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The polymorphism of HLA-DR antigens has been studied by Southern blot hybridization under conditions specific for the detection of the DRchain genes. Haplotype-specific patterns were defined with DNA from DR1, 2, 3, 4, 7, w8, w11, w12, and W13 homozygous typing cells, with restriction enzymes Eco RI, Bgl I, and Pvu II. Certain serological specificities, such as DR2, DR3, and DR7, can be encoded by distinct allelic forms of DR chain genes. The procedure of 'DNA typing' was applied to family analysis of individuals expressing only a single DR specificity upon serological typing. Three cases are described here: (1) in family GR, phenotypic DR 7 homozygotes correspond to genomic heterozygotes, and a novel DR7 allele is described: (2) in family RU, the genes corresponding to a serologically undetected (blank) DR allele were identified by restriction fragment length polymorphism (RFLP); this novel DR haplotype has an RFLP pattern similar to those of the DRw52 family, even though this specificity was not expressed on the DR-blank lymphocytes; (3) in family RG, there is no blank allele, but a homozygote RFLP situation at the DR subregion. [ABSTRACT FROM AUTHOR]

Allelic polymorphism of HLA-class II antigens plays a key role in the regulation of the immune response and in transplantation immunity. The allelic diversity of these antigens can now be analyzed at the DNA level after amplification by polymerase chain reaction. In this study we apply a simple technique based on the electrophoretic analysis of DNA heteroduplexes to the typing of HLA-DPB1 alleles. In order to increase its resolution, a group-specific amplification was used which subdivides the 19 HLA-DPB1 alleles in two non-overlapping families. A separate analysis was then performed within each group of alleles. This approach allowed an unequivocal one-step typing of the alleles belonging to group 1 which comprises few alleles of high frequency. Some group 2 alleles require, as a further step, the test with a restriction enzyme. The combination of more than one technique represents, in our opinion, the easiest way to solve the micropolymorphism of class II alleles. We conclude that this method, which is very simple, quick, and accurate and does not require probes, may become the method of choice for HLA-DPB1 typing.

Three new alloantigenic specificities of human major histocompatibility complex class II molecules have been defined by testing the reactivity of alloantisera at the molecular level. Two of these specificities identify different DR4 haplotypes. The Fe75 specificity is associated with the DR4/Dw10 haplotype and the CBC/MRG6 specificity with the DR4/DKT2 haplotype. Both are supertypic specificities and are associated with other DR specificities as well. Both specificities are carried by class II molecules belonging to the first DR subset. Together with previously described determinants, these specificities contribute to serological discrimination of the different DR4 haplotypes.

Human class II molecules were isolated from cells of a DR2/Dw2-homozygous cell line, PGF. The three mutually exclusive subsets were separated by selective binding with monoclonal antibody MCS7 and alloantisera CCB921 and KY22. The specificity involved in the binding with alloantisera was identified to be a supertypic specificity associated with DR1, 2, and w9 for CCB921 and the DQw1 specificity known to be associated with DR1, 2, w6, and w10 for KY22. The MCS7 specificity appeared to be a cross-reactive specificity related to the DRw52-like specificity. On peptide mapping, the alpha chains of MCS7- and CCB921-reactive subsets were the same, showing the pattern characteristic of DR alpha chains, whereas the beta chains were very similar to, but distinguishable from, each other. These structural features conformed to those of DR or DR-like subsets. The KY22-reactive subset was distinctive in both alpha and beta chains from the above two subsets, and it displayed peptide patterns typical to DQw1-bearing Ia molecules. Interestingly, the MCS7- and CCB921-reactive subsets both carried the DR2 specificity, as indicated by their binding to alloantiserum Fe73/22 which was proven to be DR2-specific.

Human Ia molecules were isolated from cells of LG-38, an HLA-homozygous lymphoid cell line of DR5 specificity. Three Ia subsets could be distinguished and separated by using specific alloantisera and a monoclonal antibody with polymorphic reactivity. These subsets carried the specificities DR5 and MT2, MT2 alone and MB3 alone. The structure of the three molecular species was analyzed by microfingerprinting. The subset carrying only MT2 was similar, in both the component alpha and beta chains, to the major subset carrying DR5 and MT2, whereas the subset carrying MB3 was distinct in both chains from the other two subsets. These data are compatible with our previous findings obtained for the products of two Ia loci closely linked to the DR locus and provisionally called DC and BR; they also support the conclusion that the subset carrying only MT2 is an allelic product of the BR locus, whereas the MB3 subset is an allelic product of the DC locus. MT2 appears to be a shared specificity of DR and BR loci products.

Human Ia(-like) specificities controlled by gene loci other than HLA-DR were searched for at the molecular level in cells of human B-cell-type cell lines which carry two established DR specificities. Chevalier cells of DRw3 and 7 and U698M cells of DRw2 and 4 were used. Their Ia molecules were partially purified, radioiodinated and analyzed for Ia specificities by the direct binding and sequential binding assays with a selected panel of human Ia alloantisera. It was possible in both the cell lines to define a third subset of Ia molecules carrying a new specificity in addition to two Ia subsets carrying the established DR specificities. The new specificity was detected by putative anti-DRw4 and anti-DRw7 antisera and was closely associated with DRw4 and DRw7 at population level. It was thus designated provisionally as BR4X7. These results suggest that the BR4X7 specificity is coded for by a separate Ia locus closely linked to HLA-DR locus. The determinant(s) responsible for BR4X7 was located on the small subunit of Ia molecules.