Recovery from autoimmunity of MRL/lpr mice after infection with an interleukin-2/vaccinia recombinant virus

Interleukin-2 (IL-2) is a T-cell derived molecule implicated in the clonal expansion of antigen-activated T cells and in T-cell development. IL-2 is also implicated in autoimmune disease, although its role is still controversial. Murine systemic lupus erythematosus (SLE) is a good model for human SLE as most of the immunological abnormalities in the human disease also seem to be operative in the mouse. Among SLE mice, the MRL/lpr strain develops early in life autoimmune diseases such as immune complex-mediated glomerulonephritis, arthritis and arteritis. Lymphoid abnormalities associated with those diseases in this strain are thymic atrophy and abnormal proliferation of CD3+ CD4- CD8- 'double-negative' T cells, resulting in massive generalized lymph node enlargement. We have therefore now examined the effects of IL-2 on the disease progression in MRL/lpr mice using live vaccinia recombinant viruses expressing the human IL-2 gene. Vaccinated mice showed prolonged survival, decreased autoantibody and rheumatoid factor titres, marked attenuation of kidney interstitial infiltration and intraglomerular proliferation, as well as clearance of synovial mononuclear infiltrates. Inoculation with the IL-2/vaccinia recombinant virus led, in addition, to drastic reduction of the double-negative T-cell population, improved thymic differentiation and restoration of normal values of mature cells in peripheral lymphoid organs.     

Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis.

Fas antigen is a cell-surface protein that mediates apoptosis. It is expressed in various tissues including the thymus and has structural homology with a number of cell-surface receptors, including tumour necrosis factor receptor and nerve growth factor receptor. Mice carrying the lymphoproliferation (lpr) mutation have defects in the Fas antigen gene. The lpr mice develop lymphadenopathy and suffer from a systemic lupus erythematosus-like autoimmune disease, indicating an important role for Fas antigen in the negative selection of autoreactive T cells in the thymus.     

MXH10/Mo-lpr/lpr鼠在肿瘤诊断与治疗的实验研究

MXH10/Mo-lpr/lpr鼠表现为全身性的淋巴结肿大,6周龄时淋巴结直径可达1 cm。与其亲代MRL/lpr鼠相比,MXH10/Mo-lpr/lpr鼠有较轻的肾小球肾炎、血管炎、关节炎、唾液腺炎等症状且生命周期长。基于这些特点,MXH10/Mo-lpr/lpr鼠主要用于肿瘤淋巴结转移、淋巴管给化疗药的研究,并应用于小动物CT和MRI的诊断,为临床提供早期淋巴结转移诊断奠定可靠的临床前动物模型。本文将对MXH10/Mo-lpr/lpr鼠的遗传背景、特征性表型和应用加以概述,并介绍其在肿瘤诊断与治疗方面的应用。     

Abnormal tyrosine phosphorylation on T-cell receptor in lymphoproliferative disorders

The study of human autoimmune diseases has benefited greatly from analysis of animal models. Mice that are homozygous for either the lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) mutant genes develop a disease characterized by massive lymphadenopathy and autoantibody formation. With age, the lymphoid organs in these mice are replaced with a greatly expanded population of abnormal lymphocytes. Recent work has shown that these cells are likely to be in the T-cell lineage. They rearrange and transcribe the genes for the alpha and beta subunits of the T-cell receptor (TCR) and a third, T-cell receptor-like gene, T gamma. As determined by immunofluorescence with anti-receptor antibodies the cells also express TCR on the cell surface. The murine T-cell receptor consists of the alpha and beta chains, derived from the rearranged alpha and beta genes, in non-covalent association with seven other chains; the delta chain, of relative molecular mass (Mr) 26,000 (26K), the epsilon chain (25K), a glycosylated 21K chain (gp21) which is probably the homologue of the gamma chain of T3 (CD3), a 16K homodimer (zeta) and a 21K dimer (p21). This multichain complex is thought to be the murine analogue of the human T3 complex. After activation of normal T cells by antigen or lectin, p21 is phosphorylated on tyrosine residues and gp21 is phosphorylated on serine residues. In contrast, in the gld and lpr cells, p21 is phosphorylated even in the absence of antigen or lectin, whereas gp21 is not phosphorylated.     

Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors

Autoreactive B cells are present in the lymphoid tissues of healthy individuals, but typically remain quiescent. When this homeostasis is perturbed, the formation of self-reactive antibodies can have serious pathological consequences. B cells expressing an antigen receptor specific for self-immunoglobulin-gamma (IgG) make a class of autoantibodies known as rheumatoid factor (RF). Here we show that effective activation of RF+ B cells is mediated by IgG2a-chromatin immune complexes and requires the synergistic engagement of the antigen receptor and a member of the MyD88-dependent Toll-like receptor (TLR) family. Inhibitor studies implicate TLR9. These data establish a critical link between the innate and adaptive immune systems in the development of systemic autoimmune disease and explain the preponderance of autoantibodies reactive with nucleic acid-protein particles. The unique features of this dual-engagement pathway should facilitate the development of therapies that specifically target autoreactive B cells.     

Erythrocyte receptors for Mycoplasma pneumoniae are sialylated oligosaccharides of Ii antigen type

Among the pathological effects in man following infection with Mycoplasma pneumoniae is a transient autoimmune disorder characterized by the presence of high-titre erythrocyte autoantibodies (cold agglutinins). These autoantibodies are usually directed against the carbohydrate antigen termed I (ref. 3) which consists of a branched oligosaccharide. The mechanism by which the anti-I antibodies are elicited is unknown. However, sialic acid-containing receptors have been implicated in the adherence of M. pneumoniae to erythrocytes and other cell types, and both I and the related antigen i occur on erythrocytes in sialylated form: i is the predominant antigen on fetal erythrocytes and I is predominant in adults. Anti-I antibodies might arise in M. pneumoniae infection in response to a modification of the 'self' antigen-I as a result of its interaction with this agent. Here we report our study of the specificity of the interaction of M. pneumoniae with human erythrocytes. We found that this interaction is mediated by long chain oligosaccharides of sialic acid joined by alpha 2-3 linkage to the terminal galactose residues of poly-N-acetyllactosamine sequences of Ii antigen type.     

Somatic hypermutation of an immunoglobulin transgene in kappa transgenic mice

Initial studies of somatically acquired mutations in immunoglobulin V regions from hybridomas and myelomas that are not derived from joining aberrations, suggested a controlled and specific hypermutation process, because spontaneous mutation rates observed for other genes are extremely low. Some evidence for the idea that mutations are introduced during V-gene rearrangement came from the clustering of mutations at the joining sites, from the absence of mutations in unrearranged V genes and from the low level of mutations in only partially (D-J) rearranged nonproductive heavy-chain alleles. Another model in which mutations accumulate with each cell division, rather than being introduced all at once, was supported by the finding that immunoglobulin genes of hybridomas derived from a single mouse frequently had several mutations in common, and so might be derived from the same precursor cell whose daughters then accumulated additional mutations. But the common mutations in some cases could be due to as yet unidentified related germline genes, or could represent the effect of antigen selection for certain amino acids. To try to detect hypermutation in the absence of V-gene rearrangement, we isolated B lymphocytes with endogenous heavy-chain gene mutations from transgenic mice carrying pre-rearranged kappa-transgenes. We found that these kappa-transgenes were also somatically mutated. This and other observations indicated that: ongoing rearrangement is not required for mutation; there are signals for hypermutation in the transgenes; the mutations are found only in the variable region, so the constant region may not be a target; different transgene insertion sites are compatible with hypermutations and more than one transgene is expressed in the same cell.     

IgM-autoantibodies against isologous erythrocytes also react with isologous IgG(Fc)

The mechanisms by which the adaptive immune system of animals discriminate between foreign and self components are not known. It is generally believed that antibodies are produced only in response to foreign agents. However, against this axiom there is mounting evidence that normal animals produce autoantibodies against a variety of self components. In two murine model systems, a high proportion of IgM-producing cells secrete autoantibodies. In one model, the autoantibodies are specific for isologous mouse IgG(Fc) and in the other, the autoantibodies are specific for antigens revealed on the surface of mouse erythrocytes (RBC) by proteolytic enzymes. The function of these two autoimmune responses is a mystery. Here we show that the two responses are related. The lysis of mouse RBC, modified with bromelain (brom) by IgM autoantibodies was completely inhibited by isologous IgG(Fc), and antibodies against mouse IgG(Fc) formed precipitin bands with solubilized membranes from mouse RBC. We conclude that mouse RBC membranes and IgG(Fc) have at least one antigenic determinant in common and that this determinant is the target for autoimmune responses in normal mice.     

Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice

Self-reactive B cells can be regulated by either deletion or inactivation. These manifestations of self-tolerance have been dramatically shown in transgenic mice in which the number of self-reactive cells has been artificially expanded. We have now extended these models to ask if B-cell tolerance as described for non-disease-associated antigens also operates for the targets of autoimmunity. The target we have chosen is DNA. Anti-DNA antibodies are diagnostic of certain autoimmune syndromes in humans and are a characteristic of the murine model of systemic autoimmunity, the MRl/lpr mouse. Antibodies to both single-stranded and double-stranded DNA have been implicated in disease. By generating anti-DNA transgenic mice, we have addressed the question of whether DNA-specific B cells are regulated in normal (non-autoimmune) mice. We indeed found that most transgenic B cells bind DNA, yet we failed to detect secreted anti-DNA. We suggest that as a consequence of their self-reactivity these B cells are developmentally arrested.     

雄黄对MRL／lpr小鼠脾细胞分泌干扰素-γ、白细胞介素-6的影响

目的：探讨雄黄（As4S4）对MRL／lpr狼疮鼠脾脏细胞分泌干扰素-γ（IFN-γ）、自细胞介素-6（IL-6）的影响。方法：将20周龄MRL／lpr狼疮小鼠和正常C57BL／6J小鼠无菌条件下取出脾脏,制成脾脏淋巴细胞悬液,体外经植物凝集素P（PHA-P,终浓度20mg／L）、白细胞介素-2（IL-2,终浓度10^6IU／L）常规刺激48h后,随机分组进行如下实验：（1）不同浓度As4S4溶液（6．5、12．5、25、50mg／L）作用24h;（2）As4S4对MRL／lpr小鼠和C57BL／6J小鼠作用：①PBS组：空白对照;②As4S4组：25mg／L;以上各组继续培养24h,酶联免疫吸附法（ELISA）测定各组培养液IFN-γ、IL-6表达量。结果：（1）MRL／fpr狼疮鼠PBS组IFN-γ的分泌高于C57BL／6J小鼠PBS组（P〈0．01）。（2）在MRL／加狼疮鼠中,与PBS组相比,As4S4组（25mg／L）IFN-γIL-6的分泌水平下降。（3）在C57BL／6J小鼠中,PBS组和As4S4组之间IFN-γ、IL-6浓度水平无明显差异。结论：As4S4下调MRL／lpr狼疮鼠IFN-γ、IL-6的分泌,而对C57BL／6J小鼠脾细胞IFN-γ、IL-6的分泌无明显影响。     

TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease

B cells are important in the development of autoimmune disorders by mechanisms involving dysregulated polyclonal B-cell activation, production of pathogenic antibodies, and co-stimulation of autoreactive T cells. zTNF4 (BLyS, BAFF, TALL-1, THANK) is a member of the tumour necrosis factor (TNF) ligand family that is a potent co-activator of B cells in vitro and in vivo. Here we identify two receptors for zTNF4 and demonstrate a relationship between zTNF4 and autoimmune disease. Transgenic animals overexpressing zTNF4 in lymphoid cells develop symptoms characteristic of systemic lupus erythaematosus (SLE) and expand a rare population of splenic B-Ia lymphocytes. In addition, circulating zTNF4 is more abundant in NZBWF1 and MRL-lpr/lpr mice during the onset and progression of SLE. We have identified two TNF receptor family members, TACI and BCMA, that bind zTNF4. Treatment of NZBWF1 mice with soluble TACI-Ig fusion protein inhibits the development of proteinuria and prolongs survival of the animals. These findings demonstrate the involvement of zTNF4 and its receptors in the development of SLE and identify TACI-Ig as a promising treatment of autoimmune disease in humans.     

Non-tolerance and autoantibodies to a transgenic self antigen expressed in pancreatic beta cells

Transgenic mice expressing simian virus 40 T antigen under control of the insulin gene regulatory region vary in their response to this protein. Each lineage is characteristically either tolerant to T antigen, or not, in which case autoantibodies arise with high frequency, and lymphocytes infiltrate and disrupt the pancreatic islets. Both non-tolerance and the autoimmune response appear to result from delayed onset of T antigen expression during beta cell development.     

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling

A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.     

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.     

Cbl-b regulates the CD28 dependence of T-cell activation

Whereas co-stimulation of the T-cell antigen receptor (TCR) and CD28 triggers T-cell activation, stimulation of the TCR alone may result in an anergic state or T-cell deletion, both possible mechanisms of tolerance induction. Here we show that T cells that are deficient in the adaptor molecule Cbl-b (ref. 3) do not require CD28 engagement for interleukin-2 production, and that the Cbl-b-null mutation (Cbl-b(-/-)) fully restores T-cell-dependent antibody responses in CD28-/- mice. The main TCR signalling pathways, such as tyrosine kinases Zap-70 and Lck, Ras/mitogen-activated kinases, phospholipase Cgamma-1 and Ca2+ mobilization, were not affected in Cbl-b(-/-) T cells. In contrast, the activation of Vav, a guanine nucleotide exchange factor for Rac1/Rho/CDC42, was significantly enhanced. Our findings indicate that Cbl-b may influence the CD28 dependence of T-cell activation by selectively suppressing TCR-mediated Vav activation. Mice deficient in Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis, suggesting that the dysregulation of signalling pathways modulated by Cbl-b may also contribute to human autoimmune diseases such as multiple sclerosis.     

Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation

Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ～7% of human malignancies and ～60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.     

Antigen-induced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice.

Studies on transgenic mice expressing immunoglobulins against self-antigens have shown that self-tolerance is maintained by active elimination (clonal deletion), functional inactivation (clonal anergy) of self-reactive B cells, or a combination of both. We have established and characterized a transgenic mouse line expressing an anti-erythrocyte autoantibody. In contrast to other autoantibody transgenic lines, about 50% of the animals of this transgenic line suffer from autoimmune disease, indicating a loss of self-tolerance. Here we show that peritoneal Ly-1 B cells (also known as B-1 cells) are responsible for this autoimmune disease in our transgenic mice. A few self-reactive Ly-1 B cells that have somehow escaped the deletion mechanism expand in the peritoneum because of the absence of self-antigen. These Ly-1 B cells are eliminated in vivo by apoptosis once exposed to self-antigen. On the basis of these results we propose a novel autoantibody production mechanism whereby self-reactive B cells sequestered in compartments free of self-antigens may survive, proliferate and be activated for generation of pathogenic autoantibodies in autoimmune diseases.     

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.     

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.     

XIAP deficiency in humans causes an X-linked lymphoproliferative syndrome

The homeostasis of the immune response requires tight regulation of the proliferation and apoptosis of activated lymphocytes. In humans, defects in immune homeostasis result in lymphoproliferation disorders including autoimmunity, haemophagocytic lymphohystiocytosis and lymphomas. The X-linked lymphoproliferative syndrome (XLP) is a rare, inherited immunodeficiency that is characterized by lymphohystiocytosis, hypogammaglobulinaemia and lymphomas, and that usually develops in response to infection with Epstein-Barr virus (EBV). Mutations in the signalling lymphocyte activation molecule (SLAM)-associated protein SAP, a signalling adaptor molecule, underlie 60% of cases of familial XLP. Here, we identify mutations in the gene that encodes the X-linked inhibitor-of-apoptosis XIAP (also termed BIRC4) in patients with XLP from three families without mutations in SAP. These mutations lead to defective expression of XIAP. We show that apoptosis of lymphocytes from XIAP-deficient patients is enhanced in response to various stimuli including the T-cell antigen receptor (TCR)-CD3 complex, the death receptor CD95 (also termed Fas or Apo-1) and the TNF-associated apoptosis-inducing ligand receptor (TRAIL-R). We also found that XIAP-deficient patients, like SAP-deficient patients, have low numbers of natural killer T-lymphocytes (NKT cells), indicating that XIAP is required for the survival and/or differentiation of NKT cells. The observation that XIAP-deficiency and SAP-deficiency are both associated with a defect in NKT cells strengthens the hypothesis that NKT cells have a key role in the immune response to EBV. Furthermore, by identifying an XLP immunodeficiency that is caused by mutations in XIAP, we show that XIAP is a potent regulator of lymphocyte homeostasis in vivo.