbeta-specific changes in T cell repertoire they induce, human studies of in vitro stimulation or analysis of cells from infected patients have produced inconsistent findings. Here we have evaluated the contribution of HLA allelic polymorphisms and strength of stimulus to this response. We show that there are differences in binding and presentation of the staphylococcal Sag, staphylococcal enterotoxin A (SEA), by different HLA-DR alleles. We also show that the TCR Vbeta response, previously thought to be a fixed property defining a given Sag, varies with stimulus strength such that a broader repertoire of response is seen at higher concentrations or following presentation by high-binding class II types. Responses of human Vbeta8 and Vbeta1 to SEA, Vbeta5 to SEB and of Vbeta12 and Vbeta13 to streptococcal pyrogenic exotoxin A are absolutely dependent on stimulus strength. These findings have important implications for heterogeneity in the response to Sags and the consequent differences in susceptibility to severe toxic shock.

Antigen Presentation↗
PubMed2006

The impact of glycosylation on HLA-DR1-restricted T cell recognition of type II collagen in a mouse model.

OBJECTIVE: Type II collagen (CII) is a candidate autoantigen implicated in the pathogenesis of rheumatoid arthritis (RA). Posttranslational glycosylation of CII could alter intracellular antigen processing, leading to the development of autoimmune T cell responses. To address this possibility, we studied the intracellular processing of CII for presentation of the arthritogenic glycosylated epitope CII(259-273) to CD4 T cells in macrophages from HLA-DR1-transgenic mice. METHODS: HLA-DR1-transgenic mice were generated on a class II major histocompatibility complex-deficient background, and T cell hybridomas specific for the glycosylated and nonglycosylated epitope CII(259-273) were developed. Subcellular fractionation of macrophages was used to localize CII degradation to particular compartments and to identify the catalytic subtype of proteinases involved. RESULTS: We showed that the glycosylated CII(259-273) epitope required more extensive processing than did the nonglycosylated form of the same epitope. Dense fractions containing lysosomes were primarily engaged in the processing of CII for antigen presentation, since these compartments contained 1) enzyme activity that generated antigenic CII fragments bearing the arthritogenic glycosylated epitope, 2) the antigenic CII fragments themselves, 3) CII peptide-receptive HLA-DR1 molecules, and 4) peptide/HLA-DR1 complexes that could directly activate T cell hybridomas. Degradation of CII by dense fractions occurred optimally at pH 4.5 and was abrogated by inhibitors of serine and cysteine proteinases. CONCLUSION: Processing of the arthritogenic glycosylated CII(259-273) epitope, which is implicated in the induction of autoimmune arthritis, is more stringently regulated than is processing of the nonglycosylated form of the same epitope. Mechanisms of intracellular processing of the glycosylated epitope may constitute novel therapeutic targets for the treatment of RA.

Animals↗
PubMed2006

The human prion protein residue 129 polymorphism lies within a cluster of epitopes for T cell recognition.

T cell immune responses to central nervous system-derived and other self-antigens are commonly described in both healthy and autoimmune individuals. However, in the case of the human prion protein (PrP), it has been argued that immunologic tolerance is uncommonly robust. Although development of an effective vaccine for prion disease requires breaking of tolerance to PrP, the extent of immune tolerance to PrP and the identity of immunodominant regions of the protein have not previously been determined in humans. We analyzed PrP T cell epitopes both by using a predictive algorithm and by measuring functional immune responses from healthy donors. Interestingly, clusters of epitopes were focused around the area of the polymorphic residue 129, previously identified as an indicator of susceptibility to prion disease, and in the C-terminal region. Moreover, responses were seen to PrP peptide 121-134 containing methionine at position 129, whereas PrP 121-134 [129V] was not immunogenic. The residue 129 polymorphism was also associated with distinct patterns of cytokine response: PrP 128-141 [129M] inducing IL-4 and IL-6 production, which was not seen in response to PrP 128-141 [129V]. Our data suggest that the immunogenic regions of human PrP lie between residue 107 and the C-terminus and that, like with many other central nervous system antigens, healthy individuals carry responses to PrP within the T cell repertoire and yet do not experience deleterious autoimmune reactions.

Algorithms↗
PubMed2006

Inhibition of macropinocytosis blocks antigen presentation of type II collagen in vitro and in vivo in HLA-DR1 transgenic mice.

Professional antigen-presenting cells, such as dendritic cells, macrophages and B cells have been implicated in the pathogenesis of rheumatoid arthritis, constituting a possible target for antigen-specific immunotherapy. We addressed the possibility of blocking antigen presentation of the type II collagen (CII)-derived immunodominant arthritogenic epitope CII259-273 to specific CD4 T cells by inhibition of antigen uptake in HLA-DR1-transgenic mice in vitro and in vivo. Electron microscopy, confocal microscopy, subcellular fractionation and antigen presentation assays were used to establish the mechanisms of uptake, intracellular localization and antigen presentation of CII by dendritic cells and macrophages. We show that CII accumulated in membrane fractions of intermediate density corresponding to late endosomes. Treatment of dendritic cells and macrophages with cytochalasin D or amiloride prevented the intracellular appearance of CII and blocked antigen presentation of CII259-273 to HLA-DR1-restricted T cell hybridomas. The data suggest that CII was taken up by dendritic cells and macrophages predominantly via macropinocytosis. Administration of amiloride in vivo prevented activation of CII-specific polyclonal T cells in the draining popliteal lymph nodes. This study suggests that selective targeting of CII internalization in professional antigen-presenting cells prevents activation of autoimmune T cells, constituting a novel therapeutic strategy for the immunotherapy of rheumatoid arthritis.

Amiloride↗
PubMed2005

The mechanism of superantigen-mediated toxic shock: not a simple Th1 cytokine storm.

The profound clinical consequences of Gram-positive toxic shock are hypothesized to stem from excessive Th1 responses to superantigens. We used a new superantigen-sensitive transgenic model to explore the role of TCRalphabeta T cells in responses to staphylococcal enterotoxin B (SEB) in vitro and in two different in vivo models. The proliferative and cytokine responses of HLA-DR1 spleen cells were 100-fold more sensitive than controls and were entirely dependent on TCRalphabeta T cells. HLA-DR1 mice showed greater sensitivity in vivo to two doses of SEB with higher mortality and serum cytokines than controls. When d-galactosamine was used as a sensitizing agent with a single dose of SEB, HLA-DR1 mice died of toxic shock whereas controls did not. In this sensitized model of toxic shock there was a biphasic release of cytokines, including TNF-alpha, at 2 h and before death at 7 h. In both models, mortality and cytokine release at both time points were dependent on TCRalphabeta T cells. Anti-TNF-alpha pretreatment was protective against shock whereas anti-IFN gamma pretreatment and delayed anti-TNF-alpha treatment were not. Importantly, anti-TNF-alpha pretreatment inhibited the early TNF-alpha response but did not inhibit the later TNF-alpha burst, to which mortality has previously been attributed. Splenic T cells were shown definitively to be the major source of TNF-alpha during the acute cytokine response. Our results demonstrate unequivocally that TCRalphabeta T cells are critical for lethality in toxic shock but it is the early TNF-alpha response and not the later cytokine surge that mediates lethal shock.

Animals↗
PubMed2005

HLA-C and killer cell immunoglobulin-like receptor genes in idiopathic bronchiectasis.

RATIONALE: In idiopathic bronchiectasis, lung inflammation and chronic bacterial infection lead to progressive lung damage. A possible role for natural killer (NK) cells is suggested by the observation that familial bronchiectasis occurs in a rare group of individuals with impaired HLA class I expression and consequent NK cell dysfunction. OBJECTIVE: Because the HLA-C locus and killer cell immunoglobulin-like receptors (KIRs) are of key importance for NK cell recognition, we analyzed HLA-C/KIR combinations by genotyping patients with idiopathic bronchiectasis. METHODS: Genomic DNA from 96 individuals with idiopathic bronchiectasis and 101 control subjects was analyzed by polymerase chain reaction with sequence-specific primers. High-resolution HLA-C genotyping was performed in addition to KIR analysis. RESULTS: HLA-Cw*03 alleles and, in particular, HLA-C group 1 homozygosity are associated with the presence of bronchiectasis. Analysis of the relationship between HLA-C and KIR genes suggests a shift to activatory NK cell function. CONCLUSION: This is the first demonstration of genetic susceptibility in idiopathic bronchiectasis. The association with HLA-C group 1 homozygosity, and the interplay between HLA-C and KIR genes, argue for a role for NK cells in the progressive lung damage seen in this disease. This will require further investigation using functional studies.

Bronchiectasis↗
PubMed2005

Pulmonary infection with Cryptococcus neoformans in the face of underlying sarcoidosis.

We present a case of limited pulmonary cryptococcal infection following exposure to pigeon excreta in a patient with sarcoidosis. The pathogenicity of Cryptococcus neoformans depends on the interplay between the immune status of the host and the virulence of the infecting strain. It can range from asymptomatic lung colonization in the immunocompetent host to rapidly progressive meningitis in immunocompromised patients. Immunological models of respiratory disease emphasize a distinction between infections associated with immune suppression on the one hand and diseases such as sarcoidosis believed to involve an excessive Th1-mediated immune response on the other. This case exemplifies the complex nature of immunological responses in the lung and highlights the importance of considering the possibility of co-existent fungal infection in individuals with sarcoidosis. Novel immunotherapeutic options for cryptococcal infection are discussed.

Adult↗
PubMed2005

Stat4-null non-obese diabetic mice: protection from diabetes and experimental allergic encephalomyelitis, but with concomitant epitope spread.

There is much interest in therapeutic manipulation of cytokine responses in autoimmunity, yet studies in mouse models have sometimes produced conflicting findings as to the role of particular mediators in disease. Examples include the contradictory findings regarding susceptibility to experimental allergic encephalomyelitis (EAE) or diabetes in knockout mice for various individual Th1 or Th2 cytokines or their receptors. An alternative approach to the analysis of Th1 and Th2 mechanisms in these diseases is to investigate strains carrying a null mutation for molecules involved in cytokine receptor signal transduction, signal transducer and activator of transcription (Stat4) and Stat6. Stat4 is pivotal in Th1 polarization, being activated when IL-12 binds the IL-12R and leading to the production of IFNgamma. We here report disease susceptibility in non-obese diabetic mice carrying a Stat4-null mutation. Knockout mice were almost completely protected from diabetes, only rarely showing pancreatic peri-islet infiltrates. Furthermore, there was near complete protection from the induction of EAE by either of the two encephalitogenic myelin epitopes. Despite this protection, Stat4-null mice showed clear epitope spread compared with controls during myelin oligodendrocyte glycoprotein-induced EAE as judged by T cell proliferation, although this was not associated with a strong Th1 response to the initial or spread epitope and, furthermore, there was no evidence of a switch to Th2 cytokines.

Animals↗
PubMed2005

High incidence of spontaneous disease in an HLA-DR15 and TCR transgenic multiple sclerosis model.

Multiple sclerosis (MS) is thought to involve CD4 T cell recognition of self myelin, many studies focusing on a pathogenic role for anti-myelin, HLA-DR15-restricted T cells. In experimental allergic encephalomyelitis, it is known which epitopes trigger disease and that disease is associated with determinant spread of T cell reactivity. Characterization of these events in human MS is critical for the development of peptide immunotherapies, but it has been difficult to define the role of determinant spread or define which epitopes might be involved. In this study, we report humanized transgenic mice, strongly expressing HLA-DR15 with an MS-derived TCR; even on a RAG-2 wild-type background, mice spontaneously develop paralysis. Disease, involving demyelination and axonal degeneration, correlates with inter- and intramolecular spread of the T cell response to HLA-DR15-restricted epitopes of myelin basic protein, myelin oligodendrocyte glycoprotein, and alphaB-crystallin. Spread is reproducible and progressive, with two of the epitopes commonly described in responses of HLA-DR15 patients. The fact that this pattern is reiterated as a consequence of CNS tissue damage in mice demonstrates the value of the transgenic model in supplying an in vivo disease context for the human responses. This model, encompassing pathologically relevant, spontaneous disease with the presentation of myelin epitopes in the context of HLA-DR15, should offer new insights and predictions about T cell responses during MS as well as a more stringent test bed for immunotherapies.

Animals↗
PubMed2005

Spread of T lymphocyte immune responses to myelin epitopes with duration of multiple sclerosis.

Although the primary cause of multiple sclerosis (MS) is unclear, evidence supports a role for autoimmune attack of myelin by T lymphocytes. However, it has been difficult to relate patterns of autoimmunity to pathogenesis. In mouse models, the case has been made for relapsing and remitting disease driven by epitope spread: an initial lesion leads to presentation of central nervous system antigens, in turn triggering the next wave of autoimmune T cells of different specificity, the response thus broadening. Few studies have been done to determine whether these events could be important over the longer time scale of human disease. We compared T cell responses with a panel of myelin epitopes in clinically isolated syndrome patients with a first attack, patients with MS with a mean disease duration of 0.95 years, and patients with MS having a mean disease duration of 15.9 years. T cells from patients with long-term disease recognize more myelin epitopes than patients with recent-onset disease. The epitope myelin basic protein 131-149, in particular, was more commonly recognized by patients with long-term disease. The data support the notion that the T cell response in MS broadens with time and is thus implicated in the ongoing pathogenic process. However, there was no clear correlation between disease severity and number of epitopes recognized. This may argue against a simple causal role of epitope spread in driving progression, as has been suggested in experimental allergic encephalomyelitis.

Adult↗
PubMed2005

Developing vaccines to counter bioterrorist threats.

Large and innovative research programs are underway to define the immune parameters for vaccines against a wide array of pathogens considered to represent a potential bioterrorist threat. However, the development and utilization of such vaccines presents a number of predicaments that have not previously been addressed by the field of vaccinology.

Animals↗
PubMed2004

Immune responses and tolerance to the RhD blood group protein in HLA-transgenic mice.

RhD is a major blood group and the most important target antigen in hemolytic disease of the newborn (HDN). The aims of this study were to establish a humanized mouse model of responses to the RhD protein and to test whether these could be prevented by the induction of immune tolerance. HLA-DR15 is a major restricting element for human T-helper (Th) cells specific for RhD protein, and expression of this HLA-DR transgene was found to confer on mice the ability to respond to immunization with purified RhD protein. Synthetic peptides containing dominant Th cell epitopes, previously identified from studies of human alloimmunized donors, were administered to the nasal mucosa of transgenic mice before immunization with purified RhD protein. Treatment with each of the 4 dominant peptides, RhD52-66, RhD97-111, RhD117-131, and RhD177-191, inhibited T-cell priming and prevented antibody responses to the RhD protein. The ability to induce such active tolerance offers the prospect of peptide immunotherapy as a replacement for passive immune globulin in the prophylaxis of HDN.

Animals↗
PubMed2004

Modified amino acid copolymers suppress myelin basic protein 85-99-induced encephalomyelitis in humanized mice through different effects on T cells.

A humanized mouse bearing the HLA-DR2 (DRA/DRB1*1501) protein associated with multiple sclerosis (MS) and the myelin basic protein (MBP) 85-99-specific HLA-DR2-restricted T cell receptor from an MS patient has been used to examine the effectiveness of modified amino acid copolymers poly(F,Y,A,K)n and poly-(V,W,A,K)n in therapy of MBP 85-99-induced experimental autoimmune encephalomyelitis (EAE) in comparison to Copolymer 1 [Copaxone, poly(Y,E,A,K)n]. The copolymers were designed to optimize binding to HLA-DR2. Vaccination, prevention, and treatment of MBP-induced EAE in the humanized mice with copolymers FYAK and VWAK ameliorated EAE more effectively than Copolymer 1, reduced the number of pathological lesions, and prevented the up-regulation of human HLA-DR on CNS microglia. Moreover, VWAK inhibited MBP 85-99-specific T cell proliferation more efficiently than either FYAK or Copolymer 1 and induced anergy of HLA-DR2-restricted transgenic T cells as its principle mechanism. In contrast, FYAK induced proliferation and a pronounced production of the antiinflammatory T helper 2 cytokines IL-4 and IL-10 from nontransgenic T cells as its principle mechanism of immunosuppression. Thus, copolymers generated by using different amino acids inhibited disease using different mechanisms to regulate T cell responses.

Amino Acid Sequence↗
PubMed2004

HLA class II polymorphisms determine responses to bacterial superantigens.

The excessive immunological response triggered by microbial superantigens has been implicated in the etiology of a wide range of human diseases but has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Because MHC class II presentation of superantigens to T cells is not MHC-restricted, the possibility that HLA polymorphisms could influence superantigenicity, and thus clinical susceptibility to the toxicity of individual superantigens, has received little attention. In this study, we demonstrate that binding of streptococcal and staphylococcal superantigens to HLA class II is influenced by allelic differences in class II. For the superantigen streptococcal pyrogenic exotoxin A, class II binding is dependent on DQ alpha-chain polymorphisms such that HLA-DQA1*01 alpha-chains show greater binding than DQA1*03/05 alpha-chains. The functional implications of differential binding on T cell activation were investigated in various experimental systems using human T cells and murine Vbeta8.2 transgenic cells as responders. These studies showed quantitative and qualitative differences resulting from differential HLA-DQ binding. We observed changes in T cell proliferation and cytokine production, and in the Vbeta specific changes in T cell repertoire that have hitherto been regarded as a defining feature of an individual superantigen. Our observations reveal a mechanism for the different outcomes seen following infection by toxigenic bacteria.

Amino Acid Sequence↗
PubMed2004

Disease-related epitope spread in a humanized T cell receptor transgenic model of multiple sclerosis.

While EAE has been an invaluable model for the immunopathogenesis of multiple sclerosis, it has sometimes been difficult to bridge the gap between findings and therapies in the rodent models and the cellular and molecular interactions that can be studied in the human disease. Humanized transgenic models offer a means of achieving this, through the expression of disease-implicated HLA class II molecules, co-expressed with a cognate HLA-class II-restricted, myelin-specific TCR derived from a human T cell clone implicated in disease. We have generated such a transgenic line, called line 8, that co-expresses a high level of HLA-DR15 and a human TCR specific for HLA-DR15/MBP 85-99. T cells from the transgenic line are skewed to the CD4 single-positive compartment and produce IFN-gamma in response to peptide from mylein basic protein. Mice develop a spontaneous disease phenotype, showing poverty of movement, although this rarely develops into paralysis except following immunization with peptide. On induction of paralysis by immunization with peptide, disease correlates with epitope spread to a number of additional, HLA-DR15-restricted myelin epitopes. This model should be valuable for analyzing epitope spread in a humanized immunogenetic environment and for the testing of specific immunotherapies.

Animals↗
PubMed2002

The bacterial superantigen streptococcal mitogenic exotoxin Z is the major immunoactive agent of Streptococcus pyogenes.

The gene encoding streptococcal mitogenic exotoxin Z (SMEZ) was disrupted in Streptococcus pyogenes. Despite the presence of other superantigen genes, mitogenic responses in human and murine HLA-DQ transgenic cells were abrogated when cells were stimulated with supernatant from the smez(-) mutant compared with the parent strain. Remarkably, disruption of smez led to a complete inability to elicit cytokine production (TNF-alpha, lymphotoxin-alpha, IFN-gamma, IL-1 and -8) from human cells, when cocultured with streptococcal supernatants. The potent effects of SMEZ were apparent even though transcription and expression of SMEZ were barely detectable. Human Vbeta8(+) T cell proliferation in response to S. pyogenes was SMEZ-dependent. Cells from HLA-DQ8 transgenic mice were 3 logs more sensitive to SMEZ-13 than cells from HLA-DR1 transgenic or wild-type mice. In the mouse, SMEZ targeted the human Vbeta8(+) TCR homologue, murine Vbeta11, at the expense of other TCR T cell subsets. Expression of SMEZ did not affect bacterial clearance or survival from peritoneal streptococcal infection in HLA-DQ8 mice, though effects of SMEZ on pharyngeal infection are unknown. Infection did lead to a rise in Vbeta11(+) T cells in the spleen which was partly reversed by disruption of the smez gene. Most strikingly, a clear rise in murine Vbeta4(+) cells was seen in mice infected with the smez(-) mutant S. pyogenes strain, indicating a potential role for SMEZ as a repressor of cognate anti-streptococcal responses.

Acute Disease↗