Proliferative responses to selected peptides of IA-2 in identical twins discordant for Type 1 diabetes.

BACKGROUND: The aim of the study was to define T lymphocyte reactivity to selected peptides of an islet antigen IA-2, associated with Type 1 diabetes. METHODS: We used 10 peptides selected from the IA-2 molecule due to their predicted ability to bind to HLA-DRB1*0401, a Type 1 diabetes-associated allele. We tested 21 identical twin pairs discordant for the disease and 15 control subjects and then followed them prospectively; seven non-diabetic twins developed diabetes. RESULTS: Twins of identical pairs tended to respond to different peptides suggesting that the T cell response is, to a degree, shaped by non-genetically determined factors (p<0. 0001). However, there was no difference in the T cell responses between diabetic twins and either their non-diabetic identical twins or control subjects and the response was heterogenous. T cell responses did not differ in those seven non-diabetic twins who developed diabetes from those twins who did not. T cell responses to peptide 11 (amino acids 502-514) was immunodominant in diabetic twins as well as their non-diabetic twins and controls; responses were not correlated with HLA, IA-2 antibodies, age or duration of disease. CONCLUSION: We conclude that T cell responses to selected IA-2 peptides are not genetically determined, heterogeneous, not strictly HLA controlled and did not distinguish diabetic or prediabetic twins from non-diabetic twins or controls. The identification of an immunodominant T cell response to IA-2 peptide 502-514 raises the possibility that this, or similar, epitopes may be of therapeutic value in disease prevention.

Melanoma antigen-encoding gene expression in melanocytic naevi and cutaneous malignant melanomas.

Melanoma antigen-encoding (MAGE) genes encode peptides which are expressed by a proportion of malignant melanomas (MMs) and can be recognized in vitro by autologous cytolytic T lymphocytes in a human leucocyte-associated antigen (HLA)-restricted manner. Although expression of MAGE genes has been studied in cutaneous MMs, little is known about their expression in melanocytic naevi. We studied MAGE 1, 2, 3, 4, 6 and 12 gene expression in tissue from 10 benign melanocytic naevi, 14 dysplastic melanocytic naevi, three cutaneous MMs in situ, four primary cutaneous MMs and three distant metastatic MMs. MAGE gene expression was determined with reverse transcription-polymerase chain reaction (PCR) using random hexamers to generate cDNA from total tissue homogenate RNA followed by PCR using intron-spanning, MAGE-specific primer pairs. Controls were cDNA from MAGE-expressing melanoma cell lines. Expression of HLA class 1 was used as a cDNA quality control. MAGE 2, 4, 6 and 12 gene expression was not detected in any of the lesions studied. MAGE 1 and 3 gene expression was also not detected in any of the cases of benign and dysplastic melanocytic naevi and in situ MMs. One of four primary MMs expressed the MAGE 3 gene. Two of three distant metastatic MMs also expressed the MAGE 3 gene and one of these additionally expressed the MAGE 1 gene. Thus, MAGE gene expression appears to be a late event in the evolution of MMs.

HLA genotype of patients with severe haemophilia A due to intron 22 inversion with and without inhibitors of factor VIII.

Molecular genetic studies have shown that development of antibodies to factor VIII (inhibitors) occurs most frequently in patients with severe haemophilia due to major gene lesions including inversions, stop codons and large deletions. Previous studies of HLA type were performed on inhibitor and non-inhibitor patients with diverse uncharacterized mutations which may have confounded detection of significant associations. We therefore selected a group of patients with a single mutation type, the prevalent intron 22 inversion, with or without inhibitors, to determine HLA genotype. Seventy-one such patients, 42 without and 29 with inhibitors (13 high, 9 low and 7 transient responders) were genotyped for MHC Class I HLA-A, -B, -C and Class II HLA-DQA, -DQB and -DRB loci. No strong correlation of any HLA-allele to inhibitor or non-inhibitor status was found. However, alleles of the haplotype HLA-A3, HLA-B7, HLA-C7, HLA-DQA0102, HLA-DQB0602, HLA-DR15 occurred more often in inhibitor patients. Since the alleles of this extended haplotype are common in the North European population only a very strong association would achieve statistical significance. Further studies of groups of patients similar to those studied here will be needed to confirm or exclude this association.

Human vascular endothelial cells process and present autoantigen to human T cell lines.

The effectiveness of cultured human umbilical vein endothelial cells as accessory cells for T cell activation has been investigated using T cell clones and lines derived from patients with myasthenia gravis which were specific for different epitopes on the alpha subunit of the human acetylcholine receptor. The endothelial cells were induced with IFN-gamma to express HLA-DR and -DQ at high and low levels respectively. They could then efficiently present specific peptides of the alpha subunit to an HLA-DR- and an HLA-DQw5-restricted T cell line. They could also process epitopes for both T cell lines from the full-length recombinant alpha subunit (r1-437) of the human acetylcholine receptor, where the known epitopes are 80 amino acid residues apart. The endothelial presentation of r1-437, but not of the peptides, was sensitive to chloroquine inhibition. Presentation appeared slightly less efficient (by 1.5- to 3.0-fold) with endothelial cells than with presenting cells from peripheral blood. This may reflect differences in accessory signalling since mAb blocking studies suggested that ligands for CD28 provided important accessory signalling by peripheral blood presenting cells while LFA-3 was used by endothelial cells.

Single-step allele-specific polymerase chain reaction HLA-DQ genotyping using ARMS primers.

A set of allele-specific oligonucleotide primer pairs has been developed that allows the determination of HLA-DQ genotypes in a rapid, single-step PCR. The primer set unambiguously distinguishes the 18 most common DQ haplotypes in heterozygous as well as homozygous individuals. Because of linkage disequilibrium between the DQ and DR gene loci, accurate determination of the DQ haplotypes enables the prediction of the DR serotype. HLA-DQ genotyping can be performed using DNA from any tissue, such as skin and hair roots, as well as from blood cells. It is therefore anticipated that HLA-DQ genotyping by PCR will have applications in forensic science, in the identification of individuals, in research studies of the role of DQ and DR haplotypes in the pathogenesis of autoimmune diseases, and in the rapid matching of organ donor and transplant recipient pairs.

Human CD4+ T cells proliferate to HLA-DR+ allogeneic vascular endothelium. Identification of accessory interactions.

Serially passaged human endothelial cell (EC) cultures will stimulate highly purified peripheral blood CD4+ T cells to proliferate if and only if the EC cultures are pretreated with IFN-gamma to induce de novo expression of MHC class II molecules, principally HLA-DR. HLA-DR-expressing EC alone appear sufficient to stimulate purified CD4+ T cell proliferation without the involvement of other leukocyte populations, as indicated by the following observations: (1) we find no contaminating leukocytes in our EC cultures by FACS analysis or fluorescence microscopy; specifically, there are no detectable CD45 or HLA-DR expressing cells; (2) neither the EC cultures nor the purified CD4+ T cells contain HLA-DR expressing cells detectable by polymerase chain reaction (PCR) of reverse-transcribed mRNA; (3) the stimulatory capacity of the EC cultures is maintained through serial subculture and through low-density replating, indicating that the stimulatory cell type must proliferate in culture as well as EC; and (4) in contrast to MLRs, the response to EC cultures is not inhibited by pretreatment of the stimulator cells and/or responding T cells with the monocyte toxin L-leucine-O-methyl ester. We have used mAb to investigate the role of various EC and T cell surface molecules in the T cell response. mAb to HLA-DR and CD4 inhibit proliferative responses of CD4+ T cells to EC cultures, as would be expected if T cells recognize and proliferate to IFN-gamma-induced allogeneic class II MHC molecules; whereas, also as expected, mAb to class I MHC molecules were without effect. Proliferation is also inhibited by mAbs to T cell CD2 and LFA-1 beta chain (CD18) and by mAbs to LFA-3 (CD58) and CD44, which are expressed by T cells and EC. mAb to ICAM-1 (CD54, a ligand for LFA-1) provides inconsistent inhibition, and mAb to ICAM-2, used with or without anti-ICAM-1, is not inhibitory. Because both of these mAb block adhesion of LFA-1 expressing T cells to EC, our data suggest that additional ligands for LFA-1 must be important for allogeneic proliferation. mAb to VLA-4 alpha or beta chains (CD49d, CD29) enhance proliferation, presumably through direct costimulation of the T cells by these antibodies. However, a mAb to VCAM-1, an EC ligand for VLA-4, is partially inhibitory.(ABSTRACT TRUNCATED AT 400 WORDS)

Prevention of insulin-dependent diabetes mellitus in non-obese diabetic mice by transgenes encoding modified I-A beta-chain or normal I-E alpha-chain.

Insulin-dependent diabetes mellitus (IDDM) is a disease with an autoimmune aetiology. The inbred non-obese diabetic (NOD) mouse strain provides a good animal model of the human disease and genetic analysis suggests that, as in man, at least one of the several genes controlling the development of IDDM is linked to the major histocompatibility complex. The NOD mouse does not express I-E owing to a deletion in the promoter region of the I-E alpha-chain gene, and the sequence of NOD I-A beta-chain in the first external domain is unique with His 56 and Ser 57 replacing Pro and Asp, respectively, at these positions. There has been considerable interest in the role amino acid 57 might have in conferring susceptibility to autoimmune diseases, including IDDM. The presence of a charged residue (such as Asp) at this position might affect the conformation of the peptide binding groove. But it could be assumed that Pro 56 gives rise to a different conformation of I-A beta-chain than does His 56. We therefore constructed transgenic NOD mice in which the transgene encoded a modified A beta nod with Pro 56, and studied its effect on the development of IDDM in this mouse strain. Previous studies have suggested that NOD mice expressing I-E as a result of the introduction of an I-E alpha-chain (E alpha) transgene are protected from the development of insulitis and hence IDDM. To explore further the protective effect of this molecule we constructed a second class of transgenic NOD mouse carrying an E alpha d transgene. Both transgenes protected the mice from IDDM, but this was not associated with a complete deletion of any T cells expressing commonly used T-cell receptor V beta genes.

Differing reactions of monoclonal anti-A antibodies with oligosaccharides related to blood group A.

Inhibition radioimmunoassays with blood group A-related oligosaccharides have been used to investigate the specificities of six monoclonal anti-A antibodies, three of which had been intentionally generated by immunization of mice with blood group A erythrocytes and A-active blood group substance, and three were incidentally produced following immunization of mice with human tonsil cell membranes or a human colon cancer cell line. By hemagglutination, these antibodies are highly specific for human blood group A erythrocytes. However, they differ from one another in their reaction patterns with mono- and difucosyl A antigen structures and the corresponding afucosyl sequences on Type 1 and Type 2 backbone structures. The six antibodies, together with four previously characterized anti-A monoclonal antibodies (originally raised against the receptor for epidermal growth factor) have been classified into five groups. The first two groups consist of antibodies with broad specificities for A-related structures. There are five antibodies in the first group (TL5, 29.1, A17/3D1, MH2/6D4, and MH1/5D1) reacting to varying degrees with the mono- and difucosyl A antigen structures on either type of backbone sequence. In the second group are two antibodies (A15/3D4 and A15/3D3) which are difficult to inhibit with the oligosaccharides tested, but they reacted best with monofucosyl A structure on either type of backbone. Each of the remaining three antibodies had a distinct and more restricted reaction pattern, with a specificity for the difucosyl A antigen on both types of backbone (antibody EGR/G49) or the Type 1-based mono- and difucosyl A antigen structures (antibody MAS 016c) or the Type 2-based monofucosyl A antigen structure (antibody 455). The reactions of four of the antibodies with N-acetylgalactosamine or with oligosaccharides containing the afucosyl sequence GalNAc alpha 1-3Gal suggest that they may react with certain glycoconjugates with alpha-N-acetylgalactosaminyl termini ("A-like" structures) that are unrelated to the products of the blood group A gene-specified alpha-N-acetylgalactosaminyl-transferase. Knowledge of the differing reactions of these monoclonal antibodies is important for interpreting their reactions with glycoproteins and glycolipids of diverse origins.

Monoclonal antibody (EGR/G49) reactive with the epidermal growth factor receptor of A431 cells recognizes the blood group ALeb and ALey structures.

The carbohydrate specificity of the monoclonal antibody EGR/G49, raised against the epidermal growth factor (EGF) receptor of A431 cells, has been investigated by assessing its interactions with glycoproteins and erythrocytes derived from individuals of known blood group ABH, Lewis and secretor types, and by inhibition of binding assays using structurally defined oligosaccharides. The results indicate that this antibody reacts with the difucosylated blood group structures ALeb and ALey: (formula; see text) This antibody differs from the previously described anti-EGF receptor antibody. TL5, which is directed at the terminal blood group A trisaccharide structure and reacts poorly with the ALeb/Ley structures. Since both antibodies were selected for their reactivities with the receptor for EGF, their specificities provide evidence for the presence of both the mono- and difucosylated blood group A structures on the receptor glycoprotein. These antibodies will be invaluable in the studies of the distribution and the roles of blood group related carbohydrate structures in the organisation and function of the EGF and other receptor systems.

Blood group antigens A, B, H, Lea, Leb, and I(Ma) in resting and tetragastrin stimulated gastric juice of patients with non-neoplastic diseases of the stomach.

In view of the anomalous expression of blood group and related antigens in the gastric mucosae of patients with malignant and premalignant diseases of the stomach, and the potential clinical value of their measurement, a preliminary study has been performed on the blood group antigens A, B, H, Lea, Leb, and I(Ma) in glycoprotein rich extracts of the resting and tetragastrin stimulated gastric juice of patients without evidence of gastric cancer. The aim has been to assess whether the antigenic profiles known to distinguish the gastric mucosae of secretors from those of non-secretors are reflected in the glycoproteins of gastric juice. Antigenic profiles which distinguish secretors from non-secretors were observed in the stimulated rather than the resting gastric juice as follows: the A, B or H antigens but not I(Ma) were strongly expressed in the glycoproteins of secretors, while I(Ma) was the antigen characteristic of non-secretors. On the other hand, there was considerable overlap in the Lea and Leb antigen values in the resting and stimulated gastric juice of secretors and non-secretors. Among these antigens, I(Ma) is known to appear as a neo-antigen in the gastric mucosae of secretors with malignant and premalignant diseases of the stomach. Thus this antigenic determinant is potentially a clinically useful marker in the gastric juice of 75% of the population who are secretors. The clinical value of the levels of this antigen in the gastric juice now deserves investigation.

Evidence for sialylated type 1 blood group chains on human erythrocyte membranes revealed by agglutination of neuraminidase-treated erythrocytes with Waldenström's macroglobulin IgMWOO and hybridoma antibody FC 10.2.

Haemagglutination studies have been performed with untreated and neuraminidase-treated human erythrocytes of the three Lewis antigen types Le(a-b-), Le(a+b-) and Le(a-b+) using two monoclonal antibodies, IgMWOO and FC 10.2, which were previously shown to recognize the type 1 based blood group chains: Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc/GlcNAc (for explanation of abbreviations see table IV legend). Both antibodies behaved as cold agglutinins with neuraminidase-treated but not with untreated erythrocytes of the three Lewis antigen types. Neuraminidase-treated erythrocytes of i antigen type were similarly agglutinated. This haemagglutination was specifically inhibited by the type 1 based milk oligosaccharide lacto-N-tetraose. Thus, there is strong evidence for the occurrence of sialylated type 1 chains on human erythrocyte membranes of I and i antigen types. In addition, evidence for the presence of type 1 chains which are both sialylated and fucosylated was obtained by (1) haemagglutination of Le(a+b-) erythrocytes with the monoclonal antibody 19.9; (2) increased haemagglutination of neuraminidase-treated erythrocytes with anti-H antibodies of Bombay serum; (3) increased haemagglutination of neuraminidase-treated Le(a+b-) cells with anti-Lea antibodies, and (4) the appearance of Lea antigen activity on neuraminidase-treated erythrocytes of Le(a-b+) type.

Tumour-associated and differentiation antigens on the carbohydrate moieties of mucin-type glycoproteins.

In this report the carbohydrate antigens expressed on the three oligosaccharide domains, core, backbone and peripheral, of mucin-type glycoproteins are briefly reviewed in the light of recent observations with monoclonal antibodies. These have revealed that a number of cell-surface antigens which behave as tumour-associated and differentiation antigens of man or mouse are abundantly expressed on the carbohydrate chains of a variety of secreted mucins of human and animal origins and they belong to an antigen system which also includes the major blood group antigens. Examples are given of the use of well-characterized anti-carbohydrate antibodies to derive structural information on (a) mucin-type glycoproteins of human B lymphocyte membranes, (b) the high molecular weight glycoproteins of the normal human gastric and distal-colon mucosae and (c) tumour-derived glycoproteins from these two organs. Major differences between the antigenicities of the normal stomach and distal-colon, and between their tumour-derived glycoproteins, and the important effect of the secretor status in the expression of these antigens are described. These observations have enabled a better understanding of the individual and tissue differences in the expression of tumour-associated antigens. The possibility is raised that these carbohydrate structures (many of which also occur on certain N-linked oligosaccharides and glycolipids) are components of receptor systems for endogenous ligands. More tangible evidence is cited for the role of certain structures in this family of saccharides as receptors for infective agents.

Peanut lectin and anti-Ii antibodies reveal structural differences among human gastrointestinal glycoproteins.

Human gastrointestinal glycoproteins (mucins), isolated by pepsin digestion from foetal stomachs and meconia, and from paired tumour and non-neoplastic mucosal samples of patients with gastric and colorectal carcinomas, were tested for precipitating reactions with peanut lectin (PNL) and four anti-carbohydrate antibodies (two anti-I, Ma and Low, and two anti-i, Den and Galli). There was remarkable correlation between reactivities with PNL and anti-I (Ma): both reagents reacted with non-neoplastic gastric glycoproteins of "non-secretors", but not with those of "secretors", and also with the majority of gastric tumour and meconium extracts regardless of secretor status. Colorectal tissue extracts (with the exception of one tumour extract) reacted with neither reagent. The various precipitating activities, and results of mild acid hydrolysis and affinity chromatography experiments, enable certain inferences to be made regarding the oligosaccharide moieties of gastrointestinal glycoproteins: (a) expression of PNL and anti-I (Ma) determinants in gastric glycoproteins is dependent on secretor status; (b) extracts reacting with PNL and anti-I (Ma) are mixtures of macromolecules: minor populations react with both reagents, or with PNL only; the major population lacks both determinants, or they are masked by other substitutions; (c) determinants reactive with anti-Ii sera other than anti-I (Ma) are less frequently expressed; and (d) colonic glycoproteins in their lack of PNL and Ii determinants. This suggests that there are structural differences in the oligosaccharide backbones of the two types of glycoprotein.

A monoclonal antibody against human colonic adenoma recognizes difucosylated Type-2-blood-group chains.

A monoclonal antibody C14/1/46/10 showing preferential binding to membranes of human colorectal carcinomas over normal colon mucosae was obtained by immunization of mice with extra-nuclear membranes of a human colonic adenoma. Binding and inhibition of binding assays using blood cells or glycoproteins with known blood-group activities indicated that the antibody recognizes a carbohydrate antigen co-existing with the blood-group-H determinant: Fuc alpha 1 leads to 2 Gal. Inhibition assays with structurally defined oligosaccharides showed that the antigenic determinant involves difucosylated Type-2-blood-group chains with the structure: (formula; see text)