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G T Nepom

Publications and source records attributed to G T Nepom.

At least 37 records · Page 2Linked to original sources
PubMed2000

Molecular aspects of HLA class II alphabeta heterodimers associated with IDDM susceptibility and protection.

HLA-DQ alleles are strongly associated with IDDM susceptibility and protection. Studies assessing the molecular properties of HLA-DR, a HLA class II locus in linkage disequilibrium with HLA-DQ, have made substantial contributions toward elucidating the structure and function of HLA class II molecules. Reports on the molecular properties of HLA-DQ are now following and have revealed interesting observations regarding the stability of HLA-DQ alphabeta heterodimers. Future work is expected to provide an understanding of the mechanism by which HLA-DQ is associated with IDDM susceptibility and protection.

Animals↗
PubMed1999

MHC-peptide ligand interactions establish a functional threshold for antigen-specific T cell recognition.

Antigen-specific T cell recognition is dependent on the functional density of the TCR-ligand, which consists of specific MHC molecules and a specifically bound peptide. We have examined the influence of the affinity and concentration of exogenous peptide and the density of specific MHC molecules on the proliferation of a CD4+, DQA1*0501/DQB1*0201 (DQ2.1)-restricted, HSV-2-specific T cell clone. Using antigen peptide analogs with different mutations of known DQ2-anchor residues, T cell response was reduced in an peptide-affinity and - concentration specific manner. The decrease using weaker binding peptides was gradual as stimulation with a peptide with intermediate affinity yielded intermediate T cell proliferation and the poorest binding peptide induced an even weaker T cell response. MHC class II density on the APC was modified using DQ2 homo- and heterozygous B-LCLs as APCs, however this variation of MHC concentration had no effect on T cell proliferation. We interpret this as a reflection of a low threshold for activation of the T cell clone, in which peptide-MHC avidity is the over-riding determinant of the strength of ligand signal.

Alleles↗
PubMed1999

Peptide binding affinity and pH variation establish functional thresholds for activation of HLA-DQ-restricted T cell recognition.

Peptides derived from the HSV-2 VP16 protein were utilized for studies of peptide binding to DQ0302 molecules and T cell activation at both neutral and acidic pH. The native peptide VP16 430-444 contains an Asp at position 442, binds to DQ0302 strongly, with a Kd value of 50nM at acidic pH and very weakly, with a Kd value of greater than 10 microM at neutral pH. A truncated version of 430-444, i.e., VP16 433-442, binds with an affinity 10-fold lower compared to 430-444 at acidic pH, and binding at neutral pH was barely detectable. The homologous peptide 430-444,442A has an Asp to Ala substitution at position 442 and binds to DQ0302 with a Kd similar to 433-442. The short truncated analog 433-442A binds very poorly at both acidic and neutral pH. Both the wild type 430-444 and 433-442 peptides stimulated a HSV-specific T cell clone after a brief incubation with antigen presenting cells (APC) expressing DQ0302 at acidic pH. Much higher concentrations of wild type peptides were needed to activate T cells at neutral pH. In contrast, APC pulsed with Ala-substituted peptides 430-444,442A or 433-442A at neutral pH failed to stimulate the T cell clone, while APC pulsed at acidic pH and subsequently washed led to successful T cell activation. The Ala-substituted peptide was recognized by the T cell clone at neutral pH only when it was present in the APC culture throughout the stimulation process. While the MHC-peptide complexes formed with the native peptide are stable, complexes formed with the Ala-substituted peptide had a functional t1/2 of less than 4 hr at neutral pH.

Amino Acid Sequence↗
PubMed1999

Impaired binding of a DQ2 and DQ8-binding HSV VP16 peptide to a DQA1*0501/DQB1*0302 trans class II heterodimer.

DQalpha and DQbeta trans heterodimeric HLA-DQ molecules form in individuals heterozygous for the DQ2 and DQ8 specificities. Unique functions and disease associations have been postulated for such trans-dimers, which may be different from cis-encoded DQ molecules encoded by the corresponding haplotypes. We analyzed the ability of the trans-dimer encoded by HLA-DQA1*0501/DQB1*0302 to bind a peptide antigen which interacts with DQ molecules encoded by both parental haplotypes. Markedly impaired binding was observed, consistent with both the use of different anchor residues and with changes in levels of DQ cis-dimer availability for peptide binding interactions.

Animals↗
PubMed1999

Radioimmunoassay for glutamic acid decarboxylase-65.

BACKGROUND: Glutamic acid decarboxylase-65 (GAD65), the enzyme that catalyzes the formation of gamma-aminobutyric acid (GABA), is the major autoantigen in both type 1 (insulin-dependent) diabetes and stiff-man syndrome (SMS). The observation that GAD65 autoantibodies may be present for years before the clinical onset of diabetes raises the question of when GAD65 is available to initiate an immune response to allow the formation of autoantibodies. In order to address this question it will be necessary to measure GAD65 in tissue, cells, and plasma. METHODS: A radioimmunoassay (RIA) was developed for GAD65 based on the use of a polyclonal rabbit antiserum directed to the N-terminus of GAD65. RESULTS: Using the GAD65 RIA, we have determined the GAD65 content in a human GAD65 gene transfected cell line and in beta-cell preparations from different species. The assay detects an increase of immunoreactive GAD65 after glucose-stimulation and GAD65 that is discharged from rat beta cells after their exposure to the diabetogenic agent streptozotocin. It also measures good recovery of GAD65 added to human plasma samples. CONCLUSIONS: The GAD65 RIA makes it possible to determine both cellular and extracellular levels of GAD65; this might be useful in investigating the mechanisms leading to the formation of GAD65 autoantibodies in type 1 diabetes and SMS patients.

Animals↗
PubMed1999

MHC class II tetramers identify peptide-specific human CD4(+) T cells proliferating in response to influenza A antigen.

Antigen-specific T helper cells present in peripheral blood at very low frequencies are capable of rapid clonal expansion during antigenic challenge. The exquisite specificity of this response provides for activation and expansion of a very select cohort of T cells, a feature we have used to directly identify and quantify human epitope-specific T helper cells from peripheral blood. Soluble tetramerized class II MHC molecules, loaded with an immunodominant peptide from hemagglutinin (HA) and labeled with fluorescent dyes, were constructed and used to directly identify antigen-specific T cells from influenza-immune individuals. After 7 days of proliferation in response to stimulation by HA peptide or whole influenza vaccine, cells staining positive with the HA tetramer had undergone between 6 and 9 divisions and were CD3(+), CD4(+), CD25(+), and CD8(-), characteristic of activated T helper cells responding to antigen. The HA epitope-specific component of the complex response to whole influenza vaccine represented a major subset of proliferating T helper cells. Soluble class II tetramers allow a direct approach for the analysis of immunodominant antigenic specificities. The identification of antigen-specific T helper cells in the peripheral blood provides a means for tracking the immune response against infectious agents and in autoimmune disease. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Antigens, Viral↗
PubMed1999

Immunogenetic analysis suggests different pathogenesis for obese and lean African-Americans with diabetic ketoacidosis.

OBJECTIVE: When presenting with diabetic ketoacidosis (DKA), lean and obese patients differ in their subsequent clinical course. Although lean patients tend to remain insulin dependent, most obese patients recover endogenous insulin secretion and discontinue insulin therapy. The aim of this study was to determine whether obese African-American patients with DKA could be determined to have type 1 or type 2 diabetes based on insulin secretion or the presence of immunological and genetic markers. RESEARCH DESIGN AND METHODS: This was a prospective study that analyzed the clinical characteristics, insulin secretion indices, immunological markers (islet cell, GAD, ICA512, and insulin autoantibodies), and HLA susceptibility genes (DR/DQ) in 131 patients with DKA (77 obese and 54 lean), 51 obese patients with hyperglycemia but no DKA, and 25 nondiabetic subjects. All subjects were African-American. Beta-cell function was evaluated by the C-peptide response to glucagon (1 mg i.v.) within 48 h of resolution of DKA or hyperglycemia. RESULTS: The acute C-peptide response was lower in obese DKA patients (1.0+/-0.1 ng/ml) than in obese patients with hyperglycemia (1.7+/-0.2 ng/ml, P < 0.01), but was higher than that in lean DKA patients (0.2+/-0.1 ng/ml, both P < 0.01). The overall prevalence of autoantibodies in obese subjects with DKA (17%) and obese subjects with hyperglycemia (16%) was lower than that in lean subjects with DKA (65%, P < 0.01). Obese patients with hyperglycemia and positive autoantibodies had lower rates of insulin secretion than those without antibodies. Regardless of body weight, all DKA patients with GAD autoantibodies carried the DQB1*0201 allele. However, there were no significant differences in HLA distribution between the three patient groups. CONCLUSIONS: Our results indicate that most obese African-American patients with DKA have type 2 diabetes characterized by higher insulin secretion, the absence of autoimmune markers, and a lack of HLA genetic association. In contrast, most lean African-American patients with DKA have metabolic and immunological features of type 1 diabetes. At presentation, assessment of beta-cell function and determination of autoimmune markers allow for correct classification of diabetes in African-Americans with hyperglycemic crises.

Adult↗
PubMed1998

Exceptional stability of the HLA-DQA1*0102/DQB1*0602 alpha beta protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus.

HLA-DQ alleles are closely associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM) but the immunologic mechanisms involved are not understood. Structural studies of the IDDM-susceptible allele, HLA-DQA1*0301/DQB1*0302, have classified it as a relatively unstable dimer, particularly at neutral pH. This is reminiscent of studies in the nonobese diabetic mouse, in which I-A(g7) is relatively unstable, in contrast to other murine I-A alleles, suggesting a correlation between unstable MHC class II molecules and IDDM susceptibility. We have addressed this question by analysis of dimer stability patterns among various HLA-DQ molecules. In EBV-transformed B-lymphoblastoid cell lines and PBL, the protein encoded by the IDDM-protective allele HLA-DQA1*0102/DQB1*0602 was the most SDS stable when compared with other HLA-DQ molecules, including HLA-DQA1*0102/DQB1*0604, a closely related allele that is not associated with protection from IDDM. Expression of six different HLA-DQ allelic proteins and three different HLA-DR allelic proteins in the bare lymphocyte syndrome cell line, BLS-1, revealed that HLA-DQA1*0102/DQB1*0602 is SDS stable even in the absence of HLA-DM, while other HLA class II molecules are not. These results suggest that the molecular property of HLA-DQ measured by resistance to denaturation of the alphabeta dimer in SDS may play a role in IDDM protection.

Alleles↗
PubMed1998

Structural basis of specificity and degeneracy of T cell recognition: pluriallelic restriction of T cell responses to a peptide antigen involves both specific and promiscuous interactions between the T cell receptor, peptide, and HLA-DR.

TCR engagement of peptide-MHC class II ligands involves specific contacts between the TCR and residues on both the MHC and peptide molecules. We have used molecular modeling and assays of peptide binding and T cell function to characterize these interactions for a CD4+ Th1 cell clone, ESL4.34, which recognizes a peptide epitope of the herpes simplex type 2 virus virion protein, VP16 393-405, in the context of several HLA-DR alleles. This clone responded to VP16 393-405 in proliferation and cytotoxicity assays when presented by DRB1*0402, DRB1*1102, and DRB1*1301, which share a common amino acid sequence, ILEDE, at residues 67-71 in the alpha-helical portion of the DRbeta polypeptide, but not when presented by other DR4, DR11, and DR13 alleles that are negative for this sequence. Using a panel of APCs expressing DR4 molecules that were mutagenized in vitro at individual residues within this shared epitope and using peptide analogues with single amino acid substitutions of predicted MHC and TCR contact residues, a unit of recognition was identified dependent on DRbeta residues 67-71 and relative position 4 (P4) of the VP16 393-405 peptide. The interactions of this portion of the peptide-DR ligand with the ESL4.34 TCR support a structural model for MHC-biased recognition in some Ag-specific and alloreactive T cell responses and suggest a possible mechanism for autoreactive T cell selection in rheumatoid arthritis.

Alleles↗
PubMed1998

Highly polarized HLA class II antigen processing and presentation by human intestinal epithelial cells.

The high concentration of foreign antigen in the lumen of the gastrointestinal tract is separated from the underlying lymphocytes by a single cell layer of polarized epithelium. Intestinal epithelial cells can express HLA class II antigens and may function as antigen-presenting cells to CD4(+) T cells within the intestinal mucosa. Using tetanus toxoid specific and HLA-DR-restricted T lymphocytes, we show that polarized intestinal epithelial cells directed to express HLA-DR molecules are able to initiate class II processing only after internalization of antigen from their apical surface. Coexpression of the class II transactivator CIITA in these cells, which stimulates highly efficient class II processing without the characteristic decline in barrier function seen in polarized monolayers treated with the proinflammatory cytokine gamma-IFN, facilitates antigen processing from the basolateral surface. In both cases, peptide presentation to T cells via class II molecules was restricted to the basolateral surface. These data indicate a highly polarized functional architecture for antigen processing and presentation by intestinal epithelial cells, and suggest that the functional outcome of antigen processing by the intestinal epithelium is both dependent on the cellular surface at which the foreign antigen is internalized and by the underlying degree of mucosal inflammation.

Anti-Bacterial Agents↗
PubMed1998

Immunity to T cell receptor peptides in multiple sclerosis. III. Preferential immunogenicity of complementarity-determining region 2 peptides from disease-associated T cell receptor BV genes.

Vaccination with synthetic TCR peptides from the BV5S2 complementarity-determining region 2 (CDR2) can boost significantly the frequency of circulating CD4+ peptide-specific Th2 cells in multiple sclerosis (MS) patients, with an associated decrease in the frequency of myelin basic protein (MBP)-reactive Th1 cells and possible clinical benefit. To evaluate the immunogenicity of CDR2 vs other regions of the TCR, we vaccinated seven MS patients with overlapping BV5S2 peptides spanning amino acids 1-94. Six patients responded to at least one of three overlapping or substituted CDR2 peptides possessing a core epitope of residues 44-52, and one patient also responded to a CDR1 peptide. Of the CDR2 peptides, the substituted (Y49T)BV5S2-38-58 peptide was the most immunogenic but cross-reacted with the native sequence and had the strongest binding affinity for MS-associated HLA-DR2 alleles, suggesting that position 49 is an MHC rather than a TCR contact residue. Two MS patients who did not respond to BV5S2 peptides were immunized successfully with CDR2 peptides from different BV gene families overexpressed by their MBP-specific T cells. Taken together, these results suggest that a widely active vaccine for MS might well involve a limited set of slightly modified CDR2 peptides from BV genes involved in T cell recognition of MBP.

Adult↗
PubMed1998

Modulation of peptide-dependent allospecific epitopes on HLA-DR4 molecules by HLA-DM.

Peptide binding to HLA-DR molecules in intracellular compartments is facilitated by HLA-DM molecules, present in most types of antigen-presenting cells. Allorecognition of DR specificities represents a form of T cell recognition of the MHC-peptide complex which in some cases is influenced by peptide binding. DRA and DRB*0401 (Dw4) genes were introduced into different cell types including DM-negative and DM-restored mutant cells to analyze recognition of DR4 subtypes by alloreactive T cell clones and Dw4-specific monoclonal antibodies. Distinct patterns of T cell recognition were identified: (i) deficient response to Dw4 molecules in the absence of DM expression in which T cell responses were restored by transfecting DM into the Dw4-expressing cells; and (ii) equivalent recognition of Dw4 on DM- and DM+ cells. Using several mAb to Dw4 molecules, a similar distinction was observed: a shared epitope on Dw4 and Dw14 molecules was partially DM-independent while a Dw4-specific epitope was DM-dependent and cell type-specific. Thus, a subset of both T cell and mAb allodeterminants are influenced by a DM-dependent interaction of MHC molecules with peptides, while the formation of DM-independent allodeterminants may represent direct MHC epitope recognition by the T cell receptor or an alternative peptide loading mechanism distinct from the HLA-DM pathways.

Antibodies, Monoclonal↗
PubMed1998

Promiscuous T-cell recognition of a rubella capsid protein epitope restricted by DRB1*0403 and DRB1*0901 molecules sharing an HLA DR supertype.

Two T cell clones derived from different donors with HLA-DRB1*0403 or DRB1*0901 phenotype recognize a rubella capsid peptide, C(265-273) in the context of several different HLA-DR molecules in addition to DRB1*0403 and DRB1*0901. All DR molecules restricting the T-cell clones have in common residues, R or Q at position beta 70, R at position beta 71, and E at position beta 74 in pocket '4' of the DR peptide binding groove, suggesting that a DR subregion structure or supertype, "Q/RRE" underlies the promiscuous T-cell recognition of this peptide. Single amino acid substituted analogs of peptide C(263-275) at anchor position 4 for natural residue R were tested for their ability to induce clonal T-cell cytotoxic responses. The results indicated that a positively charged residue, R or K, was required for T-cell recognition, suggesting a possible mechanism of electrostatic interactions between the negatively charged residue E at position beta 74 of these DR molecules and the positively charged residue at anchor position 4 of the peptide in T-cell recognition.

Amino Acid Sequence↗
PubMed1998

HLA-DR class II associations with rubella vaccine-induced joint manifestations.

HLA class II (HLA-DR) frequencies were examined in relation to incidence of acute arthralgia or arthritis in 283 white women who had received RA27/3 rubella vaccine (n = 146) or placebo (n = 137) postpartum. Leukocyte DNA was molecularly typed for HLA-DRB1 gene expression. Univariate analysis revealed higher frequencies of DR2 (odds ratio [OR], 4.8; 95% confidence interval [CI], 1.2-18.8) and DR5 (OR, 7.5; 95% CI, 1.5-37.5) but lower frequencies of DR4 (OR, 2.3; 95% CI, 1.1-4.9) and DR6 (OR, 2.8; 95% CI, 1.4-5.8), in rubella vaccinees compared with placebo recipients with arthropathy. Logistic regression modelling of DR, treatment, age, time postpartum, and arthropathy revealed that the odds of developing arthropathy was 1.9 times greater (95% CI, 1.07-3.44) after rubella vaccine than placebo. Risk for arthropathy (regardless of rubella vaccination) was also influenced by DR interactions: odds were 8 times greater in individuals with both DR1 and DR4 (95% CI, 1.45-44.02) and 7.1 times greater with both DR4 and DR6 present (95% CI, 1.85-27.52), suggesting that coexpression of these specificities may predispose to postpartum arthropathy.

Adolescent↗
PubMed1998

HLA-DRB1 typing in rheumatoid arthritis: predicting response to specific treatments.

OBJECTIVE: To determine the predictive value of shared epitope alleles for response to treatment in patients with rheumatoid arthritis. METHODS: Patients from our previously published triple DMARD study were tested for the presence of shared epitope alleles (DRB1 *0401, 0404/0408, 0405, 0101, 1001, and 1402). Patients who were shared epitope positive were then compared with those who were negative to see if there was a differential effect on therapeutic response. RESULTS: Shared epitope positive patients were much more likely to achieve a 50% response if treated with methotrexate-sulphasalazine-hydroxychloroquine compared with methotrexate alone (94% responders versus 32%, p < 0.0001). In contrast shared epitope negative patients did equally well regardless of treatment (88% responders for methotrexate-sulphasalazine-hydroxychloroquine versus 83% for methotrexate). Additionally, a trend toward an inverse relation of the gene dose was seen for response to methotrexate treatment (p = 0.05). CONCLUSIONS: These data suggest that determining shared epitope status may provide clinical information useful in selecting among treatment options.

Adult↗
PubMed1998

Molecular basis for HLA-DQ associations with IDDM.

Autoimmune diabetes is the clinical end point for a sequential cascade of immunologic events that occur in a genetically susceptible individual. Structural and functional analysis of the HLA class II susceptibility genes in IDDM suggests likely molecular mechanisms for several of the key steps in this cascade of autoimmune events. We outline a pathway in which the HLA-DQ genes associated with IDDM bias the immunologic repertoire toward autoimmune specificities, creating an autoimmune-prone individual, followed by amplification and triggering events that promote subsequent immune activation. There are several direct links between genetics and autoimmune disease in this pathway: the developmental maturation of T-cells in a genetically susceptible individual occurs through molecular interactions between the T-cell receptor and the HLA-peptide complex. Selection of T-cells with receptors likely to contribute to autoreactivity may preferentially occur in the context of specific HLA-DQ alleles that are diabetes prone, because of inefficiencies in the peptide-MHC structural interactions of these molecules. Subsequent activation of these T-cells in the context of recognizing islet-associated antigens can trigger a poorly regulated immune response that results in progressive islet destruction. These subsequent diabetes-specific events are also directed by specific HLA genes, most prominently by the binding of specific antigenic peptides by the disease-associated HLA molecules. In this sequential cascade, opportunities for environmental influences and modulation by non-HLA genes are identified that likely act in concert with the predominant genetic susceptibility contributed by the HLA molecules themselves. Clarification of the steps in this pathway extends our understanding of the prevailing role of HLA genes in IDDM pathogenesis and suggests opportunities to intervene at discrete initiating, disease-promoting, or regulatory steps in IDDM development.

Autoimmunity↗