Species specificity and augmentation of responses to class II major histocompatibility complex molecules in human CD4 transgenic mice.

Murine T cell responses to human class II major histocompatibility complex (MHC) molecules were shown to be a minimum of 20-70-fold lower than responses to allogeneic molecules. Transgenic mice expressing slightly below normal (75-95%) or very high (250-380%) cell surface levels of human CD4 were utilized to determine whether this was due to a species-specific interaction between murine CD4 and class II molecules. Human CD4 was shown to function in signal transduction events in murine T cells based on the ability of anti-human CD4 antibody to synergize with suboptimal doses of anti-murine CD3 antibody in stimulating T cell proliferation. In mice expressing lower levels of human CD4, T cell responses to human class II molecules were enhanced up to threefold, whereas allogeneic responses were unaltered. In mice expressing high levels of human CD4, responses to human class II molecules were enhanced at least 10-fold, whereas allogeneic responses were between one and three times the level of normal responses. The relatively greater enhancement of the response to human class II molecules in both lines argues for a preferential interaction between human CD4 and human class II molecules. In mice expressing lower levels of human CD4, responses to human class II molecules were blocked by antibodies to CD4 of either species, indicating participation by both molecules. In mice expressing high levels of human CD4, responses to both human and murine class II molecules were almost completely blocked with anti-human CD4 antibody, whereas anti-murine CD4 antibody had no effect. However, anti-murine CD4 continued to synergize with anti-CD3 in stimulating T cell proliferation in these mice. Thus, overexpression of human CD4 selectively impaired the ability of murine CD4 to assist in the process of antigen recognition. The ability of human CD4 to support a strong allogeneic response under these conditions indicates that this molecule can interact with murine class II molecules to a significant extent. Despite the fact that human CD4 appeared to be the only functional coreceptor in these mice, responses to human class II molecules were still much lower than those to murine class II alloantigens. This indicates that species-specific interactions between class II molecules and CD4 expressed on peripheral T cells are not sufficient to account for the low xenogeneic response and that intrinsic differences in T cell receptor structures or the need for species specificity in the interaction between CD4 and class II molecules during positive selection are also important.

Megabase-scale mapping of the HLA gene complex by pulsed field gel electrophoresis.

In the study of the genetic structure of mammalian chromosomes, there exists a "resolution gap" between molecular cloning experiments and meiotic linkage analyses. This gap has discouraged attempts to construct full-scale genetic maps of mammalian chromosomes. The organization of the human major histocompatibility complex was examined within this range by pulsed field gel electrophoresis. The data obtained indicate that the complex spans over 3000 kilobases and enable the construction of a megabase-scale molecular map. These results indicate that the techniques employed in DNA extraction, enzymatic digestion, electrophoresis, and hybridization are suitable for the efficient analysis of megabase regions of mammalian chromosomes and effectively bridge the resolution gap between molecular cloning and classical genetics.

Megabase scale restriction fragment length polymorphisms in the human major histocompatibility complex.

Pulsed-field gel electrophoresis of human peripheral blood lymphocyte DNA was used to ascertain the extent of megabase restriction fragment length variation in the HLA region of human chromosome 6 and to determine whether previously reported diversity was due to experimental variation or DNA polymorphism. Polymorphism was found to predominate in the vicinity of the class II DR, class III complement, and the class I A genes and to be limited or absent near the class II DP genes, the TNF genes, and the class I B and C genes. Thus, the MHC region is characterized by both fine and large-scale structural diversity.

Structure and nucleotide sequence of the heavy chain gene of HLA-DR.

We have used a 175-nucleotide-long primer extension product corresponding to the 5' end of HLA-DR alpha-chain mRNA to isolate a genomic clone from a human DNA library. The entire HLA-DR alpha gene is contained in two contiguous EcoRI fragments spanning about 7.5 kilobases (kb); most of the sequence has been determined. The 5' end of the gene is contained in a 4.4-kb fragment, and the coding segments and the 3' untranslated region are contained in a 3.1-kb fragment. The gene is split into five exons. The 5' untranslated region, the leader peptide, and the first two NH2-terminal amino acids are fused into the first exon. Exons 2 and 3 represent two extracellular coding domains of mature p34. The transmembrane domain, cytoplasmic domain, and part of the 3' untranslated region are merged into a fourth exon. The rest of the 3' untranslated region is in exon 5. The predicted amino acid sequence of mature p34, as deduced from its gene structure, has 229 residues and reveals a single potential disulfide loop (between cysteine residues 107 and 163) as well as a 22-amino acid residue membrane integrated segment (residues 193-214). Fifteen amino acids (residues 215-229) reside on the cytoplasmic side of the plasma membrane. There is considerable amino acid sequence homology between the second external domains of p34 and p29, as well as the immunoglobulin-like third domain of HLA-B7, and beta 2-microglobulin and the homologous constant region domains of the light and heavy chains of immunoglobulins.

The genomic organisation and nucleotide sequence of the HLA-SB(DP) alpha gene.

We have isolated a unique fragment of the HLA-DR alpha gene and probed human genomic DNA at low stringency to search for homologous sequences. A minimum of six non-polymorphic cross-hybridizing high molecular weight fragments were found in all DNAs examined. In order to obtain molecular clones of these cross-hybridizing fragments, we constructed lambda and cosmid libraries of human DNA and screened them at low stringency with the HLA-DR alpha gene specific subclone. We have isolated clones corresponding to each of the six fragments and, in this paper, describe those which contain the gene encoding HLA-SB(DP) alpha.

Identification of monozygotic twin chimpanzees by microsatellite analysis.

Zygosity determination is important for epidemiological, biological, obstetric, and prognostic studies in both human and nonhuman primates. In this study, microsatellite loci were used to screen a pair of chimpanzee (Pan troglodytes) twins and their parents. The twins share identical alleles at all loci tested. The probability of dizygotic origin is estimated to be 2.9 x 10(-11). Even after excluding linkage of loci on the same chromosome, the probability is still low enough (3.7 x 10(-9)) to exclude dizygotic origin. MHC typing was also done on Patr-DRB and Patr-DQB loci and the twins share identical alleles at both loci, consistent with the microsatellite results. Together these results demonstrate a monozygotic origin for the chimp twins. Our results suggest that microsatellite analysis is a powerful method for zygosity determination, which can be screened reliably and efficiently.

Transgenic HLA-DR alpha faithfully reconstitutes IE-controlled immune functions and induces cross-tolerance to E alpha in E alpha 0 mutant mice.

We have constructed transgenic mice that express the human class II MHC molecule HLA-DR alpha on a genetic background in which the equivalent endogenous gene, H-2 IE alpha, is not expressed. In these mice, DR alpha complemented the E beta chain such that tissue-specific expression of an interspecies hybrid DR alpha-E beta heterodimer was obtained. Despite 25% amino acid differences between DR alpha and E alpha, immune responsiveness to IE-controlled antigens, clonal deletion of IE-reactive T cells, and alloantigenicity were quantitatively and qualitatively indistinguishable in IE-positive mice and in mice that had integrated at least four copies of the transgene. These results demonstrate a remarkable degree of structural, regulatory, and functional conservation. They also suggest that tolerance induction involves only discrete portions of MHC molecules.

Structure, expression, and molecular mapping of a divergent member of the class I HLA gene family.

A class I gene distinct from HLA-A, -B, or -C was identified in a cosmid clone and transfected into mouse L cells. The gene, placed adjacent to the polyoma enhancer, produced a full-length class I mRNA and high levels of a 43-kDa protein in the cytoplasm. The surface expression of the gene product required its association with human beta 2-microglobulin. The protein was recognized by a xenoantiserum raised against a mixture of human B- and T-cell lines. The product was also serologically reactive with the HLA framework monoclonal antibodies. The complete nucleotide sequence of the gene was determined and a specific oligonucleotide probe was synthesized. This probe was used to identify a full-length mRNA transcript in a B-lymphoblastoid cell line (JY). The gene was mapped within a 190-kilobase Not I restriction fragment located in the telomeric portion of the human major histocompatibility complex. Distinct features of the gene include the structure of the promoter, the position of the translation initiation site, a frameshift mutation at the carboxyl terminus, the insertion of an Alu repeat element in the eighth exon, divergence in the derived amino acid sequence, and the lack of expression of the gene in some cells.

Age-associated changes in Epstein-Barr virus-induced human lymphocyte autoantibody responses.

In the present investigation we have studied the capacity of the Epstein-Barr virus (EBV), a ubiquitous human B lymphocyte polyclonal activator, to induce cultured peripheral blood lymphocytes from umbilical cords, young adults (20 to 40 yr old), and elderly adults (75 to 90 yr old) to form IgM antibodies to human IgG or human thyroglobulin. The EBV preparation used was shown to exert its B cell stimulatory effect independently of T cell-suppressor effects. The cultures were studied in limiting dilution analyses, and the data were taken to represent relative numbers of B cell precursors of autoantibody-forming cells in the 3 age groups. The results showed: 1) the EBV-inducible IgM anti-IgG and anti-thyroglobulin-producing cells increased in number from birth to old age; 2) the rise occurred at different times of life for the 2 autoantibodies, anti-IgG reactive B cells increasing between birth and young adulthood, and anti-thyroglobulin reactive B cells between young adulthood and old age; 3) the apparent relative avidity of the anti-IgG for antigen was higher in the elderly adults than in the younger adults. We believe these findings are determined by differences in the frequencies of the respective self-reactive B cells in the circulation. What physiologic or environmental factors determine the differential expansions of the human autoreactive B lymphocytes for IgG and thyroglobulin are not known. It seems possible that individual variations in the sizes of these pools may be a factor in determining susceptibility to autoimmune disease.

Use of synthetic oligonucleotide probes complementary to genes for human HLA-DR alpha and beta as extension primers for the isolation of 5'-specific genomic clones.

We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.