HLA-restricted T-cell recognition of Epstein-Barr virus-infected B cells

In mice the cytotoxic T-cell response to several types of virus is influenced by genes within the major histocompatibility complex; in particular, genetic control is exercised at the effector cell level through a requirement that virus-specific cytotoxic T cells recognise viral antigens in association with H-2K and H=2D region gene products on the surface of infected cells. In man the restriction which the analogous HLA-A, -B and -C-region gene products might place on virus-specific T-cell function is still in dispute. The earliest and most controversial evidence concerns the Epstein-Barr virus (EBV), a B lymphotropic agent which causes infectious mononucleosis (IM) and which induces an unusually vigorous T-cell response; cytotoxic T cells from IM patients' blood were shown to be EBV-specific yet, in contrast to mouse systems, apparently free of any obvious HLA restriction. Since then T-cell recognition of EBV-infected B cells has assumed particular significance as a model system for the study of cytotoxic T-cell function in man. This report describes the results of a new approach clearly indicating that HLA-A and -B region products do indeed have a role in this system.     

Strong xenogeneic HLA response in transgenic mice after introducing an alpha 3 domain into HLA B27.

The pronounced response by mouse T cells to the major histocompatibility complex (MHC) class I antigens of the same species is characterized by a relatively large fraction of responding cells. Responses to MHC class I allelles of other species are, however, generally much weaker. T lymphocytes are positively selected on thymic MHC antigens, resulting in a T-cell repertoire with strong alloreactivity. This has been explained in terms of a mouse T-cell repertoire that is not efficiently selected for recognition of HLA molecules owing to the absence of HLA in mice. Here we show that mice transgenic for HLA mount a T-cell response against allogeneic HLA that is no better than in normal mice. We decided instead to test whether the mouse accessory molecule Lyt-2 on cytotoxic T lymphocytes could interact efficiently with the alpha 3 domain of HLA. To do this, we replaced the alpha 3 domain of HLA-B27 by a murine alpha 3 domain in a gene construct used to produce transgenic mice, and then used the spleen cells from these mice to stimulate normal mouse T cells. Under these conditions cytotoxic T lymphocytes were generated with the same frequency against xenogeneic HLA-B27 determinants as against allogeneic mouse class I antigens. These findings indicate that the normally weak xeno-MHC response is due to the inefficient interaction of the murine Lyt-2 accessory molecule with HLA class I, and not to limitations of the mouse T-cell repertoire.     

Murine cells expressing an HLA molecule are specifically lysed by HLA-restricted antiviral human T cells

Class I HLA (histocompatibility locus antigen) molecules are the targets of allospecific cytolytic T lymphocytes (CTL) in graft rejection, and constitute the restricting elements necessary for the interaction between antiviral CTL and virus-infected cells. Cells expressing only one HLA in the absence of other human molecules would provide a remarkable model for studying the function of these molecules. However, HLA+ murine cells transfected with human genes are generally not lysed by allospecific human CTL, and this is ascribed to insufficient HLA expression, lack of human beta 2-microglobulin, alteration of HLA molecules or absence of receptors for human T8 or LFA1 molecules in murine cells. Here we report, for the first time, the specific lysis of virus-infected HLA+ murine cells by HLA-restricted antiviral human CTL. Therefore, these murine cells constitute an excellent model for studying the role of HLA molecules.     

MHC antigens in urine as olfactory recognition cues

The classical class I antigens of the major histocompatibility complex (MHC) are cell-surface glycoproteins which were originally discovered because they cause rapid rejection of cells or tissues grafted between unrelated individuals. These molecules are encoded by the K, D and L loci of the mouse MHC (and analogous loci in other species) which show extreme species polymorphism and a large number of alleles. In an outbreeding population 3.6 X 10(9) unique MHC class I phenotypes can be encoded by the 100 alleles at each of the K and D loci and the 6 alleles at the L locus. This level of polymorphism ensures that the cells and tissues of each unrelated individual are uniquely identified by their class I membrane-bound antigens. Like other membrane bound proteins, these class I molecules are anchored in the lipid bilayer by a hydrophobic domain encoded by exon 5. However, there have been reports of the occurrence of classical class I molecules in true solution in the blood of humans, mice, and rats. We report here that classical polymorphic class I molecules in normal rats are constitutively excreted in the urine and that untrained rats can distinguish the smell of urine samples taken from normal donors that differ only at the class I MHC locus and therefore excrete different allelomorphs of class I molecules in their urine.     

Virus-induced autoantibody response to a transgenic viral antigen

The induction of autoantibodies and their possible role in the pathogenesis of autoimmune disease are poorly understood. Involvement of infectious agents has been suspected, but direct evidence is sparse. Whether immunological unresponsiveness to self by antibody-forming B cells is maintained by clonal abortion, clonal anergy or suppression, or how the scenario of interactions between helper T cells, B cells and antigen-presenting cells is distorted in autoantibody responses, is being analysed and widely debated. To evaluate tolerance of neutralizing B-cell responses we used transgenic mice expressing the cell membrane associated glycoprotein (G) of vesicular stomatitis virus (VSV) as self-antigen. We show that autoantibodies to VSV-G cannot be induced by VSV-G in adjuvant or by recombinant vaccinia virus expressing VSV-G, but are triggered by infection with wild-type VSV. The data show that helper T-cell tolerance is crucial in maintenance of B-cell non-reactivity and that cognate T-B recognition is necessary to break tolerance of self-reactive B cells. These results may help to understand mechanisms of virus-induced autoimmunity.     

Regulation of human insulin gene expression in transgenic mice

Insulin is a polypeptide hormone of major physiological importance in the regulation of fuel homeostasis in animals (reviewed in refs 1,2). It is synthesized by the beta-cells of pancreatic islets, and circulating insulin levels are regulated by several small molecules, notably glucose, amino acids, fatty acids and certain pharmacological agents. Insulin consists of two polypeptide chains (A and B, linked by disulphide bonds) that are derived from the proteolytic cleavage of proinsulin, generating equimolar amounts of the mature insulin and a connecting peptide (C-peptide). Humans, like most vertebrates, contain one proinsulin gene, although several species, including mice and rats, have two highly homologous insulin genes. We have studied the regulation of serum insulin levels and of insulin gene expression by generating a series of transgenic mice containing the human insulin gene. We report here that the human insulin gene is expressed in a tissue-specific manner in the islets of these transgenic mice, and that serum human insulin levels are properly regulated by glucose, amino acids and tolbutamide, an oral hypoglycaemic agent.     

Prevention of diabetes in non-obese diabetic I-Ak transgenic mice

The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) with mononuclear cell infiltration of the islets of Langerhans and selective destruction of the insulin-producing beta-cells, as in humans. Most infiltrating cells are T lymphocytes, and most of these carry the CD4 antigen. Adoptive transfer of T cells from diabetic NOD mice into irradiated NOD or athymic nude NOD mice induces diabetes. Susceptibility to IDDM in NOD mice is polygenic, with one gene linked to the major histocompatibility complex class II locus, which in NOD mice expresses a unique I-A molecule but no I-E. Speculation exists as to the role of the I-A molecule in the diabetes susceptibility of NOD mice, especially regarding the significance of specific unique residues. To examine the role of the NOD I-A molecule in IDDM pathogenesis, we made NOD/Lt mice transgenic for I-Ak by microinjecting I-Ak alpha- and beta-genes into fertilized NOD/Lt eggs. Insulitis was markedly reduced and diabetes prevented in NOD/Lt mice expressing I-Ak.     

Formation of beta-amyloid protein deposits in brains of transgenic mice

Deposits of beta-amyloid are one of the main pathological characteristics of Alzheimer's disease. The beta-amyloid peptide constituent (relative molecular mass 4,200) of the deposits is derived from the beta-amyloid precursor protein (beta-APP) which is expressed in several different isoforms. The two most prevalent beta-APP isoforms are distinguished by either the presence (beta-APP751) or absence (beta-APP695) of a Kunitz serine protease inhibitor domain. Changes in the abundance of different beta-APP messenger RNAs in brains of Alzheimer's disease victims have been widely reported. Although these results have been controversial, most evidence favours an increase in the mRNAs encoding protease inhibitor-containing isoforms of beta-APP and it is proposed that this change contributes to beta-amyloid formation. We have now produced an imbalance in the normal neuronal ratio of beta-APP isoforms by preparing transgenic mice expressing additional beta-APP751 under the control of a neural-specific promoter. The cortical and hippocampal brain regions of the transgenic mice display extracellular beta-amyloid immunoreactive deposits varying in size (less than 5-50 microns) and abundance. These results suggest that one mechanism of beta-amyloid formation may involve a disruption of the normal ratio of neuronal beta-APP isoform expression and support a direct relationship between increased expression of Kunitz inhibitor-bearing beta-APP isoforms and beta-amyloid deposition.     

Isotype exclusion and transgene down-regulation in immunoglobulin-lambda transgenic mice

A given B lymphocyte makes an antibody containing either kappa- or lambda-light chains, but not both. This isotype exclusion is effected at the level of the rearrangement of the immunoglobulin gene segments, although by an unknown mechanism. An attractive possibility is that, following productive rearrangement of one of the light-chain loci, the newly synthesized light-chain polypeptide inhibits DNA rearrangement for the other isotype. To test such feedback regulation, we have created transgenic mice carrying a rearranged lambda 1-gene. By contrast with the B cells in normal newborn mice which are mainly kappa+lambda-, the B cells in the newborn transgenic mice express lambda- but not kappa-chains. We propose that the synthesis of any light chain, be it kappa or lambda, that allows expression of IgM on the cell surface results in a cessation of all V-J joining. Interestingly, the limited light-chain repertoire of the transgenic mice does not persist and most adult B cells express endogenous kappa-rearrangements and down-regulate the transgene.     

Bovine papillomavirus genome elicits skin tumours in transgenic mice

Transmission of the bovine papillomavirus-1 (BPV-1) genome through the mouse germ line results in the heritable formation of fibropapillomas of the skin, a tissue-specific phenotype analogous to that observed in natural BPV-1 infection of cattle. Oncogenesis is slow, with tumours first arising at 8-9 months of age, usually in areas prone to wounding. Extrachromosomal BPV-1 DNA is detected in all tumours, whereas normal tissues show only integrated DNA.     

血管内皮生长因子C嵌合体（CA65）转基因小鼠的建立和分析

将血管内皮生长因子C嵌合体CA65插入角蛋白14（keratin 14,K14）启动子下游,构建了嵌合体CA65转基因表达载体,通过显微注射法建立转基因小鼠.利用特异引物聚合酶链式反应法鉴定转基因小鼠的基因型后,通过逆转录聚合酶链式反应检测嵌合体CA65的表达水平,证实成功建立了皮肤特异表达嵌合体CA65转基因小鼠.对...     

Genomic imprinting determines methylation of parental alleles in transgenic mice

Mouse embryogenesis relies on the presence of both the maternal and the paternal genome for development to term. It has been proposed that specific modifications are imprinted onto the chromosomes during gametogenesis; these modifications are stably propagated, and their expression results in distinct and complementary contributions of the two parental genomes to the development of the embryo and the extraembryonic membranes. Genetic data further suggest that a substantial proportion of the genome could be subject to chromosomal imprinting, the molecular nature of which is unknown. We used random DNA insertions in transgenic mice to probe the genome for modified regions. The DNA methylation patterns of transgenic alleles were compared after transmission from mother or father in seven mouse strains carrying autosomal insertions of the same transgenic marker. One of these loci showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. This difference was observed in embryos on day 10 of gestation, but not in their extraembryonic membranes. Moreover, the methylation pattern was faithfully reversed upon each germline transmission to the opposite sex. Our findings provide evidence for heritable molecular differences between maternally and paternally derived alleles on mouse chromosomes.