Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4

Abstract

During immune responses, antigen-presenting cells (APCs) process antigens and present peptide epitopes complexed with human leukocyte antigen (HLA) molecules. CD4 cells recognize these naturally processed and presented epitopes (NPPEs) bound to HLA class II molecules. Epitope identification is important for developing diagnostic and therapeutic tools for immune-mediated diseases and providing insight into their etiology, but current approaches overlook effects of natural processing on epitope selection. We have developed a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). We applied the technique to identify NPPEs of the intracellular domain of the type 1 diabetes mellitus-associated (type 1 DMassociated) autoantigen insulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around distinct core regions. Synthetic peptides based on these regions bind HLA-DR4 and elicit primary T-cell proliferation frequently in HLA-DR4-positive type 1 DM patients, but rarely in non-HLA-DR4 patients, and in none of the HLA-DR4 nondiabetic controls we tested. This flexible, direct approach identifies an HLA allele-specific map of NPPEs for any antigen, presented by any HLA class II molecule. This method should enable a greater understanding of epitope selection and lead to the generation of sensitive and specific reagents for detecting autoreactive T cells.J. Clin. Invest. 104:1449-1457(1999 significantly enhance epitope recognition by CD4 T cells. For these reasons, we elected to develop a system for the direct identification of peptides naturally processed from specific Ag's and presented by HLA class II molecules as NPPEs recognized by CD4 T cells. We have applied the new technology to the prototypic organ-specific autoimmune disease, type 1 diabetes mellitus (DM), in which immune responses to numerous islet cell autoantigens occur on a distinctive genetic background, notably the possession of HLA-DRB1*0401, DQA1*0301/DQB1*0302 [DQ3.2] genotypes (15,16). In the present study we have focused on the islet cell autoantigen insulinoma-associated-2 (IA-2), one of a family of protein tyrosine phosphatases (PTPs) (17). Autoantibodies against these PTPs are directed toward the intracellular domain (IA-2ic), almost without exception (18), and are associated with rapid progression to diabetes in high-risk subjects (19). In the present study we identify NPPEs of IA-2ic bound to HLA-DR4 (0401) and demonstrate that synthetic peptides based on these are sensitive and specific reagents in the detection of autoreactive T cells in HLA-DR4 type 1 DM patients. MethodsSubjects. Fresh heparinized and clotted blood samples were obtained from 21 Caucasian type 1 DM patients (median age 20 years, range 6-42 years) recruited a median of 8 weeks after diagnosis (range 1-28 weeks). All had acute onset of symptoms, ketosis, and required insulin from diagnosis. Samples were also col...

IFN-γ and IL-10 islet-antigen-specific T cell responses in autoantibody-negative first-degree relatives of patients with type 1 diabetes

“…Peptide panel The peptide panel was derived from that used by Arif et al [25] and included IA-2 and proinsulin peptides naturally processed and presented by HLA-DR4 (previously identified by Peakman et al [32] and Arif et al [25], respectively), as well as three 'promiscuous epitopes' from tetanus toxoid used as controls for the specificity of the T cell response [25,33]. Table 1 summarises the peptides used and their sequences.…”

The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment

“…While the decreasing proinsulin-specific responses and the increasing of IGRP [265][266][267][268][269][270][271][272][273] reactivity are reminiscent of NOD mouse data (6), the finding of a similar phenomenon for GAD and IA-2 is unparalleled. This is not surprising, since GAD and IA-2 are thought to be important target Ags in human type 1 diabetes (15,17,26,(37)(38)(39) but not in the NOD mouse (40 -43). Consideration of additional epitopes poorly recognized at type 1 diabetes onset (17,18) may reveal further specificities becoming immunodominant at later time points.…”

The role of T-cells in the pathogenesis of Type 1 diabetes: From cause to cure

“…Another study identified naturally processed and presented epitopes of the intracellular domain of the Type 1 diabetes-associated autoantigen IA-2, presented by HLA-DR4 (DRB1*0401) [65]. IA-2ic-derived peptides were eluted from HLA-DR4 that constituted six sets of peptides nested around distinct core regions.…”