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Biomedical subjects

Gerald T Nepom

Publications and source records attributed to Gerald T Nepom.

17 recordsLinked to original sources
PubMed2003

Differential antigen sensitivity and costimulatory requirements in human Th1 and Th2 antigen-specific CD4+ cells with similar TCR avidity.

The differentiation of naive CD4(+) Th cells into Th1 and Th2 phenotypes is influenced by cytokines, concentration of Ag, accessory molecules, and the affinity of the MHC-TCR interaction. To study these factors in human memory T cells, T cell lines with Th1 or Th2 phenotypes specific for the peptide hemagglutinin (HA)(307-319) in the context of DRB1*0401 were established from the peripheral blood of an individual previously vaccinated for influenza virus. Flow cytometric analysis with fluorescent-labeled MHC class II tetramers was used to analyze TCR avidity: the Th2 line bound the HLA-DR*0401-HA(307-319) tetramers with higher mean avidity, although the range of binding avidity largely overlapped with the Th1 line. High-affinity Th1 and Th2 lines were established for further study by FACS sorting. When activated with plate-bound HLA-DR*0401-HA(307-319) monomers, the Th1 line proliferated and produced IFN-gamma without additional costimulation whereas the Th2 line required the addition of soluble anti-CD28 Ab to induce proliferation and IL-5 production, but this requirement could be overcome with high concentrations of plate-bound monomer alone. IL-2 production was dependent on costimulation in both cell lines. These findings demonstrate that upon antigenic rechallenge, Th1 and Th2 cells differ in their response to Ag-specific stimulation. Th2 cells were sensitive to the strength of signal to a greater degree than Th1 cells and required costimulation through CD28 for maximal proliferation. These distinctions between Th1 and Th2 activation are not consistent with a simple avidity model of Ag recognition and indicate both qualitative and quantitative differences in determining cell lineage commitment.

Cell Differentiation↗
PubMed2003

Binding of outer surface protein A and human lymphocyte function-associated antigen 1 peptides to HLA-DR molecules associated with antibiotic treatment-resistant Lyme arthritis.

OBJECTIVE: To assess the binding of outer surface protein A (OspA) and human lymphocyte function-associated antigen 1 (hLFA-1) peptides to 5 major histocompatibility complex (MHC) molecules. METHODS: Peptide binding to the MHC molecules was determined by in vitro binding assays, and binding was correlated with the frequencies of the 5 MHC molecules in patients with treatment-resistant Lyme arthritis. RESULTS: The HLA-DRB1*0401 molecule bound both OspA(163-175) and hLFA-1alpha(L330-342) well. Although the magnitude of the binding was less, the DRB1*0404 molecule also showed binding of both peptides. The DRB1*0101 molecule bound OspA(163-175) well, but hLFA-1alpha(L330-342) only weakly; the DRB1*0801 or *1101 molecule bound both peptides weakly, if at all. The magnitude of OspA(163-175) binding correlated well with the frequencies of the DRB1 alleles in patients with treatment-resistant arthritis, but the binding of hLFA-1alpha(L330-342) showed only an association with the DRB*04 alleles. CONCLUSION: These correlations support the hypothesis that OspA(163-175) is the critical epitope in triggering antibiotic treatment-resistant Lyme arthritis. However, the inability of the DRB*0101 molecule to bind hLFA-1alpha(L330-342) suggests that this peptide may not be a relevant autoantigen, at least in DRB1*0101-positive patients.

Antigens, Surface↗
PubMed2003

Low-avidity recognition by CD4+ T cells directed to self-antigens.

Self-reactive T cells populate the peripheral immune system, and likely form the reservoir from which autoreactive cells are derived. We analyzed a panel of self and non-self peptides presented by HLA-DR4, a class II molecule associated with autoimmunity, by immunization of mice transgenic for HLA-DR4. Significant structural avidity for T cell recognition, as measured by MHC class II tetramer binding to CD4(+) T cells was only observed in mice immunized with the non-self antigens. T cell hybridomas were generated from mice immunized with the naturally processed self-peptide hGAD65 (552-572) and also from mice immunized with an influenza-derived non-self epitope (HA 306-318). T cells specific for the self peptide failed to bind tetramers and exhibited low functional avidity as measured by the peptide concentration required to reach half-maximum proliferation values. In contrast, T cells specific for the non-self HA (306-318) peptide exhibited high structural and functional avidity profiles. As recently described in studies of murine CD8(+) T cell function, the predominance of low avidity recognition of self-peptide epitopes may be a characteristic feature of CD4(+) T cells responding to autoantigens.

Amino Acid Sequence↗
PubMed2003

HLA tetramer-based artificial antigen-presenting cells for stimulation of CD4+ T cells.

Current evidence suggests that immunotherapy for cancer or infectious diseases will require the activation of CD4(+) T cells in addition to the activation of cytotoxic CD8(+) T cells. To complement and overcome some of the limitations of dendritic-cell-based vaccines and ex vivo expansion of human T cells, we sought to engineer artificial antigen-presenting cells (aAPCs) for the stimulation of antigen-specific human CD4(+) T cells. We have designed a variety of aAPCs using magnetic beads as a scaffold on which to coat HLA-peptide tetrameric complexes along with costimulatory molecules such as anti-CD28. Here, we tested various forms of conjugation of the tetramers onto beads, characterized the relative concentration of antigen available on the surface of the beads, and evaluated the ability of different types of beads to promote activation of antigen-specific CD4(+) T cells. We find that an indirect coating of HLA-peptide tetramers on beads via an anti-Class II antibody provides specific stimulation of antigen-specific CD4(+) T cells.

Antigen-Presenting Cells↗
PubMed2002

Differential presentation of group A streptococcal superantigens by HLA class II DQ and DR alleles.

Superantigens have been implicated as pivotal mediators of severe invasive group A streptococcal (GAS) infections, by virtue of their potent immunostimulatory activity. HLA polymorphism has been suggested to influence the susceptibility to severe invasive GAS infection. Here we studied the influence of allelic and isotypic variation of HLA class II molecules on GAS superantigen-induced immune responses using cells derived from patients with bare lymphocyte syndrome, untransfected or transfected with various HLA class II alleles. Significantly higher proliferative responses were detected when streptococcal pyrogenic exotoxin (Spe) A was presented by cells expressing DQA1*0101/DQB1*0302 (DQ3.2), as compared to cells expressing DR1, DR4, or DR5 alleles (p=0.0002-0.01). In contrast to SpeA, SpeC was preferentially presented by DR4 as compared to DQB1*03 (p=0.04). In agreement with the proliferation results, a significantly higher frequency of IL-2-, TNF-alpha-, TNF-beta-, and IFN-gamma-producing cells was detected when SpeA was presented by HLA class II DQB1*03 alleles as compared to DR4 (p=0.0002-0.04). Binding experiments showed a high affinitybinding of SpeA to both class II DR4 and DQB1*0302, and there was no significant difference in SpeA binding affinity between the two alleles. The data confirm the effect of allelic polymorphism in superantigen responses and show that different superantigens are preferentially presented by distinct class II alleles.

Alleles↗
PubMed2002

HLA class II tetramers: tools for direct analysis of antigen-specific CD4+ T cells.

Immunotherapies for human autoimmune and immune-mediated diseases are proliferating rapidly, and with these changes comes the opportunity to monitor patients for immune responses to therapy based on early surrogate markers for clinical responses. Class II tetramers have the potential to serve as these sorts of markers for immune monitoring, and thereby assist with patient management, therapy selection, and improved outcomes. However, important issues of TCR avidity require resolution, because much is still unknown regarding location, quantitation, and characterization of the human T cell response. Opportunities for application of tetramer technologies in the near future will enable both clinical progress and the development of new insights into human CD4+ T cell biology in vivo.

CD4-Positive T-Lymphocytes↗
PubMed2002

Mutational analysis of critical residues determining antigen presentation and activation of HLA-DQ0602 restricted T-cell clones.

Three different HLA-DQ0602 restricted T-lymphocyte clones (clones 5, 44, and 48) specific for two different Herpes simplex virus type 2 (HSV-2) VP16 peptides were used in a series of proliferation assays with BLS-1 cell lines expressing mutated HLA-DQ0604 molecules as APC. Up to four residues in the peptide-binding region of DQ0604 were replaced by the respective DQ0602 residue. For all three clones, residue beta70 played a crucial role in TCR recognition; beta30 and beta57 were important, although beta86 was less significant. Clone 5 and 48, specific to the HSV-2 VP16 369--379 peptide, responded to the same mutated DQ0604 molecules. Both clones could be stimulated only when the antigen presenting DQ molecule contained the DQ0602-like Gly at position beta70. Stimulation of clone 44, which recognized a different HSV-2 VP16 epitope (VP16 40-50), was less restricted. Molecular homology modeling showed that the beta70Arg of DQ0604 partially covered the peptide around P5/P6. Interactions of beta70 with residues from the antigen-peptide and polymorphic residues at positions beta30 and beta57 can modulate this effect. Supported by molecular modeling data, we conclude that the distinct molecular topography of DQ0602 is not contributed by a single residue, but rather the interactions of various polymorphic DQ residues with particular antigenic peptides.

Antigen Presentation↗
PubMed2002

Transcript array analysis in rheumatology.

Transcript array analysis is a novel technique that examines the expression of thousands of genes simultaneously. Transcript array analyses are being used to clarify the diagnosis and prognosis of malignancies, and to understand the underlying pathogenesis of complex human disorders such as the rheumatic diseases. In this review, the authors will outline the use of transcript arrays to simultaneously assess gene activation of hundreds or thousands of genes, and their potential use in understanding and managing rheumatic disorders. The authors focus on the use of transcript arrays to confirm and refine disease diagnoses, to generate new hypotheses regarding pathophysiology of rheumatic diseases, and to the possible profiling of patients with respect to their likely response to therapies.

Animals↗
PubMed2002

Therapy of autoimmune diseases: clinical trials and new biologics.

Successful clinical efficacy trials have been recently completed for a growing array of new biologics. Prospects are improving for rational therapeutic interventions that potently interrupt adaptive immune response pathways. By contrast, disappointing results with antigen-based therapeutics highlight the difficulties of achieving tolerance in the context of active autoimmunity. Combination immunotherapy is probably necessary to sequentially or simultaneously inhibit inflammation, uncouple innate immune pathways, interrupt or ablate the adaptive memory response and promote antigen-specific tolerance in a suitable immunological context.

Antibodies, Monoclonal↗
PubMed2002

Modulation of T cell response to hGAD65 peptide epitopes.

Human CD4 T cell responses to an epitope of hGAD65 (GAD = glutamic acid decarboxylase), residues 555-567, are modulated by interaction with an altered peptide ligand containing modifications at TCR contact residues. Using different HLA-DR4 molecules with polymorphisms at sites corresponding to peptide binding pockets p1 and p9, we tested the effect of additional modifications in the altered peptide ligand (APL) designed to increase the avidity of the MHC-peptide interaction and therefore the efficiency of TCR signaling. Modification of the peptide or the MHC molecule which enhanced the p1 interaction also enhanced the antagonist activity of the modified APL. In contrast, modifications at p9 led to a reversal in APL function, resulting in agonist activity. Molecular homology modeling of these MHC-peptide interactions suggests a structural basis for this functional dichotomy in which topographically remote variations lead to unique interaction effects.

Epitopes, T-Lymphocyte↗
PubMed2002

Defining antigen-specific responses with human MHC class II tetramers.

Antigen-specific CD4(+) T lymphocytes play key roles in the orchestration of immune responses, including the initiation and amplification of allergic and immune-mediated disorders. Direct detection and quantitation of these cells is made possible by the use of HLA class II tetramers, soluble recombinant forms of a complex of HLA molecule and antigenic peptide that bind the antigen-specific T-cell receptor used for T-cell recognition. By using flow cytometry with fluorescent tetramers, specific T cells can be identified, recovered, and analyzed for functional markers and transcripts, helping to characterize the lineage and commitment program for individual lymphocytes. Several newly emerging uses for tetramers in clinical immunology are under development for patient management or prognosis when the number or nature of antigen-specific T cells can be clinically important. Tetramers might be useful as patient-monitoring tools for evaluating response to immunotherapy, providing a surrogate marker for the immunologic response foreshadowing a clinical response to either immune stimulation, suppression, or deviation therapeutic modalities.

Antigens↗
PubMed2002

Genetics of rheumatoid arthritis: is there a scientific explanation for the human leukocyte antigen association?

Human leukocyte antigen genes associated with rheumatoid arthritis are commonly found in the unaffected population, implying that causal mechanisms of disease involve interactions between these genes and other factors. A variety of approaches-genetic, structural, and immunologic-are used to explore possible molecular interactions that may contribute to understanding the basis for this disease association. The specific relation between human leukocyte antigen-DR4 alleles and rheumatoid arthritis remains one of the strongest and most thoroughly studied examples of human leukocyte antigen risk genes among human autoimmune disorders.

Alleles↗
PubMed2002

Detection of GAD65-specific T-cells by major histocompatibility complex class II tetramers in type 1 diabetic patients and at-risk subjects.

Soluble HLA-DR401 or -DR404 tetramers containing a peptide corresponding to an immunodominant epitope from human GAD65 were used to analyze peripheral blood T-cells of newly diagnosed type 1 diabetic patients and at-risk subjects. Peripheral blood mononuclear cells were expanded on antigen-presenting cells presenting GAD65 peptide and subsequently activated with specific plate-bound class II-peptide monomers. T-cell activation defined in flow cytometry by CD4(high) and/or CD25 markers were observed in all type 1 diabetic patients and some at-risk subjects, but not in normal control subjects. The activated T-cells stained positive with tetramers containing the GAD65 epitope 555-567. Tetramer-positive cells were CD4(high) T-cells with high avidity for an immunodominant GAD65 T-cell epitope. Phenotyping of T-cells utilizing HLA class II tetramers provides a new tool to characterize the autoimmune response in type 1 diabetes.

Adolescent↗
PubMed2002

Successful prospective prediction of type 1 diabetes in schoolchildren through multiple defined autoantibodies: an 8-year follow-up of the Washington State Diabetes Prediction Study.

OBJECTIVE: Almost 90% of type 1 diabetes appears in individuals without a close family history. We sought to evaluate the best current predictive strategy, multiple defined autoantibodies, in a long-term prospective study in the general population. RESEARCH DESIGN AND METHODS: Autoantibodies to pancreatic islets (islet cell antibodies [ICAs]) and defined autoantibodies (d-aab) to human GAD, IA2/ICA512, and insulin were tested in 4,505 Washington schoolchildren. Eight years later, 3,000 (67%) subjects were recontacted, including 97% of subjects with any test >99th percentile. RESULTS: Six subjects developed diabetes (median interval 2.8 years), all from among the 12 individuals with multiple d-aab, representing 50% positive predictive value (95% CI 25-75%) and 100% sensitivity (58-100%). Among the others, diabetes occurred in 0 of 6 with one d-aab plus ICA, 0 of 26 with ICA only, 0 of 7 with one d-aab equaling the 99th percentile and another d-aab equaling the 97.5th percentile, 0 of 86 with one d-aab, and 0 of 2,863 with no d-aab or ICA. Adjusted for verification bias, multiple d-aab were 99.9% specific (99.86-99.93%). At this age, new d-aab seldom appeared. Once present, d-aab usually persisted regardless of disease progression, although less so for insulin autoantibodies. Insulin secretion by sequential glucose tolerance testing remained normal in four multiple d-aab subjects not developing diabetes. Of children developing diabetes, five of six (83%) would be included if HLA-DQ genotyping preceded antibody testing, but HLA-DQ did not explain outcomes among high-risk subjects, even when considered along with other genetic markers. CONCLUSIONS: Multiple d-aab were established by age 14 years and prospectively identified all schoolchildren who developed type 1 diabetes within 8 years.

Adolescent↗