Peptides bound to class I molecules are 8-10 amino acids long, and possess a binding motif representative of peptides that bind to a given class I allele. In the only published study of naturally processed peptides bound to class II molecules (mouse I-Ab and I-Eb), these peptides were longer (13-17 amino acids) and had heterogenous carboxy terminals but precise amino-terminal truncations. Here we report the characterization of acid-eluted peptides bound to HLA-DR1 by high-performance liquid chromatography, mass spectrometry and microsequencing analyses. The relative molecular masses of the peptides varied between 1,602 and 2,996 (13-25 residues), the most abundant individual M(r) values being between 1,700 and 1,800, corresponding to an average peptide length of 15 residues. Complete sequence data were obtained for twenty peptides derived from five epitopes, of which all but one were from self proteins. These peptides represented sets nested at both the N- and C-terminal ends. Binding experiments confirmed that all of the isolated peptides had high affinity for the groove of DR1. Alignment of the peptides bound to HLA-DR1 and the sequences of 35 known HLA-DR1-binding peptides revealed a putative motif. Although peptides bound to class II molecules may have some related features (due to the nonpolymorphic HLA-DR alpha-chain), accounting for degenerate binding to different alleles, particular amino acids in the HLA-DR beta-chains presumably define allelic specificity of peptide binding.

Surnmal~Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DRS. Using the complementary techniques of mass spectrometry and Edman microsequencing, >200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10--34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino-and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (>85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.MH C class I and II molecules are membrane-bound glycoproteins that present processed antigen to T cells and initiate an immune response (1). Crystallographic analysis of several class I molecules identified a groove composed of two ct helices supported by an eight-strand/J-pleated sheet containing electron-dense material that represents bound antigenic peptide (2-4). Several groups have characterized the complex mixtures of acid-extracted class I-bound peptides by HPLC fractionation and sequencing (5-9). The majority of these peptides were 8-11 amino acids long and possessed a binding motif characteristic of peptides that bind to a given class I allele.The characterization of naturally processed peptides bound to class II molecules provides an approach towards understanding both antigen processing and peptide binding events in vivo. The stability of class II molecules requires peptide binding (10, 11); however, the precise class II molecule--peptide contacts that provide this energy are not yet well defined. Identification of naturally processed peptides extracted and sequenced from class II molecules revealed that the bound peptides were longer (13-25 residues) than those bound to class I (12-15) and nested at the amino-and/or carboxyterminal ends, suggesting that the peptide binding groove on class II molecules is open at both ends (13,15). Although only a limited number of source proteins were reported, peptides derived from both endogenous proteins and exogenous serum proteins were identified. The association constants (measured by competitive inhibition) for several of these peptides were in the nanomolar range, c...

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...

An unusual monoclonal antibody (MARB4) directed against HLA-B27 that reacts with only

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR,B chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define-binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301.