New HLA DNA polymorphisms associated with autoimmune diseases

Certain class II determinants of the human histocompatibility locus antigens (HLA) have been implicated in the aetiology of several autoimmune diseases, including rheumatoid arthritis (RA) and insulin-dependent diabetes mellitus (IDDM). HLA-Dw4 was the first HLA determinant found to be significantly increased in RA patients compared with controls, while Dw4 and Dw3 were found to be significantly increased in IDDM patients. When the HLA-DR system was defined, RA patients were found to have an increased frequency of DR4 and IDDM patients an increased incidence of both DR4 and DR3 compared with controls. As the HLA-Dw specificities are narrower than the serologically defined DR specificities, it was of specific interest to the present study that Dw4, Dw10, Dw13, Dw14, Dw15 and DKT2 are included in DR4. We describe here new restriction fragment length polymorphisms (RFLPs) and, together with the newly described serologically defined DQ specificity TA10, test their prevalence and associations in controls and diseased patients. We find that the newly characterized DNA bands are present at a much higher frequency in RA and IDDM patients than in controls. These findings may lead to a greater understanding of the pathogenesis of such diseases.     

Identification of susceptibility loci for insulin-dependent diabetes mellitus by trans-racial gene mapping

INSULIN-dependent (type I) diabetes mellitus (IDDM) follows an autoimmune destruction of the insulin-producing beta-cells of the pancreas. Family and population studies indicate that predisposition is probably polygenic. At least one susceptibility gene lies within the major histocompatibility complex and is closely linked to the genes encoding the class II antigens, HLA-DR and HLA-DQ (refs 3, 4). Fine mapping of susceptibility genes by linkage analysis in families is not feasible because of infrequent recombination (linkage disequilibrium) between the DR and DQ genes. Recombination events in the past, however, have occurred and generated distinct DR-DQ haplotypes, whose frequencies vary between races. DNA sequencing and oligonucleotide dot-blot analysis of class II genes from two race-specific haplotypes indicate that susceptibility to IDDM is closely linked to the DQA1 locus and suggest that both the DQB1 (ref. 7) and DQA1 genes contribute to disease predisposition.     

Insulin-IGF2 region on chromosome 11p encodes a gene implicated in HLA-DR4-dependent diabetes susceptibility

A class of alleles at the VNTR (variable number of tandem repeat) locus in the 5' region of the insulin gene (INS) on chromosome 11p is associated with increased risk of insulin-dependent diabetes mellitus (IDDM), but family studies have failed to demonstrate linkage. INS is thought to contribute to IDDM susceptibility but this view has been difficult to reconcile with the lack of linkage evidence. We thus investigated polymorphisms of INS and neighbouring loci in random diabetics, IDDM multiplex families and controls. HLA-DR4-positive diabetics showed an increased risk associated with common variants at polymorphic sites in a 19-kilobase segment spanned by the 5' INS VNTR and the third intron of the gene for insulin-like growth factor II (IGF2). As INS is the major candidate gene from this region, diabetic and control sequence were compared to identify all INS polymorphisms that could contribute to disease susceptibility. In multiplex families the IDDM-associated alleles were transmitted preferentially to HLA-DR4-positive diabetic offspring from heterozygous parents. The effect was strongest in paternal meioses, suggesting a possible role for maternal imprinting. Our results strongly support the existence of a gene or genes affecting HLA-DR4 IDDM susceptibility which is located in a 19-kilobase region of INS-IGF2. Our results also suggest new ways to map susceptibility loci in other common diseases.     

A trans-acting class II regulatory gene unlinked to the MHC controls expression of HLA class II genes

Class II (or Ia) antigens are highly polymorphic surface molecules which are essential for the cellular interactions involved in the immune response. In man, these antigens are encoded by a complex multigene family which is located in the major histocompatibility complex (MHC) and which comprises up to 12 distinct alpha- and beta-chain genes, coding for the HLA-DR, -DQ and -DP antigens. One form of congenital severe combined immunodeficiency (SCID) in man, which is generally lethal, is characterized by an absence of HLA-DR histocompatibility antigens on peripheral blood lymphocytes (HLA class II-deficient SCID). In these patients, as reported here, we have observed an absence of messenger RNA for the alpha- and beta-chains of HLA-DR, -DQ and -DP, indicating a global defect in the expression of all class II genes. Moreover, the lack of expression of HLA class II mRNAs could not be corrected by gamma-interferon, an inducer of class II gene expression in normal cells. Family studies have established that the genetic defect does not segregate with the MHC. We conclude, therefore, that the expression of the entire family of class II genes is normally controlled by a trans-acting class II regulatory gene which is unlinked to the MHC and which is affected in the patients. This gene controls a function or a product necessary for the action of gamma-interferon on class II genes.     

Expression and function of CD4 in a murine T-cell hybridoma

The CD4 (T4) antigen was originally described as a phenotypic marker specific for helper T cells, and has recently been shown to be the receptor for the human immunodeficiency virus (HIV). Functional studies using monoclonal antibodies directed at CD4 and major histocompatibility complex (MHC) class II molecules led to the suggestion that CD4 binds to the MHC class II molecules expressed on stimulator cells, enhancing T-cell responsiveness by increasing the avidity of T cell-stimulator cell interaction and/or by transmitting a positive intracellular signal. But recent evidence that antibodies to CD4 inhibit T-cell responsiveness in the absence of any putative ligand for CD4 has been interpreted as suggesting that antibody-mediated inhibition may involve the transmission of a negative signal via the CD4 molecule instead. We have infected a murine T-cell hybridoma that produces interleukin 2 (IL-2) in response to human class II HLA-DR antigens with a retroviral vector containing CD4 cDNA. The resulting CD4-expressing hybridoma cell lines produce 6- to 20-fold more IL-2 in response to HLA-DR antigens than control cell lines. Furthermore, when antigen levels are suboptimal, the response of the cell lines is entirely CD4-dependent. The data presented here clearly demonstrate that CD4 can enhance T-cell responsiveness and may be crucial in the response to suboptimal levels of antigen.     

A minimum of four human class II alpha-chain genes are encoded in the HLA region of chromosome 6

The major histocompatibility complex (MHC) in man, also called the HLA region, is located on the short arm of chromosome 6 and encodes antigens involved in immunological processes. The class II HLA antigens consist of two noncovalently associated polypeptide chains, one of molecular weight 34,000 (alpha) and the other of molecular weight 29,000 (beta). The extensive polymorphism of the beta chain(s) has allowed the genetic mapping of the corresponding beta gene(s) to the HLA-DR region. cDNA clones for the HLA-DR alpha chain have been used to map the non-polymorphic DR alpha-chain gene to chromosome 6 using mouse-human somatic cell hybrids. Similarly, the DR alpha-chain gene has been mapped to the short arm of chromosome 6 centromeric to the HLA-A, -B and -C loci by in situ hybridization experiments. We isolated a cDNA clone that is related to the DR alpha chain and encodes the class II antigen DC alpha chain. We describe here how this DC alpha clone was used to find two or three additional alpha-chain genes by cross-hybridization and how HLA-antigen loss mutants of a human lymphoblastoid cell line (LCL) were used to ascertain that these additional class II antigen alpha-chain genes are also located in the HLA region.     

A combination of a particular HLA-DP beta allele and an HLA-DQ heterodimer confers susceptibility to coeliac disease

Coeliac disease is an autoimmune disease of the intestinal mucosa, elicited by ingestion of wheat gluten in genetically susceptible individuals. Susceptibility to coeliac disease has been associated with the serologically defined variants DR3 and DR7 of the class II antigens encoded by the HLA-D region. Three related class II antigens, each consisting of an alpha and a beta glycoprotein chain, have been identified and are designated HLA-DR, HLA-DQ, and HLA-DP. These highly polymorphic transmembrane proteins bind peptides derived from the processing of foreign antigens and present them to T lymphocytes; they also influence the specificity of the mature T-cell repertoire. The role of HLA-DP polymorphism in susceptibility has not been as fully explored as that of the other class II antigens because of the complexity of the primed lymphocyte typing (PLT) method for determining DPw specificities. Here we use a new DNA-based method of HLA-DP typing to analyse the distribution of DP beta alleles in a group of coeliac disease patients and healthy controls. Two specific DP beta alleles (DPB4.2 and DPB3) are increased in the patient population. Comparison of the DP beta sequences suggests that the polymorphic residues at position 69 and at 56 and 57 may be critical in conferring susceptibility. Further, the contribution of the susceptible DP beta alleles appears to be independent of linkage to the previously reported DR3 and DR7 markers for coeliac disease. The distribution of DQ alpha and beta alleles in patients suggests that a specific DQ heterodimer may be responsible for the observed DR associations. Individuals with both this DQ antigen and a specific DP beta allele are at increased risk for coeliac disease.     

Processing pathways for presentation of cytosolic antigen to MHC class II-restricted T cells.

Antigens presented to CD4+ T cells derive primarily from exogenous proteins that are processed into peptides capable of binding to class II major histocompatibility complex (MHC) molecules in an endocytic compartment. In contrast, antigens presented to CD8+ T cells derive mostly from proteins processed in the cytosol, and peptide loading onto class I MHC molecules in an early exocytic compartment is dependent on a transporter for antigen presentation encoded in the class II MHC region. Endogenous cytosolic antigen can also be presented by class II molecules. Here we show that, unlike class I-restricted recognition of antigen, HLA-DR1-restricted recognition of cytosolic antigen occurs in mutant cells without a transporter for antigen presentation. In contrast, DR1-restricted recognition of a short cytosolic peptide is dependent on such a transporter. Thus helper T-cell epitopes can be generated from cytosolic antigens by several mechanisms, one of which is distinct from the classical class I pathway.     

A novel HLA class II molecule (DR alpha-DQ beta) created by mismatched isotype pairing

Human major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins composed of non-covalently associated alpha and beta chains. Only isotype-matched alpha-beta associations have been described in man; these can occur either by cis- or trans-complementation (HLA-DR, DQ, DP). Here evidence is provided for the existence of a new type of hybrid molecule (DR alpha-DQ beta) arising by mixed-isotype pairing in human B-cell lines. Class II isotype-mismatched heterodimers have been recently reported in the mouse after transfection of class II genes, and our data demonstrate that such interisotypic pairing can occur in untransfected cells. This crosspairing greatly enhances the repertoire of the class II antigens that regulate immune responses and leads us to reconsider the HLA-disease association.     

High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR

Staphylococcal enterotoxins A-E (refs 1-3), toxic shock toxin (TST-1) (ref. 1), a product of Mycoplasma arthritidis and the Mls antigens provoke dramatic T-cell responses. All are extremely potent polyclonal mitogens stimulating a large proportion of both murine and human CD4+ and CD8+T cells although activity is tightly restricted by major histocompatibility complex (MHC) class II antigens. The murine T-cell response to staphylococcal enterotoxin B (SEB) has recently been shown to involve only those T cells expressing T-cell receptor V beta 3, 8.1, 8.2 and 8.3 domains, a situation which closely mimics the response to Mls antigens. This paper examines the initial events in SEA and SEB T-cell activation and shows that MHC restriction results from a direct high affinity binding by intact SEA and SEB to the same site on MHC class II HLA-DR antigens.     

A malaria T-cell epitope recognized in association with most mouse and human MHC class II molecules

An ideal vaccine should elicit a long lasting immune response against the natural parasite, both at the T- and B-cell level. The immune response should occur in all individuals and be directed against determinants that do not vary in the natural parasite population. A major problem in designing synthetic peptide vaccines is that T cells generally recognize peptide antigens only in association with one or a few of the many variants of major histocompatibility complex (MHC) antigens. During the characterization of epitopes of the malaria parasite Plasmodium falciparum that are recognized by human T cells, we analysed a sequence of the circumsporozoite protein, and found that synthetic peptides corresponding to this sequence are recognized by T cells in association with many different MHC class II molecules, both in mouse and in man. This region of the circumsporozoite protein is invariant in different parasite isolates. Peptides derived from this region should be capable of inducing T-cell responses in individuals of most HLA-DR types, and may represent good candidates for inclusion in an effective anti-malaria peptide vaccine.     

50例中国人IX因子基因的限制性片段长度多态性

用IX因子基因内探针F9(VⅢ)对TaqI,BamHI和EcoRI酶切的50例中国人基因组DNA进行杂交分析。结果表明,所有个体经TaqI酶切的杂交片段为4.5kb和1.8kb,BamHI和EcoRI酶切的杂交片段分别为23kb和5.0 kb。基因组DNA样本中未发现限制性片段长度多态性(RFLP),这与欧美国家的民族群体中存在着IX因子基因内TaqI和BamHI的RFLP的结论不同。造成不同种族间DNA水平差异的原因,很可能与长期在不同地理环境中的进化适应有关。     

Prevention of autoimmune insulitis by expression of I-E molecules in NOD mice

The NOD (non-obese diabetic) mouse spontaneously develops insulin-dependent diabetes mellitus (IDDM) characterized by autoimmune insulitis, involving lymphocytic infiltration around and into the islets followed by pancreatic beta (beta) cell destruction, similar to human IDDM. Genetic analysis in breeding studies between NOD and C57BL/6 mice has demonstrated that two recessive genes on independent chromosomes contribute to the development of insulitis. One of the two recessive diabetogenic genes was found to be linked to the major histocompatibility complex (MHC). This is of interest, because the NOD strain has a unique class II MHC: it does not express I-E molecules as no messenger RNA for the alpha-chain of I-E is visible in Northern blot analysis; I-A molecules are not detected with any available monoclonal antibodies or by allo-reactive or autoreactive T-cell clones, although their expression is demonstrated with a conventional antiserum to Ia antigens. To examine whether the unusual expression of class II MHC molecules may be responsible for the development of autoimmune insulitis, we attempted to express I-E molecules in NOD mice selectively, without introducing other genes on chromosome 17 by using I-E-expressing C57BL/6 (B6(E alpha d)) transgenic mice. We report here that the expression of I-E molecules in NOD mice can prevent the development of autoimmune insulitis.     

Binding of labelled influenza matrix peptide to HLA DR in living B lymphoid cells

T cells recognize protein antigens as fragments (peptides) held in a defined binding site of class I or class II major histocompatibility (MHC) molecules. The formation of complexes between various immunologically active peptides and different MHC molecules has been demonstrated directly in binding studies between the peptides and solubilized, purified molecules of class II MHC. Studies with intact cells, living or fixed, have not directly demonstrated the binding of the peptides to MHC molecules on antigen-presenting cells, but the formation of such complexes has been shown indirectly through the capacity of antigen-presenting cells to stimulate specific T cells. Here we report evidence that supports directly the binding of radiolabelled influenza matrix peptide 17-29 to products of the human class II MHC locus HLA-DR, on living homozygous B-cell lines, and we show that the kinetics of such binding is much faster with living cells than with fixed cells. Furthermore, whereas the peptide reacts with HLA-DR molecules of all alleles, it binds preferentially to DR1, the restricting element in antigen presentation.     

Structural relationship of the SB beta-chain gene to HLA-D-region genes and murine I-region genes

The major histocompatibility complex (MHC) regulates several aspects of the immune response. Class II antigens of the MHC control cellular interactions between lymphocytes. In man, at least three class II antigens (DR, DC and SB), consisting of distinct alpha- and beta-chains, are encoded in the HLA complex. Sequence analysis has established that the DR and DC antigens are the respective structural counterparts of the murine I-E and I-A antigens. Molecular cloning of the SB beta-chain gene has now enabled us to define its relationship to other class II genes. The DR, DC and SB beta genes have diverged from each other to the same extent. In murine DNA and in cloned genes from the I region, the best hybridization of SB beta DNA is with the E beta 2 sequence. E beta 2 may belong to a complete gene (E' beta) because first domain sequences were found adjacent to it.     

Correlation between a DNA restriction fragment length polymorphism and C4A6 protein

The fourth component of complement (C4) in man, is coded for by two separate but closely linked loci (C4A and C4B) within the major histocompatibility region (MHC), on the short arm of chromosome 6. Like class I and II loci of this region, the C4 genes are highly polymorphic with more than 30 alleles, including null alleles, assigned to the two loci. This extensive polymorphism, based mainly on electrophoretic mobility, provides a useful marker for studies of disease susceptibility. Several disorders, including systemic lupus erythematosus and type I diabetes, show associations with C4 phenotypes. We have used the technique of Southern with a C4 specific probe to examine the genomic DNA of individuals typed for C4 by protein electrophoresis. We have identified 10.7 and 3.8 kilobase (kb) BglII restriction fragments in each of 9 unrelated individuals with a C4A6 allele, and in none of 22 unrelated individuals in whom this allele was not expressed. This clear correlation of restriction fragment length polymorphism with C4 phenotype provides a precise basis for analysis of C4 polymorphism. It is likely to be of value in clinical investigations of autoimmune disease.     

Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding

Class II major histocompatibility complex (MHC) molecules are heterodimeric cell surface glycoproteins which bind and present immunogenic peptides to T lymphocytes. Such peptides are normally derived from protein antigens internalized and proteolytically degraded by the antigen-presenting cell. Class I MHC molecules also bind immunogenic peptides, but these are derived from proteins synthesized within the target cell. Whereas class I molecules seem to bind peptides in the endoplasmic reticulum, class II molecules are thought to bind peptides late in transport. Intracellular class II molecules associate in the endoplasmic reticulum with a third glycoprotein, the invariant (I) chain, which is proteolytically removed before cell surface expression of the alpha beta class II heterodimer. It has been suggested that the I chain prevents peptides from associating with class II molecules early in transport. Preventing such binding until the class II molecules enter an endosomal compartment could maintain the functional dichotomy between class I and class II MHC molecules. We have examined the ability of I chain-associated HLA-DR5 molecules to bind a well characterized influenza haemagglutinin-derived peptide (HAp). The results show that whereas mature HLA-DR alpha beta dimers effectively bind this peptide, the I chain-associated form does not.     

Interaction between CD4 and class II MHC molecules mediates cell adhesion

The CD4 glycoprotein is expressed on T-helper and cytotoxic lymphocytes which are restricted to class II major histocompatibility complex (MHC) antigens on target cells. Antibody inhibition studies imply that CD4 acts to increase the avidity of effector-target cell interactions. These observations have led to the speculation that CD4 binds to a monomorphic class II antigen determinant, thereby augmenting low affinity T-cell receptor-antigen interactions. However, no direct evidence has been presented indicating that CD4 and class II molecules interact. To address this issue, we have used a vector derived from simian virus 40 (SV40) to express a complementary DNA (cDNA) encoding the human CD4 glycoprotein. When CV1 cells expressing large amounts of the CD4 protein at the cell surface are incubated with human B cells bearing MHC-encoded class II molecules, they are bound tightly to the infected monolayer, whereas mutant B cells which lack class II molecules fail to bind. Furthermore, the binding reaction is specifically inhibited by anti-class II and anti-CD4 antibodies. Thus, the CD4 protein, even in the absence of T-cell receptor-antigen interactions, can interact directly with class II antigens to function as a cell surface adhesion molecule.     

Cell-cell adhesion mediated by CD8 and MHC class I molecules

CD4 and CD8 are cell-surface glycoproteins expressed on mutually exclusive subsets of peripheral T cells. T cells that express CD4 have T-cell antigen receptors that are specific for antigens presented by major histocompatibility complex class II molecules, whereas T cells that express CD8 have receptors specific for antigens presented by MHC class I molecules (reviewed in ref. 1). Based on this correlation and on the observation that anti-CD4 and anti-CD8 antibodies inhibit T-cell function, it has been suggested that CD4 and CD8 increase the avidity of T cells for their targets by binding to MHC class II or MHC class I molecules respectively. Also, CD4 and CD8 may become physically associated with the T-cell antigen receptor, forming a higher-affinity complex for antigen and MHC molecules, and could be involved in signal transduction. Cell-cell adhesion dependent CD4 and MHC II molecules has recently been demonstrated. To determine whether CD8 can interact with MHC class I molecules in the absence of the T-cell antigen receptor, we have developed a cell-cell binding assay that measures adhesion of human B-cell lines expressing MHC class I molecules to transfected cells expressing high levels of human CD8. In this system, CD8 and class I molecules mediate cell-cell adhesion, showing that CD8 directly binds to MHC class I molecules.     

插入Tn21质粒的分离和链霉素抗性基因质粒的组建

在携带R100.1和pSO101质粒的Lc2640细胞中分离到重组质粒pXZ2712,该质粒对链霉素、氯化汞、磺胺和四环索有抗性。用仅带有Tn21两个末端的质粒pXZ2作为探针进行分子杂交,发现两者有同源性,说明质粒pXZ2712是由Tn21插入质粒pSC101所组成的。以pXZ2712质粒中分离到的带有链霉素抗性基因的片段,组建了带有抗链霉素基因的pXZ6(14.5kb)、pYP1(9.7kb)、pYP22(9.1kb)和pYP4(4.0kb)等质粒载体。