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Serologic, cellular, and molecular evidence supports the concept of extreme complexity within the HLA-D region. To study the complexity and fine specificity of the HLA-D region at the level of T -cell recognition, a panel of T-cell clones was generated against alloantigens associated with HLA-DRI through -DRw8. After initial screening of more than 800 clones, 89 representative lines were selected for extensive testing against 204 unrelated stimulator cells. Clone-by-clone correlation analyses were performed to test whether any clones recognized similar or identical epitopes. In addition, clonal reactivity patterns were correlated with known HLA specificities. Twelve clusters of clones were identified with similar reactivity patterns using clone-by-clone correlation analysis. Some groups were significantly correlated with specificities associated with various D-region haplotypes; others had no significant correlation with any defined D-region specificity. Five general types of clones obtained in our study can be categorized as follows: (1) Those recognizing epitopes clearly demonstrating a primary association with the classically defined D-region molecules against which the clones were primed. (2) Clones recognizing epitopes associated with one of the priming antigens and also with another unrelated D-region specificity. (3) Clones detecting epitopes which showed significant correlation with D-region molecules totally different from those against which they were originally primed. (4) Clones with limited reactivity in population studies and no correlation with defined D-region molecules. (5) Clones recognizing class I-associated epitopes. [ABSTRACT FROM AUTHOR]

To study the fine specificity of the HLA-D region, a panel of human T-lymphocyte clones (TLCs) was generated against alloantigens associated with HLA-DR1 through DRw8. HLA-DR-homozygous peripheral blood lymphocytes (PBLs) were stimulated with DR-heterozygous PBLs in primary mixed lymphocyte cultures for 4 days. Blasts were cloned by limiting dilution at 0.3 cells/well in the presence of 20% T-cell growth factor and irradiated stimulator cells. Viable clones were subsequently tested in proliferation assays against the original stimulator and a limited panel of stimulators bearing relevant DR specificities. Initial primings produced approximately 800 clones; some recognized DR-associated antigens, 70 recognized only their original stimulator, and approximately 50% were nonresponsive. Analysis on extended stimulator panels revealed alloantigenic complexity within similar DR-associated antigens as recognized by TLCs. The data are consistent with evidence that extreme heterogeneity exists within the HLA-D region. [ABSTRACT FROM AUTHOR]

The HLA-DNA gene of the human major histocompatibility complex (MHC) produces RNA transcripts of unusual size compared to the 1.3 kilobase (kb) transcripts of other class II genes. Several cDNA clones derived from HLA-DNA transcripts were isolated, including a full-length 3.5 kb cDNA named DN1. Sequence analysis of DN1 demonstrated that the 3.5 kb transcript is fully spliced, contains a long 3' untranslated region, and encodes an apparently normal class II α chain. Two other cDNA clones, DN2 and DN7, revealed an alternative splice that resulted from the use of a splice acceptor upstream of the second exon encoding the α 1 domain. The translation product of this alternative transcript would share the signal sequence of the DNα chain but would otherwise be unrelated. Removal of the signal sequence would yield a highly basic peptide of 42 amino acids. RNA was prepared by in vitro transcription of DN1 and DN2, and was used to direct protein synthesis in a rabbit reticulocyte system. The product of DN1 had an apparent relative mass of 25 000, whereas the product of DN2 was a peptide with a relative mass of about 6000. The alternatively spliced RNA represents about 1/10 of the transcripts from the HLA-DNA gene in the cells tested, which included transformed B-cell lines as well as normal blood cells and thymus cells. [ABSTRACT FROM AUTHOR]

We have studied the complexity and fine specificity of the HLA-D region using a panel of T lymphocyte clones generated against alloantigens associated with HLA-DR1 through DRw8. After extensive testing in population studies, 89 clones were tested in proliferation assays with 14 families. Segregation patterns were analyzed for haplotype associations by calculating sequential lod scores to test the likelihood that genes encoding epitopes detected by TLCs were linked to HLA genes. Four general categories were identified: (1) clonal responses that segregated with the same HLA-D region haplotype in all informative pedigrees; (2) clonal responses that segregated with HLA in all pedigrees but not always with the same haplotype; (3) clonal responses that segregated with HLA in some families but failed to segregate in others or produced equivocal results; (4) clonal responses that did not segregate with HLA haplotypes. [ABSTRACT FROM AUTHOR]