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Sequence analysis has identified multiple alleles at two loci that encode for the DR2 specificity. The loci, DRB1 and DRB5, are in linkage disequilibrium which can extend to alleles of the DQ loci. Serologic, cellular, and sequence-specific oligonucleotide probe (SSOP) typing techniques have been used to identify the DR2 haplotypes. In this report, we have characterized by SSOP typing and cDNA/DNA sequence analyses the combinatorial diversity of DR2 haplotypes. Cells were selected on the basis of unique serologic reactivity, unique associations of alleles of DR and DQ loci, and/or presence in populations which have not been extensively characterized for HLA diversity. An asymmetric polymerase chain reaction (PCR) amplification was applied to rapidly screen unique cells and to characterize DNA sequence in conjunction with more conventional cDNA sequence analysis. The sequence data confirm the lack of a DRB5 locus in the DR2"LUM" specificity, the unexpected association of DRB1*1602 and DRB5*010 alleles in a nonCaucasoid population, and the association of the allele DRB1*1503 with DRB5*0101 in black African, African American and native American individuals. The DRB1*1503 and DRB5*0101 alleles were identified in an unusual haplotype, DR2,DQ2. The combinatorial diversity of the DR2 haplotypes is extended by these studies in nonCaucasoid populations.

Definition of peptide binding motifs for DR molecules has proven difficult as the peptides that bind to a DR molecule have shown extensive variability at putative motif positions. Recent studies suggest that specific peptide anchor residues (motif positions) and specific DR residues can differ in importance for peptide binding to a DR molecule. To assess further the relevance of individual peptide anchor residues, the binding of serial alanine-substituted analogs of influenza virus hemagglutinin (HA) 306-318 and human myelin basic protein (MBP) 152-165 to a panel of transfected wild-type DR molecules was examined. This analysis included DR molecules from a wide range of allelic families and, unlike most earlier studies, multiple members of single DR allelic families. The data show that different peptide residues serve as critical anchors for binding to different DR molecules. For example, MBP binding to DR(alpha, beta 1*0303) required peptide residues F154 (i), R159 (i + 5) and R162 (i + 8). In contrast, MBP binding to DR(alpha, beta 1*0102) required peptide residues I153 (i) and L156 (i + 3). More importantly, the combination of critical anchor residues in HA and MBP differed for binding to a single DR molecule [e.g. V309 (i) for HA and I153 (i) and L156 (i + 3) for MBP binding to DR(alpha, beta 1*0102)]. Although the location of the binding pocket in each DR molecule compared to the DR (alpha, beta 1 *0101) crystal is expected to be similar and suggests a common extended DR binding motif, the present results suggest that the relative importance of individual peptide anchor residues and of the corresponding DR binding pockets will differ for each DR/peptide complex.

Chimeric TCR transfectants expressing human extracellular sequences and murine intracellular/transmembrane sequences were generated to analyze the trimolecular interaction between myelin basic protein (MBP) autoantigen, HLA, and a TCR isolated from a patient with multiple sclerosis. Chimeric transfectants responded to TCR activation by CD3- and TCRBV22S1-specific mAbs and by superantigen. Additionally, chimeric transfectants responded to autoantigen-specific activation with MBP 152-165 when presented by DR(alpha,beta1*1301) independent of the CD4 adhesion molecule. Transfectants did not respond to Ag presented by other HLA-DR molecules, including the closely related DR(alpha,beta1*1302). In peptide-binding studies with a panel of serial alanine-substituted MBP peptides, HLA contact residues necessary for anchoring MBP 152-165 to DR(alpha,beta1*1301) were also defined: 154 (F), 159 (R), and 162 (R). The chimeric TCR transfectant's differential response to a similar panel of MBP analogues defined residues that interact with the TCR: 153 (I), 155 (K), 156 (L), 160 (D), and 161 (S). Analysis of molecular interactions, such as those described in this work, may be central to developing new strategies for suppressing Ag-specific responses in human autoimmune disease.

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