Infectivity and insertional mutagenesis of endogenous retrovirus in autoimmune NZB and B/W mice.

Murine leukaemia virus has been suggested to contribute to both autoimmune disease and leukaemia in the NZB mouse and in the (NZB × NZW) F1 (abbreviated B/W) mouse. However, with apparently only xenotropic but no ecotropic virus constitutively expressed in these mice, few mechanisms could explain the aetiology of either disease in either mouse strain. Because pseudotyped and/or inducible ecotropic virus may play a role, we surveyed the ability of murine leukaemia virus in NZB, NZW and B/W mice to infect and form a provirus. From the spleen of NZB mice, we isolated circular cDNA of xenotropic and polytropic virus, which indicates ongoing infection by these viruses. From a B/W lymphoma, we isolated and determined the complete sequence of a putative ecotropic NZW virus. From B/W mice, we recovered de novo endogenous retroviral integration sites (tags) from the hyperproliferating cells of the spleen and the peritoneum. The tagged genes seemed to be selected to aid cellular proliferation, as several of them are known cancer genes. The insertions are consistent with the idea that endogenous retrovirus contributes to B-cell hyperproliferation and progression to lymphoma in B/W mice.

Cell-intrinsic expression of TLR9 in autoreactive B cells constrains BCR/TLR7-dependent responses.

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9-double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus.

APOBEC3 enzymes restrict marginal zone B cells.

In general, a long-lasting immune response to viruses is achieved when they are infectious and replication competent. In the mouse, the neutralizing antibody response to Friend murine leukemia virus is contributed by an allelic form of the enzyme Apobec3 (abbreviated A3). This is counterintuitive because A3 directly controls viremia before the onset of adaptive antiviral immune responses. It suggests that A3 also affects the antibody response directly. Here, we studied the relative size of cell populations of the adaptive immune system as a function of A3 activity. We created a transgenic mouse that expresses all seven human A3 enzymes and compared it to WT and mouse A3-deficient mice. A3 enzymes decreased the number of marginal zone B cells, but not the number of follicular B or T cells. When mouse A3 was knocked out, the retroelement hitchhiker-1 and sialyl transferases encoded by genes close to it were overexpressed three and two orders of magnitude, respectively. We suggest that A3 shifts the balance, from the fast antibody response mediated by marginal zone B cells with little affinity maturation, to a more sustained germinal center B-cell response, which drives affinity maturation and, thereby, a better neutralizing response.

Introduction: mechanisms of tissue injury in autoimmune diseases.

This issue of Seminars in Immunopathology is devoted to the most recent developments in our understanding of the mechanisms leading to tissue injury in autoimmune diseases. These include rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, autoimmune liver diseases, inflammatory bowel diseases, autoimmune skin diseases, autoimmune uveitis, and autoinflammatory diseases. This impressive account of basic and clinical research in a wide spectrum of immunological disorders provides the reader with a comprehensive view of the common and unique features of these diverse conditions. It may also provide one with many new ideas for therapeutic intervention in the natural course of these autoimmune syndromes.

Insertional hypermutation in mineral oil-induced plasmacytomas.

Unless stimulated by a chronic inflammatory agent, such as mineral oil, plasma cell tumors are rare in young BALB/c mice. This raises the questions: What do inflammatory tissues provide to promote mutagenesis? And what is the nature of mutagenesis? We determined that mineral oil-induced plasmacytomas produce large amounts of endogenous retroelements--ecotropic and polytropic murine leukemia virus and intracisternal A particles. Therefore, plasmacytoma formation might occur, in part, by de novo insertion of these retroelements, induced or helped by the inflammation. We recovered up to ten de novo insertions in a single plasmacytoma, mostly in genes with common retroviral integration sites. Additional integrations accompany tumor evolution from a solid tumor through several generations in cell culture. The high frequency of de novo integrations into cancer genes suggests that endogenous retroelements are coresponsible for plasmacytoma formation and progression in BALB/c mice.

Crystal structure determination of anti-DNA Fab A52.

A52 is a murine monoclonal antibody isolated from autoimmune New Zealand Black/New Zealand White F1 mice that recognizes single and double stranded DNA. This mouse strain spontaneously develops systemic lupus erythematosus-like symptoms and has served as a model for that disease for many years. The 1.62 Å crystal structure of the A52 Fab fragment reveals an H3 complementarity determining region with four closely spaced arginine residues, creating a positively charged surface to accommodate bound DNA.

L chain allelic inclusion does not increase autoreactivity in lupus-prone New Zealand Black/New Zealand White mice.

L chain allelic inclusion has been proposed as a B cell tolerance mechanism in addition to clonal deletion, clonal anergy, and receptor editing. It is said to rescue autoreactive B cells from elimination by diluting out the self-reactive BCR through the expression of a second innocuous L chain. In autoimmune animals, such as lupus-prone mice, allelically included B cells could be activated and produce pathogenic autoantibodies. We have previously shown that anti-DNA hybridomas from diseased New Zealand Black/New Zealand White F1 mice exhibit nearly perfect allelic exclusion. In the current study, we have analyzed single B cells from these and from nonautoimmune mice. In addition, we have cloned and expressed the Ig variable regions of several L chain-included B cells in cell culture. We find that although the number of L chain-included B cells increases as a result of receptor editing, the majority of such cells do not retain an autoreactive HxL chain combination and, therefore, allelic inclusion in itself does not serve as a B cell tolerance mechanism in these autoimmune mice.

B cell tolerance and positive selection in lupus.

Systemic lupus erythematosus is considered a prototype of systemic autoimmune diseases; however, despite considerable advances in recent years in the understanding of basic mechanisms in immunology, little progress has been made in elucidating the etiology and pathogenesis of this disease. This even holds for inbred mice, such as the lupus-prone New Zealand Black/New Zealand White F(1) mice, which are all genetically programmed to develop lupus at a predetermined age. This frustrating state of affairs calls for a fundamental change in our scientific thinking and the opening of new directions in lupus research. In this study, we suggest that intrinsic B cell tolerance mechanisms are not grossly impaired in lupus-prone mice, but that an unusually strong positive selection event recruits a small number of autoreactive B cells to the germinal centers. This event could be facilitated by nucleic acid-protein complexes that are created by somatic changes in the susceptible animal.

BAFF/APRIL inhibition decreases selection of naive but not antigen-induced autoreactive B cells in murine systemic lupus erythematosus.

BAFF inhibition is a new B cell-directed therapeutic strategy for autoimmune disease. Our purpose was to analyze the effect of BAFF/APRIL availability on the naive and Ag-activated B cell repertoires in systemic lupus erythematosus, using the autoreactive germline D42 H chain (glD42H) site-directed transgenic NZB/W mouse. In this article, we show that the naive Vκ repertoire in both young and diseased glD42H NZB/W mice is dominated by five L chains that confer no or low-affinity polyreactivity. In contrast, glD42H B cells expressing L chains that confer high-affinity autoreactivity are mostly deleted before the mature B cell stage, but are positively selected and expanded in the germinal centers (GCs) as the mice age. Of these, the most abundant is VκRF (Vκ16-104*01), which is expressed by almost all IgG anti-DNA hybridomas derived from the glD42H mouse. Competition with nonautoreactive B cells or BAFF/APRIL inhibition significantly inhibited selection of glD42H B cells at the late transitional stage, with only subtle effects on the glD42H-associated L chain repertoire. However, glD42H/VκRF-encoded B cells were still vastly overrepresented in the GC, and serum IgG anti-DNA Abs arose with only a slight delay. Thus, although BAFF/APRIL inhibition increases the stringency of negative selection of the naive autoreactive B cell repertoire in NZB/W mice, it does not correct the major breach in B cell tolerance that occurs at the GC checkpoint.

An autoimmune disease prevented by anti-retroviral drugs.

BACKGROUND: Both Aicardi-Goutières syndrome, a Mendelian mimic of congenital infection, and the autoimmune disease systemic lupus erythematosus can result from mutations in the gene encoding the enzyme Trex1. In mice, the absence of Trex1 causes severe myocarditis. The enzyme is thought to degrade endogenous retroelements, thus linking them to autoimmune disease. However, inhibition of reverse transcription by the inhibitor zidovudine (AZT) did not ameliorate the disease, weakening the link to retroelements. FINDINGS: Here, we show that two other FDA-approved drugs that inhibit reverse transcriptase can ameliorate the myocarditis in Trex1-null mouse. CONCLUSIONS: The result suggests that retroelements contribute to this hereditary form of autoimmunity, and that treatment with retroelement inhibitors might ameliorate Aicardi-Goutières syndrome in humans.

B-cell anergy is maintained in anti-DNA transgenic NZB/NZW mice.

Clonal anergy has been well recognized as an important mechanism of B cell immunologic tolerance. However, the properties of anergic B cells and especially their role in the development of autoimmune disease in susceptible animals have been controversial. Here we show that low-affinity anti-DNA anergic B cells populate the mature B-cell compartment in the mouse spleen in excessive numbers and display paradoxical behavior in response to a combined B-cell receptor/TLR9 activation. Surprisingly, B-cell anergy was maintained in aged NZB/NZW F1 mice that develop a systemic lupus erythematosus (SLE)-like autoimmune disease. In several parameters of anergy, such as calcium mobilization and antibody secretion, the lupus-prone mice appeared more anergic than their non-autoimmune counterparts. We conclude that low-affinity anergic B cells are unlikely to serve as precursors for the high-affinity autoreactive B cells that give rise to pathogenic anti-DNA auto-antibodies in SLE.

Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir.

Raltegravir is a recently, Food and Drug Administration-approved, small-molecule drug that inhibits retroviral integrase, thereby preventing HIV DNA from inserting itself into the human genome. We report here that the activity profile of raltegravir on the replication of murine leukemia virus is similar to that for HIV, and that the drug specifically affects autoimmune disease in mice, in which endogenous retroelements are suspected to play a role. While NZW and BALB/c mice, which do not succumb to autoimmune disease, are not affected by raltegravir, lupus-prone (NZBxNZW) F(1) mice die of glomerulonephritis more than a month earlier than untreated mice. Raltegravir-treated NZB mice, which share the H-2 haplotype with BALB/c mice, but which are predisposed to autoimmune hemolytic anemia, develop auto-antibodies to their red blood cells >3 months earlier than untreated mice of the same strain. Because nonautoimmune mice are not affected by raltegravir, we consider off-target effects unlikely and attribute the exacerbation of autoimmunity to the inhibition of retroviral integrase.

Murine B cell response to TLR7 ligands depends on an IFN-beta feedback loop.

Type I IFNs play an important, yet poorly characterized, role in systemic lupus erythematosus. To better understand the interplay between type I IFNs and the activation of autoreactive B cells, we evaluated the effect of type I IFN receptor (IFNAR) deficiency in murine B cell responses to common TLR ligands. In comparison to wild-type B cells, TLR7-stimulated IFNAR(-/-) B cells proliferated significantly less well and did not up-regulate costimulatory molecules. By contrast, IFNAR1(-/-) B cells did not produce cytokines, but did proliferate and up-regulate activation markers in response to other TLR ligands. These defects were not due to a difference in the distribution of B cell populations or a failure to produce a soluble factor other than a type I IFN. Instead, the compromised response pattern reflected the disruption of an IFN-beta feedback loop and constitutively low expression of TLR7 in the IFNAR1(-/-) B cells. These results highlight subtle differences in the IFN dependence of TLR7 responses compared with other TLR-mediated B cell responses.

Autoreactive anti-DNA transgenic B cells in lupus-prone New Zealand black/New Zealand white mice show near perfect L chain allelic exclusion.

Recent work on B cell tolerance and autoimmunity has suggested the L chain allelic inclusion is a property of autoreactive B cells and is closely linked to receptor editing. Allelic inclusion could rescue autoreactive B cells from clonal deletion by reducing their effective BCR surface density. We have investigated this phenomenon in anti-DNA producing hybridomas, derived from different strains of Ig gene-targeted, lupus-prone NZB/NZW mice. Our results indicate that isotype and allelic exclusion was strictly maintained in most high- and low-affinity, edited and nonedited, anti-DNA transgenic B cells. However, a substantial fraction of the anti-DNA hybridomas expressed a very restricted set of nonproductively rearranged L chain mRNA, in addition to the productive anti-DNA L chain. The aberrant L chains could have a role in the selection and survival of autoreactive B cells in these autoimmune mice.

Peripheral B cell receptor editing may promote the production of high-affinity autoantibodies in CD22-deficient mice.

CD22-deficient mice are characterized by B cell hyperactivity and autoimmunity. We have constructed knock-in CD22-/- mice, expressing an anti-DNA heavy (H) chain (D42), alone or combined with Vkappa1-Jkappa1 or Vkappa8-Jkappa5 light (L) chains. The Ig-targeted mice produced a lupus-like serology that was age- and sex-dependent. High-affinity IgG autoantibodies were largely dependent on the selection of B cells with a particular H/L combination, in which a non-transgenic, endogenous L chain was assembled by secondary rearrangements through the mechanism of receptor editing. Moreover, we present evidence that these secondary rearrangements are very prominent in splenic peripheral B cells. Since CD22 is primarily expressed on the surface of peripheral B cells, we propose a model for the development of a lupus-like autoimmune disease by a combination of peripheral receptor editing and abnormal B cell activation.

Treatment with a laminin-derived peptide suppresses lupus nephritis.

The role of DNA as the target for pathogenic lupus autoantibodies in systemic lupus erythematosus is equivocal and renal damage may be due to cross-reactivity of lupus Abs with glomerular components. We have previously shown that lupus autoantibodies bind to the laminin component of the extracellular matrix. In the present work, we have analyzed the fine specificity of the interaction of pathogenic murine lupus autoantibodies with this molecule and the effect of inhibiting their binding to laminin during the course of the disease. We have found that pathogenic murine lupus autoantibodies react with a 21-mer peptide located in the globular part of the alpha-chain of laminin. Immunization of young lupus-prone mice with this peptide accelerated renal disease. Analysis of transgenic, congenic, and RAG-1(-/-) mice confirmed the importance of this epitope in the pathogenesis of lupus renal disease. We have synthesized a panel of peptides that cross-react with the anti-laminin Abs and have found that the binding of lupus autoantibodies to the extracellular matrix could be inhibited in vitro by some of these competitive peptides. Treatment of MRL/lpr/lpr mice with these peptides prevented Ab deposition in the kidneys, ameliorated renal disease, and prolonged survival of the peptide-treated mice. We suggest that laminin components can serve as the target for lupus Abs. The interaction with these Ags can explain both the tissue distribution and the immunopathological findings in lupus. Moreover, inhibition of autoantibody binding to the extracellular matrix can lead to suppression of disease.

Short-lived plasmablasts and long-lived plasma cells contribute to chronic humoral autoimmunity in NZB/W mice.

The current view holds that chronic autoimmune diseases are driven by the continuous activation of autoreactive B and T lymphocytes. However, despite the use of potent immunosuppressive drugs designed to interfere with this activation the production of autoantibodies often persists and contributes to progression of the immunopathology. In the present study, we analyzed the life span of (auto)antibody-secreting cells in the spleens of NZB x NZW F1 (NZB/W) mice, a murine model of systemic lupus erythematosus. The number of splenic ASCs increased in mice aged 1-5 mo and became stable thereafter. Less than 60% of the splenic (auto)antibody-secreting cells were short-lived plasmablasts, whereas 40% were nondividing, long-lived plasma cells with a half-life of >6 mo. In NZB/W mice and D42 Ig heavy chain knock-in mice, a fraction of DNA-specific plasma cells were also long-lived. Although antiproliferative immunosuppressive therapy depleted short-lived plasmablasts, long-lived plasma cells survived and continued to produce (auto)antibodies. Thus, long-lived, autoreactive plasma cells are a relevant target for researchers aiming to develop curative therapies for autoimmune diseases.

Autoimmune NZB/NZW F1 mice utilize B cell receptor editing for generating high-affinity anti-dsDNA autoantibodies from low-affinity precursors.

We have previously constructed knock-in (C57BL/6xBALB/c) F1 mice, each expressing an anti-DNA heavy (H) chain (D42), combined with one of three different light (L) chains, namely Vkappa1-Jkappa1, Vkappa4-Jkappa4 or Vkappa8-Jkappa5. All of these H/L chain combinations bind DNA with similar affinity and fine specificity. However, while mice carrying Vkappa1-Jkappa1-transgenic L chain were tolerized almost exclusively by L chain receptor editing, the mice expressing Vkappa8-Jkappa5 L chains utilized clonal anergy as their principal mechanism of B cell tolerance. Vkappa4-Jkappa4 targeted mice exhibited an intermediate phenotype. In the present study, these three H/L chain combinations were backcrossed onto the autoimmune NZB/NZW F1 mice. We find that the mechanism of clonal anergy is abrogated in these mice, but that receptor editing is maintained. Moreover, diseased NZB/NZW mice utilize L chain secondary rearrangements for the generation of high-affinity, anti-dsDNA-producing B cells from low-affinity precursors. The edited B cell clones are not deleted or anergized in the autoimmune animal; rather they are selected for activation, class-switching and affinity maturation by somatic mutation. These results suggest that B cell receptor editing plays an important role not only in tolerance induction, but also in generating high-affinity autoreactive B cells in autoimmune diseases.

The efficiency of B cell receptor (BCR) editing is dependent on BCR light chain rearrangement status.

Anti-DNA knock-in mice serve as models for studying B cell tolerance mechanisms to a ubiquitous antigen. We have constructed six strains of double transgenic (C57BL/6xBALB/c)F1 mice, each expressing an unmutated or somatically mutated anti-DNA heavy (H) chain, combined with one of three different light (L) chains, namely V(kappa)1-J(kappa)1, V(kappa)4-J(kappa)4 and V(kappa)8-J(kappa)5. In vitro analysis of the various Ig H/L chain combinations showed that all had a similar specificity for single-stranded DNA and double-stranded DNA, but that antibodies encoded by the mutated H chain had higher affinities for the autoantigen. None of the targeted mouse strains exhibited significant levels of serum anti-DNA activity. However, while B cells from mice carrying the V(kappa)1-J(kappa)1 transgenic L chains were tolerized almost exclusively by L chain receptor editing in an affinity-independent manner, the mice expressing V(kappa)8-J(kappa)5 L chains have utilized affinity-dependent clonal anergy as their sole mechanism of B cell tolerance. V(kappa)4-J(kappa)4 targeted mice exhibited an intermediate phenotype with respect to these two mechanisms of B cell tolerance. Our results suggest that receptor editing is the preferred mechanism of B cell tolerance and that the efficiency of L chain editing is directly related to the number of available J(kappa) segments on the expressed V(kappa) allele.