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.     

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.     

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.     

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.     

Transfer of diabetes in mice prevented by blockade of adhesion-promoting receptor on macrophages

Insulin-dependent diabetes mellitus (IDDM) is a disease with an autoimmune aetiology. The non-obese diabetic mouse is a good spontaneous animal model of the human disease, with IDDM developing in 50-80% of female mice by the age of 6 months. The disease can be transferred by splenic T cells from diabetic donors and is prevented by T-cell depletion. The mechanism(s) by which the beta cell is specifically destroyed is not known, but T cells and macrophages have both been implicated, based on the presence of macrophages in the infiltrated islet and the ability of chronic silica treatment to prevent disease. The monoclonal antibody 5C6 is specific for the myelomonocytic adhesion-promoting type-3 complement receptor (CR3 or CD11b/CD18) and does not bind to T cells. Here we show that blockade of macrophage CR3 in vivo prevents intra-islet infiltration by both macrophages and T cells and inhibits development of IDDM. We conclude that both T cells and macrophages have an essential role in the onset of IDDM.     

Type 1 diabetes in mice is linked to the interleukin-1 receptor and Lsh/Ity/Bcg genes on chromosome 1.

Human type 1 (insulin-dependent) diabetes is a common auto-immune disease of the insulin-producing beta cells of the pancreas which is caused by both genetic and environmental factors. Several features of the genetics and immunopathology of diabetes in nonobese diabetic (NOD) mice are shared with the human disease. Of the three diabetes-susceptibility genes, Idd-1 -3 and -4 that have been mapped in mice to date, only in the case of Idd-1 is there any evidence for the identity of the gene product: allelic variation within the murine immune response I-A beta gene and its human homologue HLA-DQB1 correlates with susceptibility, implying that I-A beta is a component of Idd-1. We report here the mapping of Idd-5 to the proximal region of mouse chromosome 1. This region contains at least two candidate susceptibility genes, the interleukin-1 receptor gene and Lsh/Ity/Bcg, which encodes resistance to bacterial and parasitic infections and affects the function of macrophages.     

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.     

Innate immunity and intestinal microbiota in the development of Type 1 diabetes

Type 1 diabetes (T1D) is a debilitating autoimmune disease that results from T-cell-mediated destruction of insulin-producing beta-cells. Its incidence has increased during the past several decades in developed countries, suggesting that changes in the environment (including the human microbial environment) may influence disease pathogenesis. The incidence of spontaneous T1D in non-obese diabetic (NOD) mice can be affected by the microbial environment in the animal housing facility or by exposure to microbial stimuli, such as injection with mycobacteria or various microbial products. Here we show that specific pathogen-free NOD mice lacking MyD88 protein (an adaptor for multiple innate immune receptors that recognize microbial stimuli) do not develop T1D. The effect is dependent on commensal microbes because germ-free MyD88-negative NOD mice develop robust diabetes, whereas colonization of these germ-free MyD88-negative NOD mice with a defined microbial consortium (representing bacterial phyla normally present in human gut) attenuates T1D. We also find that MyD88 deficiency changes the composition of the distal gut microbiota, and that exposure to the microbiota of specific pathogen-free MyD88-negative NOD donors attenuates T1D in germ-free NOD recipients. Together, these findings indicate that interaction of the intestinal microbes with the innate immune system is a critical epigenetic factor modifying T1D predisposition.     

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.     

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.     

Abolition of specific immune response defect by immunization with dendritic cells

Murine cytotoxic T (Tc)-cell responses to various antigens are controlled by immune response (Ir) genes mapping in the major histocompatibility complex (H-2). The genes responsible are those encoding the class I and class II H-2 antigens. The H-2 I-Ab mutant mouse strain bm12 differs from its strain of origin, C57BL/6 (H-2b), only in three amino acids in the I-A beta bm12 class II H-2 molecule. As a consequence, female bm12 mice are Tc-cell nonresponders to the male antigen H-Y and do not reject H-Y disparate skin grafts. We now report that bm12 mice generate strong H-Y-specific Tc cells following priming in vivo and restimulation in vitro with male bm12 dendritic cells (DC). Female bm12 mice primed with male DC also reject male skin grafts. Furthermore, we demonstrate that only responder cell populations containing a mixture of L3T4+ (T-helper (Th) phenotype) and Lyt 2+ (Tc phenotype) T lymphocytes generate H-Y-specific Tc cells. These data imply an essential role for Th cells, activated by DC as antigen-presenting cells (APC), in changing H-Y-nonresponder bm12 mice into H-Y responders. Priming and restimulation with DC allows the triggering of a T-cell repertoire not demonstrable by the usual modes of immunization. This principle might be used to overcome other specific immune response defects.     

Identification of the T-cell and Ia contact residues of a T-cell antigenic epitope

The precise molecular structure of the antigenic determinant recognized by the T-cell receptor of the CD4-positive cell has not been completely resolved. A major advance in our understanding of this issue has been made by our demonstration of a direct association between an immunogenic peptide and a purified Ia molecule. The most likely and economical hypothesis is that antigen binds directly to an Ia molecule creating the antigenic determinant and that this antigen-Ia complex is recognized by the T-cell receptor. We examined in detail a determinant of hen egg-white lysozyme (HEL) contained in the tryptic fragment HEL(46-61), recognized by T cells in H-2k strains of mice. This peptide binds with a Kd of approximately 3 microM to I-Ak molecules. We have already ascertained that (1) the 10-mer HEL(52-61) is the shortest stimulatory peptide; (2) the Leu56 residue, the only residue different from mouse lysozyme, is responsible for the immunogenicity; (3) the Leu56 and Tyr53 residues are critical for recognition by the T-cell receptor and (4) HEL(46-61) generates multiple determinants when it associated with the I-Ak molecule. If antigen and Ia interact, the antigen must have two features: it must bind to an Ia molecule and also interact with the T-cell receptor. The two sites do not appear to be laterally separable in this peptide and are therefore probably composed of distinct but interspersed amino-acid residues. We have now identified the three residues of HEL(52-61) that contact the T-cell receptor and three other residues that contact the I-Ak molecule. From modelling studies we also propose that HEL(52-61) assumes an alpha-helical conformation as it is bound to I-Ak and recognized by the T-cell receptor.     

Development of specific suppressor cells in hypoinsulinaemic mice

Mice and rats injected with alloxan or streptozotocin develop permanent diabetes, characterised by deficient insulin production. It has been demonstrated that hypoinsulinaemia in mice leads to significant loss of lymphatic tissue, and these diabetic animals cannot develop contact sensitivity or efficient graft rejection. Administration of insulin partially restored these responses and also caused an increase in the weight of the thymus and spleen. Similar suppression of T cell-dependent phenomena has been observed in surgically pancreatectomised rats. Lymphocytes of these hypoinsulinaemic animals show significantly decreased in vitro responses to plant lectins and generate only low levels of cytotoxic effector cells. We previously showed that cells of normoglycaemic oxazolone-sensitised mice cannot transfer significant contact sensitivity reactions into diabetic recipients indicating that the milieu of hyperglycaemic insulin deficient animals cannot support all the activity of immune T cells. By mixing immunised T cells from control and diabetic mice and transferring the mixtures into normal recipients we now show that the non-supportive millieu in diabetic animals may be due to active suppression rather than to athrepsis.     

Aurora-A对2型糖尿病小鼠模型中胰岛细胞增殖的影响

为研究Aurora-A在2型糖尿病小鼠中对胰岛细胞增殖的影响,通过Western blot、免疫组织化学染色、增殖细胞核抗原(proliferating cell nucler antigen,PCNA)染色等方法研究了Aurora-A在2型糖尿病小鼠胰腺组织中的表达以及抑制AuroraA后糖尿病小鼠胰岛细胞的增殖情况。将32只雄性C57BL/6J小鼠随机分为4组,每组8只,分别为空白组、高脂饮食组、模型组和治疗组。模型组和治疗组采用高脂饲料(high fat diet,HFD)联合链脲佐霉素(Streptozotocin,STZ)腹腔注射造模,造模成功后治疗组以Aurora-A抑制剂连续灌胃2周;模型组则以等量的生理盐水灌胃。经药物干预后采用PCNA染色检测各组小鼠胰岛细胞的增殖情况。结果表明:与空白组相比,Aurora-A在模型组胰腺组织中明显表达升高;与模型组相比,治疗组小鼠经Aurora-A抑制剂治疗后胰岛细胞增殖率明显降低。可见,Aurora-A在2型糖尿病小鼠胰腺组织中表达升高;并对胰岛细胞的增殖起着重要的作用。     

Binding of immunogenic peptides to Ia histocompatibility molecules

Most cellular interactions essential for the development of an immune response involve the membrane glycoproteins encoded in the major histocompatibility gene complex. The products of the I region, the class II histocompatibility molecules (Ia molecules), are essential for accessory cells such as macrophages to present polypeptide antigens to helper T cells. This interaction, antigen presentation, is needed for T-cell recognition of the antigen and its consequent activation. How the Ia molecules regulate the immune response during antigen presentation is not known, although it is commonly thought to result from their association with the presented antigen. Recent studies, including the elucidation of the structure of the T-cell receptor, favour recognition of a single structure, an antigen-Ia complex. Here we report attempts to determine whether purified Ia glycoproteins have an affinity for polypeptide antigens presented by intact cells in an Ia-restricted manner. We first identified the epitope of a peptide antigen involved in presentation. Several laboratories have shown that globular proteins are altered (processed) in intracellular vesicles of the antigen-presenting cell before antigen presentation. A major component of the T-cell response is directed toward determinants found in the unfolded or denatured molecule, and our laboratory has shown that the determinant of the hen-egg lysozyme protein (HEL), presented in H-2k mice to T cells, is a sequence of only 10 amino acids. This portion resides in an area of the native molecule partially buried inside the molecule, in a beta-sheet conformation. To be presented, intact or native HEL must first be processed in acidic intracellular vesicles. Having isolated the peptide responsible for T-cell recognition of HEL, we sought a physical association of this peptide with purified, detergent-solubilized I-Ak molecules from B-hybridoma cells. We have found such an association, which may explain the role of the Ia glycoproteins in cellular interactions.     

Quantitation of antigen-presenting cell MHC class II/peptide complexes necessary for T-cell stimulation

The number of specific complexes formed between peptide and the class II major histocompatibility complex (MHC) molecules expressed by an antigen-presenting cell (APC) after exposure to protein antigens is unknown, as is the number that activates T cells. Presentation of foreign peptides by APC takes place when many class II molecules may be occupied by autologous peptides. We have now estimated the number of specific peptide/class II complexes per APC by quantitative immunoprecipitation of I-Ak after pulsing the APC with stimulatory levels of a radioactive immunogenic peptide derived from hen egg-white lysozyme protein. T cells were activated by APC that expressed as few as 210-340 specific peptide/class II complexes (0.1% of the I-Ak molecules). These figures were confirmed using anti-CD3 antibody bound to latex beads as an alternative activating ligand. This low number explains the simultaneous presentation of multiple foreign antigens, even in the face of peptide competition.     

Autoimmune diabetes as a consequence of locally produced interleukin-2.

During cell differentiation in the thymus, self-reactive T cells can be generated. The majority of these seem to be deleted after intrathymic encounter with the relevant autoantigen. As all self antigens are unlikely to be present in the thymus, some autoreactive T cells may escape censorship. Here we study the fate of these cells using transgenic mice expressing the class I molecule H-2Kb (Kb) in the insulin-producing beta-cells of the pancreas. These mice were crossed with mice transgenic for genes encoding a Kb-specific T-cell antigen receptor (TCR) which could be detected using a clonotype-specific monoclonal antibody. Although T cells expressing the highest level of transgenic TCR were deleted intrathymically in double-transgenic mice, Kb-specific T cells were detected in the periphery. These cells caused the rejection of Kb-expressing skin grafts, but ignored islet Kb antigens even after priming. But when double-transgenic mice were crossed with transgenic mice expressing the lymphokine interleukin-2 in the pancreatic beta-cells, there was a rapid onset of diabetes. These results indicate that autoreactive T cells that ignore self antigens may cause autoimmune diabetes when provided with exogenous 'help' in the form of interleukin-2.     

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.     

Signal for T-cell differentiation to a CD4 cell lineage is delivered by CD4 transmembrane region and/or cytoplasmic tail.

Mature T cells express either CD4 or CD8 on their surface. Most helper T cells express CD4, which binds to class II major histocompatibility complex (MHC) proteins, and most cytotoxic T cells express CD8, which binds to class I MHC proteins. In the thymus, mature CD4+CD8- and CD4-CD8+ T cells expressing alpha beta T-cell antigen receptors (TCR) develop from immature thymocytes through CD4+CD8+ alpha beta TCR+ intermediates. Experiments using mice transgenic for alpha beta TCR suggest that the specificity of the TCR determines the CD4/CD8 phenotype of mature T cells. These results, however, do not indicate how a T cell differentiates into the CD4 or CD8 lineage. Here we show that the CD4 transmembrane region and/or cytoplasmic tail mediates the delivery of a specific signal that directs differentiation of T cells to a CD4 lineage. We generated transgenic mice expressing a hybrid molecule composed of the CD8 alpha extracellular domains linked to the CD4 transmembrane region and cytoplasmic tail. We predicted that this hybrid molecule would bind to class I MHC proteins through the extracellular domains but deliver the intracellular signals characteristic of CD4. By crossing our transgenic mice with mice expressing a transgenic alpha beta TCR specific for a particular antigen plus class I MHC protein, we were able to express the hybrid molecule in developing thymocytes expressing the class I MHC-restricted TCR. Our results show that the signal transduced by the hybrid molecule results in the differentiation of immature thymocytes expressing a class I-restricted TCR into mature T cells expressing CD4.     

Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1

The evolutionary conservation of T lymphocyte subsets bearing T-cell receptors (TCRs) using invariant alpha-chains is indicative of unique functions. CD1d-restricted natural killer T (NK-T) cells that express an invariant Valpha14 TCRalpha chain have been implicated in microbial and tumour responses, as well as in auto-immunity. Here we show that T cells that express the canonical hValpha7.2-Jalpha33 or mValpha19-Jalpha33 TCR rearrangement are preferentially located in the gut lamina propria of humans and mice, respectively, and are therefore genuine mucosal-associated invariant T (MAIT) cells. Selection and/or expansion of this population requires B lymphocytes, as MAIT cells are absent in B-cell-deficient patients and mice. In addition, we show that MAIT cells are selected and/or restricted by MR1, a monomorphic major histocompatibility complex class I-related molecule that is markedly conserved in diverse mammalian species. MAIT cells are not present in germ-free mice, indicating that commensal flora is required for their expansion in the gut lamina propria. This indicates that MAIT cells are probably involved in the host response at the site of pathogen entry, and may regulate intestinal B-cell activity.