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.     

An explanation for the protective effect of the MHC class II I-E molecule in murine diabetes

Insulin-dependent diabetes mellitus is widely believed to be an autoimmune disease. Recent onset diabetics show destruction of insulin-secreting pancreatic beta-cells associated with a lymphocytic infiltrate (insulitis), with autoantibodies to beta-cells being found even before the onset of symptoms. Susceptibility to the disease is strongly influenced by major histocompatibility complex (MHC) class II polymorphism in both man and experimental animal models such as the non-obese diabetic (NOD) mouse. As MHC class II molecules are usually associated with dominant immune responsiveness, it was surprising that introduction of a transgenic class II molecule, I-E, protected NOD mice from insulitis and diabetes. This could be explained by a change either in the target tissue or in the T cells presumed to be involved in beta-cell destruction. Recently, several studies have shown that I-E molecules are associated with ontogenetic deletion of T cells bearing antigen/MHC receptors encoded in part by certain T-cell receptor V beta gene segments. To determine the mechanism of the protective effect of I-E, we have produced cloned CD4+ and CD8+ T-cell lines from islets of recently diabetic NOD mice. These cloned lines are islet-specific and pathogenic in both I-E- and I-E+ mice. Both CD4+ and CD8+ cloned T cells bear receptors encoded by a V beta 5 gene segment, known to be deleted during development in I-E expressing mice. Our data provide, therefore, an explanation for the puzzling effect of I-E on susceptibility to diabetes in NOD mice.     

Direct evidence for the contribution of the unique I-ANOD to the development of insulitis in non-obese diabetic mice

Insulin-dependent diabetes mellitus is characterized by the infiltration of lymphocytes into the islets of Langerhans of the pancreas (insulitis) followed by destruction of insulin-secreting beta-cells leading to overt diabetes. The best model for the disease is the non-obese diabetic (NOD) mouse. Two unusual features of the class II major histocompatibility complex (MHC) of the NOD mouse are the absence of I-E and the presence of unique I-A molecules (I-ANOD), in which aspartic acid at position 57 of the beta-chain is replaced by serine. This feature is also found in the HLA-DQ chain of many Caucasians with insulin-dependent diabetes mellitus. We have previously reported that the expression of I-E prevents the development of insulitis in NOD mouse. Here we report that the expression of I-Ak (A alpha kA beta k) in transgenic NOD mice can also prevent insulitis, and that this protection is seen not only when the I-A beta-chain has aspartic acid as residue 57, but also when this residue is serine. These results show that the single amino-acid substitution at position 57 of the I-A beta-chain from aspartic acid to serine is not sufficient for the development of the disease.     

Identification and mapping to chromosome 1 of a susceptibility locus for periinsulitis in non-obese diabetic mice

Insulin-dependent diabetes mellitus (IDDM) is a polygenic disease caused by autoimmune destruction of insulin-producing beta cells in the islets of Langerhans. Its onset is preceded by a long and variable period in which lymphoid cells infiltrate the pancreas but first remain outside the islets (peri-insulitis) before invading them (insulitis). Among susceptibility loci, only the major histocompatibility complex (MHC) has been clearly assigned. Genetic study of the nonobese diabetic (NOD) mouse model for insulin-dependent diabetes mellitus has revealed genetic linkage of insulitis and of early onset diabetes with two non-MHC loci mapping to chromosome 3 and 11 respectively. Here we report a close association of periinsulitis with a third non-MHC locus mapping to chromosome 1. Successive stages in the progression of diabetic disease thus appear to be controlled by distinct genes or sets of genes.     

Antigen presenting function of class II MHC expressing pancreatic beta cells

Class II major histocompatibility complex (MHC) gene expression in the mouse is generally limited to thymic epithelium and bone marrow-derived cells such as B lymphocytes and cells of the macrophage/dendritic cell lineage (M phi/DC). Class II-bearing B lymphocytes and M phi/DC possess antigen presenting cell (APC) function; that is, they can stimulate T lymphocytes reactive to either antigen plus MHC or foreign MHC alone. To assess whether non-bone-marrow-derived cells can acquire APC function and elicit graft rejection through expression of class II, we studied transgenic pancreatic islet beta cells that express a foreign class II (I-E) molecule. In vivo, grafts of I-E+ transgenic islets into I-E- naive hosts are not rejected unless the host is primed by an injection of I-E+ spleen cells. In vitro, the I-E+ beta cells are unable to stimulate T lymphocytes reactive to I-E plus a peptide antigen. Paradoxically, they induce antigen specific unresponsiveness in the T cells. We propose that expression of class II on non-lymphoid cells may serve as an extrathymic mechanism for maintaining self tolerance.     

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.     

Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice

A fundamental question about the pathogenesis of spontaneous autoimmune diabetes is whether there are primary autoantigens. For type 1 diabetes it is clear that multiple islet molecules are the target of autoimmunity in man and animal models. It is not clear whether any of the target molecules are essential for the destruction of islet beta cells. Here we show that the proinsulin/insulin molecules have a sequence that is a primary target of the autoimmunity that causes diabetes of the non-obese diabetic (NOD) mouse. We created insulin 1 and insulin 2 gene knockouts combined with a mutated proinsulin transgene (in which residue 16 on the B chain was changed to alanine) in NOD mice. This mutation abrogated the T-cell stimulation of a series of the major insulin autoreactive NOD T-cell clones. Female mice with only the altered insulin did not develop insulin autoantibodies, insulitis or autoimmune diabetes, in contrast with mice containing at least one copy of the native insulin gene. We suggest that proinsulin is a primary autoantigen of the NOD mouse, and speculate that organ-restricted autoimmune disorders with marked major histocompatibility complex (MHC) restriction of disease are likely to have specific primary autoantigens.     

Epithelial cells expressing aberrant MHC class II determinants can present antigen to cloned human T cells

The first step in the induction of immune responses, whether humoral or cell mediated, requires the interaction between antigen-presenting cells and T lymphocytes restricted at the major histocompatibility complex (MHC). These cells invariably express MHC class II molecules (HLA-D region in man and Ia in mouse) which are recognized by T cells of the helper/inducer subset in association with antigen fragments. Interestingly, in certain pathological conditions, for example in autoimmune diseases such as thyroiditis and diabetic insulitis, class II molecules may be expressed on epithelial cells that normally do not express them. We speculated that these cells may be able to present their surface autoantigens to T cells, and that this process may be crucial to the induction and maintenance of autoimmunity. A critical test of this hypothesis would be to determine whether epithelial cells bearing MHC class II molecules (class II+ cells) can present antigen to T cells. We report here that class II+ thyroid follicular epithelial cells (thyrocytes) can indeed present viral peptide antigens to cloned human T cells.     

Structural polymorphism of I-E subregion antigens determined by a gene in the H-2K to I-B genetic interval

THE generation of immune responses in mice is influenced by Ir genes located in the I region of the major histocompatibility complex (MHC)(1). In some instances maximum responses require complementation by two genes, one in the I-A or I-B and the other in the I-E or I-C subregion(2,3). The effects of these genes are thought to be mediated by Ia alloantigens, which are cell surface molecules whose expression is controlled by the I region(4). This is based on the observations that anti-Ia sera inhibit in vitro immune responses(5,6), and soluble factors that enhance in vitro immune responses express Ia alloantigenic determinants(7,9). Jones et al.(10), using two-dimensional gel electrophoresis, observed that the expression of I-E subregion antigens is controlled by two genes, one in the I-A subregion, the other in the I-E subregion, and that the polymorphism of these antigens is influenced by an I-A subregion gene. As an explanation, the authors proposed that only one of the two polypeptide chains present in I-E immunoprecipitates is an I-E subregion product, the second being a product of the I-A subregion. Antisera obtained by cross-immunisation of I-E subregion-disparate strains of mice immunoprecipitates a molecular complex consisting of two chains, designated alpha and beta, with molecular weights of 32,000 and 29,000 respectively(11-14). Previous studies suggested that I-E antigens isolated from B10.A(5R) and B10.D2 mice had identical alpha-chains but different (beta)-chains(15). However, as these mice differed at multiple genetic regions, it was not possible to show which I subregion(s) determined the polymorphism of the E(beta) chain. Therefore, we investigated the effects of the I-A subregion on the polymorphism of I-E subregion antigens. We have now shown by peptide mapping that the I-E subregion polymorphism which Jones et al. found to be controlled by the I-A subregion probably reflects structural polymorphism of beta-chains controlled by an I-A subregion gene.     

T-cell tolerance by clonal anergy in transgenic mice with nonlymphoid expression of MHC class II I-E

T-cell reactivity to the class II major histocompatibility complex I-E antigen is associated with T-cell antigen receptors containing the V beta gene segments V beta 17a and V beta 5. Mice expressing I-E with the normal tissue distribution (on B cells, macrophages, dendritic cells and thymic epithelium) induce tolerance to self I-E by clonal deletion in the thymus. By contrast, we find that transgenic INS-I-E mice that express I-E on pancreatic beta-cells, but not in the thymus or peripheral lymphoid organs, are tolerant to I-E but have not deleted V beta 5- and V beta 17a-bearing T cells. Moreover, whereas T-cell populations from nontransgenic mice proliferate in response to receptor crosslinking with V beta 5- and V beta 17a-specific antibodies, T cells from INS-I-E mice do not. Thus, our experiments provide direct evidence that T-cell tolerance by clonal paralysis does occur during normal T-cell development in vivo.     

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.     

Dysmyelination in transgenic mice resulting from expression of class I histocompatibility molecules in oligodendrocytes.

Major histocompatibility complex (MHC) molecules are not normally expressed in the central nervous system (CNS). However, aberrant expression has been observed in multiple sclerosis lesions and could contribute to the destruction of myelin or the myelinating cells known as oligodendrocytes. The mechanism of cell damage associated with aberrant MHC molecule expression is unclear: for example, overexpression of class I and class II MHC molecules in pancreatic beta cells in transgenic mice leads to nonimmune destruction of the cells and insulin-dependent diabetes mellitus. We have generated transgenic mice that express class I H-2Kb MHC molecules, under the control of the myelin basic protein promoter, specifically in oligodendrocytes. Homozygous transgenic mice have a shivering phenotype, develop tonic seizures and die at 15-22 days. This phenotype, which we term 'wonky', is due to hypomyelination in the CNS, and not to involvement of the immune system. The primary defect appears to be a shortage of myelinating oligodendrocytes resulting from overexpression of the class I MHC molecules.     

Deletion of self-reactive thymocytes occurs at a CD4+8+ precursor stage

As T cells develop in the thymus, they become tolerant of self-antigens. A major advance in the understanding of how this process occurs was the direct demonstration that cells bearing autoreactive T-cell receptors (TCRs) are physically eliminated from the population of functionally mature T cells present in both the thymus and periphery. We have sought to determine the developmental stage at which autoreactive T cells are eliminated by examining the expression of V beta 17a anti-I-E TCRs under various experimental conditions. In vivo antibody blockage of the CD4 molecule on developing thymocytes of I-E+ C57BR mice was found to inhibit the deletion of V beta 17a-bearing cells from the CD4-8+ single positive thymocyte subset. This result provides strong evidence that deletion of potentially autoreactive T cells occurs at a CD4+8+ precursor stage, that the non-clonally distributed accessory molecules (CD4, CD8) are significant participants in the self-recognition process that leads to clonal elimination, and that thymic class II major histocompatibility complex (MHC) molecules can influence the repertoire of CD4-8+ cells.     

Unexpected expression of a unique mixed-isotype class II MHC molecule by transfected L-cells

Class II (Ia) major histocompatibility complex (MHC) molecules are heterodimeric integral membrane proteins composed of non-covalently linked alpha and beta glycoprotein chains. Studies of both normal cells and L-cell transfectants have shown that neither alpha- nor beta-chains are found on the cell surface alone, and that alpha beta dimers are required for membrane expression. In both mouse and man, several distinct non-allelic alpha and beta genes exist. Analysis of Ia molecules by immunoprecipitation and two-dimensional gel electrophoresis has demonstrated apparently selective association of particular pairs of the various alpha- and beta-chains to form the expressed class II isotypes I-A and I-E (mouse) or DQ, DP and DR (human). Because the various alpha- or beta-chains encoded by distinct loci exist in many allelic forms within a species, such specific pairing suggests a special role for isotypically conserved regions of each chain in the association process. In attempting to localize such putative assembly-controlling regions using the technique of DNA-mediated gene transfer, various combinations of murine alpha and beta genes were introduced into L-cells. Here we report the unexpected observation, following transfection, of mixed-isotype (Ad beta Ea/k alpha) molecules on the L-cell membrane and document that the formation of this pair is strongly influenced by allelic polymorphism of the A beta chain.     

Progression of autoimmune diabetes driven by avidity maturation of a T-cell population

For unknown reasons, autoimmune diseases such as type 1 diabetes develop after prolonged periods of inflammation of mononuclear cells in target tissues. Here we show that progression of pancreatic islet inflammation to overt diabetes in nonobese diabetic (NOD) mice is driven by the 'avidity maturation' of a prevailing, pancreatic beta-cell-specific T-lymphocyte population carrying the CD8 antigen. This T-lymphocyte population recognizes two related peptides (NRP and NRP-A7) in the context of H-2Kd class I molecules of the major histocompatibility complex (MHC). As pre-diabetic NOD mice age, their islet-associated CD8+ T lymphocytes contain increasing numbers of NRP-A7-reactive cells, and these cells bind NRP-A7/H-2Kd tetramers with increased specificity, increased avidity and longer half-lives. Repeated treatment of pre-diabetic NOD mice with soluble NRP-A7 peptide blunts the avidity maturation of the NRP-A7-reactive CD8+ T-cell population by selectively deleting those clonotypes expressing T-cell receptors with the highest affinity and lowest dissociation rates for peptide-MHC binding. This inhibits the local production of T cells that are cytotoxic to beta cells, and halts the progression from severe insulitis to diabetes. We conclude that avidity maturation of pathogenic T-cell populations may be the key event in the progression of benign inflammation to overt disease in autoimmunity.     

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.     

Positive selection of antigen-specific T cells in thymus by restricting MHC molecules

Thymus-derived lymphocytes (T cells) recognize antigen in the context of class I or class II molecules encoded by the major histocompatibility complex (MHC) by virtue of the heterodimeric alpha beta T-cell receptor (TCR). CD4 and CD8 molecules expressed on the surface of T cells bind to nonpolymorphic portions of class II and class I MHC molecules and assist the TCR in binding and possibly in signalling. The analysis of T-cell development in TCR transgenic mice has shown that the CD4/CD8 phenotype of T cells is determined by the interaction of the alpha beta TCR expressed on immature CD4+8+ thymocytes with polymorphic domains of thymic MHC molecules in the absence of nominal antigen. Here we provide direct evidence that positive selection of antigen-specific, class I MHC-restricted CD4-8+ T cells in the thymus requires the specific interaction of the alpha beta TCR with the restricting class I MHC molecule.     

Thymic selection process induced by hybrid antibodies

Thymus-derived (T) lymphocytes using the alpha beta T-cell antigen receptor (TCR) recognize fragmented antigen in conjunction with surface molecules encoded by genes of the major histocompatibility complex (MHC). Peripheral T lymphocytes preferentially see antigen presented by self rather than by foreign MHC molecules, and autoreactive T lymphocytes are deleted. Thus, the peripheral T-lymphocyte repertoire is skewed towards recognition of antigen in the context of self-MHC and towards tolerance to self-antigens. During T-lymphocyte development in the thymus, this repertoire is formed by the interaction of TCR with MHC molecules resulting in positive and negative selection phenomena. Hybrid antibodies (HAbs) that carry binding sites to the TCR and to a surface marker on another cell can engage all T lymphocytes regardless of their specificity. It should be possible to mimic selection processes in normal animals with HAb that specifically link members of a TCR family to MHC molecules on the thymic stroma. We have probed T-lymphocyte development with HAbs linking V beta 8-positive TCR to either class I or class II MHC products in thymic organ culture. Thymocytes exposed to either HAb in an early stage of maturation respond with a significant increase in the frequency of V beta 8-carrying cells. At a later stage of development V beta 8-positive thymocytes are depleted. These results illustrate the succession of positive and negative selection in the developing thymus of normal mice.     

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.