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

During immune responses, antigen-presenting cells (APCs) process antigens a nd present peptide epitopes complexed with human leukocyte antigen (HLA) mo lecules. CD4 cells recognize these naturally processed and presented epitop es (NPPEs) bound to HLA class II molecules. Epitope identification is impor tant for developing diagnostic and therapeutic tools for immune-mediated di seases and providing insight into their etiology, but current approaches ov erlook effects of natural processing on epitope selection. We have develope d a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). W e applied the technique to identify NPPEs of the intracellular domain of th e type 1 diabetes mellitus-associated (type 1 DM-associated) autoantigen in sulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around dist inct core regions. Synthetic peptides based on these regions bind HLA-DR4 a nd 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-D R4 nondiabetic controls we tested. This flexible, direct approach identifie s 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 ep itope selection and lead to the generation of sensitive and specific reagen ts for detecting autoreactive T cells.