Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65).

T cell recognition of autoantigens is critical to progressive immune-mediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulin-dependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4(+) T cell response in IDDM.

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.

Epstein-Barr virus uses HLA class II as a cofactor for infection of B lymphocytes.

Infection of B lymphocytes by Epstein-Barr virus (EBV) requires attachment of virus via binding of viral glycoprotein gp350 to CD21 on the cell surface. Penetration of the cell membrane additionally involves a complex of three glycoproteins, gH, gL, and gp42. Glycoprotein gp42 binds to HLA-DR. Interference with this interaction with a soluble form of gp42, with a monoclonal antibody (MAb) to gp42, or with a MAb to HLA-DR inhibited virus infection. It was not possible to superinfect cells that failed to express HLA-DR unless expression was restored by transfection or creation of hybrid cell lines with complementing deficiencies in expression of HLA class II. HLA class II molecules thus serve as cofactors for infection of human B cells.

Immunogenetic similarities between patients with Felty's syndrome and those with clonal expansions of large granular lymphocytes in rheumatoid arthritis.

OBJECTIVE: Patients with chronic clonal proliferation of large granular lymphocytes (LGL leukemia) often have splenomegaly, neutropenia, and rheumatoid arthritis (RA), thereby resembling the manifestations observed in patients with Felty's syndrome. The present study sought to determine whether patients with these disorders represent 2 distinct subsets of neutropenic RA. METHODS: Prospective cohort study of outpatients attending clinics in university and private hospitals and in offices of private practice physicians. Twenty-two patients with Felty's syndrome and 22 patients with LGL leukemia, 10 of whom had RA, were studied. HLA genotyping was performed on peripheral blood mononuclear leukocyte genomic DNA. RESULTS: Nineteen of the 22 patients with Felty's syndrome (86%) were DR4 positive. Nine of the 10 patients with LGL leukemia plus RA were also DR4 positive. In contrast, only 4 of the 12 patients with LGL leukemia without RA (33%) were DR4 positive, a frequency that was within the normal range. CONCLUSION: The finding of an equally high prevalence of DR4 in patients with Felty's syndrome and in those with LGL leukemia plus RA suggests that both disorders have a similar immunogenetic basis and are parts of a single disease process rather than 2 separate disorders.

Modulation of HLA-DQ binding properties by differences in class II dimer stability and pH-dependent peptide interactions.

Intermolecular interactions between peptides and class II molecules intracellularly are likely to be influenced by different pH conditions in different endosomal compartments. We compared the pH-dependent binding of peptides to HLA-DQ alleles 3.1 and 3.2, two well-studied human class II molecules with limited structural variability but different genetic associations with autoimmune diseases. Binding of the "promiscuous" binding peptide 34P3A as well as that of several allele-specific peptides were optimal for DQ3.2 at relatively acidic pH, compared with DQ3.1, with a pH optimum for DQ3.2 at pH 5.5 and for DQ3.1 at pH 6.5. A specific polymorphism at residue 57 of the DQ molecule accounted for some, but not all, of this difference. When the intrinsic stability of the class II dimer was assessed using partially denaturing gel electrophoresis after incubation at different pH, the DQ3.2 molecule demonstrated relative instability at neutral pH compared with DQ3.1, again suggesting a preference for peptide binding at relatively acidic conditions for HLA-DQ3.2. Interestingly, this preference for binding at acidic pH was not found for a class II-associated invariant chain peptide. Intrinsic alphabeta dimer stability and the pH optima for peptide binding studied in this report are likely to be significant determinants for allelic differences in DQ binding profiles in vivo.

Critical contribution of beta chain residue 57 in peptide binding ability of both HLA-DR and -DQ molecules.

Position 57 in the beta chain of HLA class II molecules maintains an Asp/non-Asp dimorphism that has been conserved through evolution and is implicated in susceptibility to some autoimmune diseases. The latter effect may be due to the influence of this residue on the ability of class II alleles to bind specific pathogenic peptides. We utilized highly homologous pairs of both DR and DQ alleles that varied at residue 57 to investigate the impact of this dimorphism on binding of model peptides. Using a direct binding assay of biotinylated peptides on whole cells expressing the desired alleles, we report several peptides that bind differentially to the allele pairs depending on the presence or absence of Asp at position 57. Peptides with negatively charged residues at anchor position 9 bind well to alleles not containing Asp at position 57 in the beta chain but cannot bind well to homologous Asp-positive alleles. By changing the peptides at the single residue predicted to interact with this position 57, we demonstrate a drastically altered or reversed pattern of binding. Ala analog peptides confirm these interactions and identify a limited set of interaction sites between the bound peptides and the class II molecules. Clarification of the impact of specific class II polymorphisms on generating unique allele-specific peptide binding "repertoires" will aid in our understanding of the development of specific immune responses and HLA-associated diseases.

Prognostic implications of HLA genotyping in the early assessment of patients with rheumatoid arthritis.

Current methods and approaches for the use of HLA markers in the assessment of rheumatoid arthritis (RA) are not optimal. Improved strategies for application of HLA susceptibility genetic typing in patients were evaluated and a new system for rapid determination of these RA susceptibility alleles was developed. Retrospective data summarizing the prevalence of HLA susceptibility alleles in patients with distinct clinical outcomes was analyzed to estimate the sensitivity and specificity of HLA genetic testing as a prognostic marker for erosive disease. A rapid allele specific DNA hybridization assay was performed on an automated instrument using a solid phase nonradioactive hybridization and detection system. Depending on the patient population being tested, from 70-80 percent of patients with progressive erosive disease carry one or more of the DR4 cluster of RA susceptibility genes (DRB1*0401, 0404, 0405). Sensitivity is increased by including other shared epitope positive alleles, but at the expense of specificity. The rapid automated genetic testing system correctly identified each of more than 200 samples tested, with no false positives. HLA genetic testing for RA susceptibility alleles can be performed rapidly and accurately. Prognosis for erosive disease can be facilitated in the patient with early pre-erosive RA using HLA testing in combination with other clinical assessment variables.

Deficient antigen-presenting cell function in multiple genetic complementation groups of type II bare lymphocyte syndrome.

The absence of HLA class II gene expression in type II bare lymphocyte syndrome (BLS) results from defective transcriptional activation of class II histocompatibility genes. Genetic studies have revealed that distinct defects in multiple trans-acting factors result in the immunodeficient BLS phenotype. We studied antigen-presenting cell (APC) function in DR-transfected BLS cells derived from multiple complementation groups. Each BLS cell line displayed the same defective APC phenotype: an inability to mediate class II-restricted presentation of exogenous protein antigens, and structurally altered class II alpha beta dimers. Expression of the HLA class II-like genes DMA and DMB, previously implicated in antigen presentation, was reduced or absent in the BLS cells. Fusion of BLS cells with cell line 721.174, which has a genomic deletion of HLA class II genes, coordinately restores class II structural gene and DM gene expression and a wild-type APC phenotype. Thus each of the molecular defects that silences class II structural gene transcription also results in a defective APC phenotype, providing strong evidence for coregulation of these two functionally linked pathways.

HLA and T cell receptor beta-chain DNA polymorphisms identify a distinct subset of patients with pauciarticular-onset juvenile rheumatoid arthritis.

OBJECTIVE: To evaluate and extend upon a reported association of a T cell receptor (TCR) V beta coding region polymorphism with pauciarticular-onset juvenile rheumatoid arthritis (JRA). METHODS: TCR V beta 6.1 genotypes and haplotypes in JRA and control groups were determined by DNA amplification. RESULTS: Haplotypes of the V beta 6.1 gene which encode a nonfunctional form of V beta 6.1 were significantly associated with pauciarticular JRA in patients possessing the HLA-DQA1*0101 allele (P = 0.0073). CONCLUSION: A TCR V beta gene segment in the vicinity of V beta 6.1, possibly V beta 6.1, is apparently involved in the pathogenesis of pauciarticular-onset JRA in DQA1*0101-positive individuals.

Erosive rheumatoid factor negative and positive rheumatoid arthritis are immunogenetically similar.

OBJECTIVE: The relationship between rheumatoid factor positive (RF+) and rheumatoid factor negative (RF-) rheumatoid arthritis (RA) is controversial. We sought to determine whether the HLA genes conferring susceptibility for erosive RF+RA are also prevalent in patients with erosive RF-RA. METHODS: DNA-based HLA typing for DRB1, DQB1, and DPB1 was performed on 16 consistently RF--patients with erosive RA. RESULTS: Thirteen of 16 (81%) RF-RA patients had the HLA susceptibility genes DRB1 *0401, *0404, or *0101, which are associated with RF+RA, as compared to 46% of normal controls (p = 0.017). By contrast, no associations with HLA-DQB1 and HLA-DPB1 alleles were apparent. CONCLUSION: Specific HLA susceptibility alleles are prevalent in patients with erosive RA, regardless of RF status, suggesting a similar immunogenetic basis for RA in these patients.

The role of the major histocompatibility complex in autoimmunity.

In the case of many autoimmune diseases, HLA genes are the genes most closely associated with disease susceptibility. Recent major advances in the ability to determine particular HLA genotypes in individuals now allow us to identify the precise alleles most closely associated with disease. Rheumatoid arthritis, long known to be associated with HLA-DR4, provides a good model for review of this progress, demonstrating how methodologic advances have led to an improved understanding of the immunogenetic basis of this disease, with implications for both pathogenesis and potential therapeutic interventions.

HLA-DR alleles and sterile ulcerative keratitis.

Human leukocyte antigen-DR typing was performed on 18 unrelated white patients with sterile ulcerative keratitis (SUK) to determine whether these patients share common immunogenetic susceptibility genes. There was no statistically significant increase in any DR allele among the entire group of SUK patients. There was a trend in the frequency of DR1 in the rheumatoid arthritis (RA) patients (5 of 8, 63%) versus the non-RA patients (1 of 10, 10%), which was not statistically significant, possibly due to the small number of patients in the study. Screening patients with RA without known SUK from our RA register revealed one DR1-positive patient with an inactive peripheral marginal melt. These findings suggest a possible relationship between DR1 and RA sterile corneal melting, which will need to be confirmed with a larger study.

Prediction of susceptibility to rheumatoid arthritis by human leukocyte antigen genotyping.

Recent methodologic advances now allow the precise identification of HLA genes in individuals. Population studies using these methods have pinpointed the HLA alleles strongly associated with rheumatoid arthritis (RA). This article summarizes the current status of these RA-associated HLA genes in disease susceptibility and uses that information to derive estimates of risk ratios for prediction of disease in an individual.

Juvenile rheumatoid arthritis and HLA: report of the Park City III workshop.

The workshop held during the Park City Meeting was directed toward developing a consensus about HLA associations in juvenile rheumatoid arthritis (JRA). Most agreement was achieved in pauciarticular JRA where the strongest associations were with the HLA-DRB1 alleles as is also the case in IgM rheumatoid factor positive polyarticular disease. In addition, HLA-DP associations are being identified although roles for linked genes are still possible. The critical nucleotides among HLA genes are not known; however, disease specific mutations have not been shown.

Psychosomatic musculoskeletal pain in childhood: clinical and psychological analyses of 100 children.

The clinical and psychological findings on 100 children with psychosomatic musculoskeletal pain seen at a major pediatric rheumatology referral center are reported. Most (76%) were female, median age was 13 years, and median duration of symptoms was 1 year. Multiple painful sites were common (66%). The pain was constant (63%) or intermittent (37%); 45% had hyperesthesia, and almost all maintained a cheerful affect when complaining of severe pain. Two predominant abnormal family milieu were seen. One was cohesive, stable, and organized, but intolerant of separation and individuation. The other was chaotic, emotionally unsupportive, with high levels of conflict. Members of the cohesive family type reported significantly less distress than members of chaotic families. Enmeshment between mother and child was common in both family types. Although frequently viewed as bright, most of these children had normal intelligence, and some had unrecognized academic difficulty. These children, compared with those with arthritis, had a significantly lower global well-being score. Clinical depression was unusual (11%). Most (97%) responded favorably to intensive physical and occupational therapy along with individual or family psychotherapy; 78% become symptom free or fully functional. Children with these signs and symptoms should have full psychological evaluations and respond well to treatment directed toward decreasing pain and restoring function.

The immunogenetics of juvenile rheumatoid arthritis.

Recent major advances in understanding the genetic structure of the human leukocyte antigen (HLA) region and how HLA molecules contribute to immune responses have been paralleled by more precise identification of specific HLA genes conferring susceptibility to the various forms of juvenile rheumatoid arthritis (JRA). This article presents current models for HLA-associated autoimmune disease susceptibility and summarizes the HLA Class II alleles currently known to be associated with JRA: primarily DR8, DR5, DR6, and DPw2.1 in pauciarticular onset JRA; and DR4 in rheumatoid factor-positive polyarticular onset JRA. Rheumatoid factor-negative polyarticular onset JRA and systemic onset JRA are variously associated with several of these same genes. Gene interactions and the clinical utility of HLA typing in this disease are also discussed.

HLA and T cell receptor polymorphisms in pauciarticular-onset juvenile rheumatoid arthritis.

The immunogenetic basis of pauciarticular-onset juvenile rheumatoid arthritis is unclear. We therefore analyzed the HLA and T cell receptor genes present in a clinically well-defined group of patients. We found that the DR8 haplotype contributes most of the HLA-associated risk, although alleles at other loci contribute independently. A candidate disease-associated T cell receptor polymorphism, in contrast, was not identified in this population. Mechanistic implications of these findings are discussed.