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Celiac disease has one of the strongest associations with HLA (human leukocyte antigen) class II markers of the known HLA-linked diseases. This association is primarily with the class II serologic specificities HLA-DR3 and -DQw2. We previously described a restriction fragment length polymorphism (RFLP) characterized by the presence of a 4.0-kilobase Rsa I fragment derived from an HLA class II beta-chain gene, which distinguishes the class II HLA haplotype of celiac disease patients from those of many serologically matched controls. We now report the isolation of this beta-chain gene from a bacteriophage genomic library constructed from the DNA of a celiac disease patient. Based on restriction mapping and differential hybridization with class II cDNA and oligonucleotide probes, this gene was identified as one encoding an HLA-DP beta chain. This celiac disease-associated HLA-DP beta-chain gene was flanked by HLA-DP alpha-chain genes and, therefore, was probably in its normal chromosomal location. The HLA-DP alpha-chain genes of celiac disease patients also were studied by RFLP analysis; 84% of HLA-DR3, -DQw2 patients had a 16-kb Xba I fragment that was present in only 36% of HLA-DR3, -DQw2 controls. Moreover, 79% of these patients had both alpha- and beta-chain polymorphisms in contrast to 27% of controls. Thus, celiac disease is associated with a subset of HLA-DR3, -DQw2 haplotypes characterized by HLA-DP alpha- and beta-chain gene RFLPs. Within the celiac-disease patient population, the joint segregation of these HLA-DP genes with those encoding the serologic specificities HLA-DR3 and -DQw2 indicates: (i) that the class II HLA haplotype associated with celiac disease is extended throughout the entire HLA-D region, and (ii) that celiac-disease susceptibility genes may reside as far centromeric on this haplotype as the HLA-DP subregion.

Tryptic peptide map analysis by high-pressure liquid chromatography of DR4- associated DR beta chains revealed limited structural variation within DR beta polypeptides. Comparison of 3H-leucine-labelled tryptic peptide maps of Dw4 and Dw14 homozygous cells identified distinct peaks corresponding to Dw4 and Dw14-associated DR beta polypeptides. HPLC analysis of cell line 256, heterozygous for two DR4-related specificities, Dw4 and Dw14, displayed both peptides, corresponding to the variable Dw4 and Dw14 chromatograms. This observation was confirmed using a deletion mutant cell line derived from 256 lacking Dw4-associated class II genes. The observed peptide variation correlated precisely with predicted nucleotide-derived amino acid sequences implicating amino acids 66-71 of the DR beta chain as contributing to HLA-D structural and functional polymorphisms.

Analysis of individual human leukocyte antigen (HLA) genes associated with rheumatoid arthritis was performed using oligonucleotide probes to identify putative susceptibility genes highly associated with disease. Among DR4-positive rheumatoid arthritis patients, two distinct DR-beta genes, Dw4 and Dw14, appear to be individual susceptibility alleles. Linked HLA genes at the DQ locus do not appear to contribute to this genetic risk. Among non-DR4 rheumatoid arthritis patients, a specific DNA sequence was identified that is shared by the Dw14 susceptibility gene and a majority of the non-DR4 rheumatoid arthritis patients. This sequence defines a specific small genetic element encoding a "Dw14 epitope" that appears to be a rheumatoid arthritis-associated risk factor indicative of a common genetic pathway in both DR4 and non-DR4-associated disease.

HLA-DR4, Dw4-associated haplotypes associated with IDDM and JRA were compared using genomic DNA restriction fragment analysis to distinguish among DQ beta and alpha alleles linked to DR4. DQ beta polymorphisms that subdivide the HLA-DQw3 specificity into DQ3.1 and 3.2 alleles were identified. More than 90% of DR4+ IDDM patients express one of these alleles, DQ3.2; restriction enzyme mapping indicates that the presence of this allele also accounts for the genomic fragment patterns previously reported in IDDM. Furthermore, haplo-identical siblings of DQ3.2 IDDM patients also carry the DQ3.2 allele, regardless of clinical presentation. In contrast, DR4+ JRA patients show no allelic preference at DQ beta, implicating different HLA genetic contributions in these two DR4-associated diseases.

The polymorphism of HLA class II molecules expressing the serologically defined alloantigen DQw3 was studied using cloned proliferative T lymphocytes. Two clones, IG9 and IC3, were selectively primed against DQw3-associated determinants and tested against a panel of 92 HLA-D homozygous cells. Both clones were specific for DQw3, but each showed a distinct response pattern. Clone IG9 recognized a DQw3-associated determinant expressed on a subset of DR4 and DR5 haplotypes and on all DRw6, 7, w8, and w9 haplotypes tested. In contrast, clone IC3 recognized a distinct DQw3-associated determinant expressed only on a subset of DR4 haplotypes. In monoclonal antibody inhibition experiments, anti-DQ, but not anti-DR or anti-DP antibodies, blocked reactivity of both clones IG9 and IC3, further demonstrating that the determinants defined by these clones are associated with DQ molecules. In DNA hybridization studies using a DQ beta probe, a correlation was observed between restriction site polymorphisms in the DQ beta gene, designated DQw"3.1" and "3.2," and the expression of the T-cell-defined IG9 and IC3 determinants. It is, thus, possible to demonstrate by cloned T-cell reactivity functionally relevant recognition sites on DQw3+ molecules that are associated with structural polymorphisms defined by molecular and genomic analysis.

The HLA genetic region consists of a large multigene complex which includes a number of highly homologous alpha and beta genes encoding class II polypeptides, clustered in three major loci, DP, DQ, and DR. Analysis of genomic polymorphisms at each of these loci is of considerable interest due to the role of particular structural polymorphisms in immune function, but this analysis has been hampered by difficulty in distinguishing between such highly homologous loci. We have identified locus-specific and exon-specific class II gene sequences in order to produce synthetic oligonucleotide probes which hybridize specifically to DQ beta genes. Two such oligonucleotide probes are described which are specific for the beta 1 and beta 2 exons of DQ (DC) beta, which identify DQ beta genes in digests of cellular DNA and which can be used to characterize restriction sites flanking the two oligonucleotide-specific regions. By sequentially hybridizing these probes in modified Southern analyses, we have been able to generate a tentative "restriction map" of a newly identified DQ beta allele from digests of total genomic DNA. This oligonucleotide mapping technique discriminates between two HLA-DQw3+ alleles, DQ3.1 and DQ3.2, permitting the recognition of structural polymorphisms with DQ beta which are highly associated with type I diabetes mellitus.

Two-dimensional gel electrophoresis of immunoprecipitated human HLA-DR antigens from cells expressing the HLA-DR4 haplotype shows distinct clustering of beta-chain patterns. Six unique electrophoretic variants were observed among 17 HLA-DR4 homozygous cell lines (HCL) analyzed. These patterns correlate precisely with the HLA-D phenotype of the HCL donor as determined by reactivity in mixed lymphocyte culture. All DR4 HCL that belong to one of the well-defined HLA-D antigen groups (Dw4, Dw10, LD "40", LD "DYT", LD "KT2", or LD "TAS") have identical DR beta-chain patterns; DR4 HCL belonging to different HLA-D antigen groups do not. The concordance of the functional expression in mixed lymphocyte culture of a specific D phenotype with a distinct DR beta-chain pattern on gel analysis provides a direct structural basis for understanding the genetic control of HLA-D polymorphisms; HLA-D specificities as revealed by T-cell recognition in mixed lymphocyte culture thus might be accounted for by DR beta-chain polymorphisms. The extent of this beta-chain diversity within a single DR haplotype may aid in understanding variations in Ia-regulated functions, such as Ir gene control and certain disease susceptibilities.

Retroviral vectors with an internal cytomegalovirus major immediate-early gene enhancer/promoter regulating HLA class II gene expression were used to transfer HLA cDNA into human EBV-transformed B-lymphoblastoid cell lines. HLA-DQ2 beta and DQ3.2 beta cDNA were transferred into DQ3.2 and DQ2 homozygous lymphoblastoid cell lines, respectively. Serologic analysis of the infected cell lines with allospecific mAb demonstrated surface expression of these exogenous DQ molecules implying that DQ alpha-chains from DR3, DQ2-positive cells can pair with DQ3.2 beta-chains and, similarly, DQ alpha-chains from DR4, DQ3.2-positive cells can pair with DQ2 beta-chains. Immunoprecipitation of the introduced DQ3.2 beta molecule resulted in co-purification of the allotype-mismatched endogenous DQ2 alpha polypeptide. We also show that vectors with a cytomegalovirus major immediate-early gene enhancer/promoter result in higher levels of expression of the transduced gene compared to previously described HLA vectors with either the SV-40 early enhancer/promoter or the lymphotropic papovavirus-enhanced SV-40 promoter. Although deletion of HLA cDNA did sometimes occur in the process of generating virus-producing clones, the HLA cDNA is stably maintained in virus-producing clones, once it is generated. This retroviral expression system is a highly efficient way to study HLA gene function.