Two-dimensional gel electrophoresis of immunoprecipitated human HLA-DR antigens from cells expressing the HLA-DR4 haplotype shows distinct clustering of (3-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, DwlO, LD "40", LD "DYI", LD "KT2", or LD "TAS") have identical DR (-chain . Comprehensive populations studies with DR4-associated HTC have led to the description of several distinct DR4-associated HLA-D antigens, including Dw4, DwlO, LD "40", LD "DYT", and LD "KT2" (4-8). There are several possible explanations for these apparent DR4 "splits." First, HLA-Dw4 and DR4 may be the products of different loci, each coding for distinct "D region"-associated antigens. A second possibility is that Dw4 and DR4 are products of a single gene, the primary product of which is recognized by DR4 alloantisera, but through post-translational modification sufficient phenotype variation occurs to cause the differences detected in MLC by T cells. In a third model, the haplotypes expressing Dw4, DwlO, LD 40, LD DYT, and LD KT2 may be encoded by different alleles but share a common crossreactive or "supertypic" determinant recognized by DR4 alloantisera.To answer such questions, we have begun a detailed analysis of the two-dimensional gel electrophoresis patterns of DR antigens expressed by B-lymphoblastoid cell lines (HCL) derived from HLA-DR4 homozygous donors. Since most of the polymorphism attributed to DR molecules has been correlated with structural heterogeneity of the DR (&chain (9-13), we have prepared cell lines from donors of HLA-D homozygous typing cells representing each of the distinct HLA-D antigens associated with DR4 and have studied the relationships among isolated DR4 ,3-chain polypeptides. Our results demonstrate a striking correlation between the functionally distinct HLA-D phenotypes of individual HCL and the two-dimensional gel patterns of the DR ,B chains isolated from these HCL.MATERIALS AND METHODS HCL. Seventeen B-lymphoblastoid cell lines homozygous for an HLA-DR4-associated D locus were used in this study (Table 1). The specificities Dw4, Dw10, LD 40, LD DYT, LD KT2, and DB3 have been defined by the Eighth Workshop (5), by Reinsmoen and Bach (4), and by Nose et al. (2). In addition, we have used HCL derived from a unique HTC, "TAS," described by Amar et al. (20), that is mutually reactive in MLC with cells from each of the other clusters and that may represent yet another apparent split of the DR4 haplotype.Established B-lymphoblastoid cell lines were available for some of the above HTC, otherwise new B-lymphoblastoid lines were prepared from cryopreserved HTC by cocultivation with Epstein-Barr virus (EBV) as described (21). Briefly, 1-2 X 106