The ability of a peptidomimetic (SC-67655) to block the peptide binding site of the rheumatoid arthritis-linked human leukocyte antigen encoded by the DRB1*0401 allele was evaluated. The inhibitor bound to purified DRB1*0401 molecules with an affinity similar to that of the well-characterized peptide ligand HA307-319. Cell binding assays demonstrated that, in contrast to the promiscuous HA307-319 peptide, the peptidomimetic was highly specific for DRB1*0401. The inhibitor also blocked functional T cell responses to peptide antigens but did not block T cell proliferation in response to protein antigens. Furthermore, it did not appear to be taken up by cells. An analog of the peptidomimetic that was conjugated to a signal peptide sequence did inhibit a T cell proliferative response to protein antigen. Thus, the peptidomimetic must be taken up by cells to block the presentation of peptides derived from protein antigens. These findings have implications for the rational development of inhibitors that block the class II peptide binding groove for the treatment of autoimmune diseases.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease caused by the mold Aspergillus fumigatus. We previously reported that the majority of T cell clones (TCC) isolated from three ABPA patients, and specific for a dominant Ag of A. fumigatus, Asp f 1, were IL-4-producing CD4+ Th2 cells capable of responding to Ag in association with the HLA-DR subtypes DRB1*1501, *1503, and *1601 for HLA-DR2, and DRB1*1101, *1104, and *1202 for HLA-DR5. In the present study we extended the previous findings to determine whether the observed restriction with the HLA-DR2/5 subtypes held importance in a larger patient population. Serotyping revealed that 16 of 18 ABPA patients were either HLA-DR2, HLA-DR5, or both. Compared with a normal control population, the frequencies of HLA-DR2 (50 vs 22.3%) and HLA-DR5 (44.4 vs 19.8%) were significantly increased in these ABPA patients. Genotype analyses of an additional 15 patients identified the same HLA-DR subtypes previously shown functional for Asp f 1 Ag presentation. The relative avidities of Asp f 1 peptides for the purified HLA-DR subtypes, DRB1*1501 (functional) and DRB1*1502 (nonfunctional), were examined to determine whether differential binding to the HLA-DR subtypes explains successful Ag presentation. Similar low binding avidities were detected for both HLA-DR subtypes, indicating that the functionality cannot be simply explained by differences in binding affinities. Thus, the limited number and their role in Ag presentation emphasizes the possibility that the six identified HLA-DR subtypes are important in the pathophysiology of ABPA.

OBJECTIVE To identify critical residues involved in the binding of a selective peptide to DRB1*0401. METHODS The binding of peptides to native or site-directed mutant DR molecules was evaluated using enzyme-linked immunosorbent assay and flow cytometry. RESULTS Amino acid substitutions at DR and peptide residues, which were predicted to contribute to interactions within the DR p4 pocket, had the greatest effects on the specificity of binding. CONCLUSION Differences in the peptide-binding repertoires of DR molecules may contribute to associations with autoimmune diseases.

A peptide display library was evaluated as a means to identify peptide binding motifs for class II molecules. Peptides expressed as part of a soluble fusion protein with a maltose binding protein (malE) were produced by Escherichia coli. Constructs containing the high-affinity binding influenza hemagglutinin peptide 307W–319 (mal-HA) or the low-affinity binding tetanus toxoid peptide 830–843 (mal-TT) were used as controls. mal-HA, but not mal-TT, inhibited synthetic biotinylated-HA peptide from binding to purified DR4 Dw4 molecules in a dose-dependent manner. The fusion-peptide presentation system was also evaluated for its ability to induce antigen-specific T cell proliferation. DR4 Dw4+ B cells pulsed with mal-HA, but not mal-TT, induced dose-dependent proliferation of an HA-specific DR4 Dw4-restricted T cell line to the same extent as synthetic HA peptide. Using this type of peptide display library, it may be possible to determine the antigenic specificity of T cell clones isolated from patients with autoimmune diseases.

Naturally processed peptides from immunoaffinity-purified HLA-DRB1*0401, -DRB1*0404 (rheumatoid arthritis (RA)-associated), and -DRB1*0402 (non-RA-associated) molecules were analyzed by capillary liquid chromatography and mass spectrometry. The molecular weights observed for more than 60 eluted peptides from each HLA-DR protein ranged from 788 to 3535 atomic mass units, corresponding to peptides 7 to 32 amino acids in length. Sequencing of more than 60 of the abundant peptides revealed nested sets of peptides that were derived from only 12 different proteins. The majority of these proteins were membrane-associated (HLA class I, class II, and Ig molecules). Synthetic peptides, corresponding to endogenous peptide sequences, bound with high affinity (5 to 80 nM) to the HLA-DR molecules from which they were eluted. In addition, most were promiscuous binding peptides in that they also bound to other HLA-DR molecules. Truncations of eluted peptide sequences and alanine scanning mutational analysis of a Mycobacterium leprae peptide were used to identify the peptide residues involved in binding to DRB1*0404 and DRB1*0402 molecules. Furthermore, an invariant chain peptide was eluted from the DRB1*0402 molecules but not from the RA-associated molecules. The lack of invariant chain peptides from DRB1*0401 and DRB1*0404 molecules may contribute to the loading of autoantigen peptides into these molecules and to their association with disease.