anti-DNA Ab Research Articles

Molecular and structural basis of anti-DNA antibody specificity for pyrrolated proteins

Anti-DNA antibodies (Abs), serological hallmarks of systemic lupus erythematosus (SLE) and markers for diagnosis and disease activity, show a specificity for non-nucleic acid molecules, such as N-pyrrolated proteins (pyrP) containing Nε-pyrrole-L-lysine (pyrK) residues. However, the detailed mechanism for the binding of anti-DNA Abs to pyrP remains unknown. In the present study, to gain structural insights into the dual-specificity of anti-DNA Abs, we used phage display to obtain DNA-binding, single-chain variable fragments (scFvs) from SLE-prone mice and found that they also cross-reacted with pyrP. It was revealed that a variable heavy chain (VH) domain is sufficient for the recognition of DNA/pyrP. Identification of an antigenic sequence containing pyrK in pyrP suggested that the presence of both pyrK and multiple acidic amino acid residues plays important roles in the electrostatic interactions with the Abs. X-ray crystallography and computer-predicted simulations of the pyrK-containing peptide-scFv complexes identified key residues of Abs involved in the interaction with the antigens. These data provide a mechanistic insight into the molecular basis of the dual-specificity of the anti-DNA Abs and provide a basis for therapeutic intervention against SLE.

Cutting Edge: TLR2 Signaling in B Cells Promotes Autoreactivity to DNA via IL-6 Secretion.

Autoantibodies to chromatin and dsDNA are a hallmark of systemic lupus erythematosus (SLE). In a mouse model of monogenic human SLE caused by DNASE1L3 deficiency, the anti-DNA response is dependent on endosomal nucleic acid-sensing TLRs TLR7 and TLR9. In this study, we report that this response also required TLR2, a surface receptor for microbial products that is primarily expressed on myeloid cells. Cell transfers into lymphopenic DNASE1L3-deficient mice showed that TLR2 was required for anti-DNA Ab production by lymphocytes. TLR2 was detectably expressed on B cells and facilitated the production of IL-6 by B cells activated in the presence of microbial products. Accordingly, treatment with broad-spectrum antibiotics or Ab-mediated blockade of IL-6 delayed the anti-DNA response in DNASE1L3-deficient mice. These studies reveal an unexpected B cell-intrinsic role of TLR2 in systemic autoreactivity to DNA, and they suggest that microbial products may synergize with self-DNA in the activation of autoreactive B cells in SLE.

#4760 LUPUS NEPHRITIS RELAPSE AFTER IMMUNOSUPPRESSION WITHDRAWAL: A SPANISH MULTICENTER STUDY

Abstract Background and Aims In lupus nephritis (LN), therapeutic schemas for remission induction are well known but data about duration of maintenance treatment are limited. There is no agreement between clinical practice guidelines about duration and withdrawal of therapy, and literature regarding this topic is scarce. Recently, a RCT has observed that immunosuppression discontinuation after 2‒3 years was related to LN relapse when compared with immunosuppression maintenance. However, immunosuppressive agents are related to severe adverse events and toxicity. We conducted this study with the aim to evaluate the incidence of LN relapse in a cohort of patients with discontinuation of immunosuppressive treatment after a first LN flare. We also aimed to determine factors associated to the presence of renal relapse in this population. Method Multicenter retrospective observational study including patients with biopsy proven LN who have received immunosuppressive treatment that was subsequently discontinued. Patients on prednisone at doses lower than 5 mg per day were allowed to be included. Patients were diagnosed between 1990 and 2018. The study was conducted in Spain. Relapse was defined according to Malvar et al criteria. Results 113 patients were included, mean age was 34.11 ±14.56 years-old and 85.8% were women. Serum creatinine at the time of LN diagnosis was 1.09±0.63 mg/dL, and proteinuria 3.16±2.53g/24h, ANA were positive in 85.84%, and anti-DNA Abs in 69.91%. Serum C3 and C4 levels were 72.51±44.06 and 13.04±11.27 respectively. 55.75% presented with class IV LN, 12.39% with class III or V LN, and 8.85% with class II. Mixed forms were less frequent. In a follow up period of 172.53±162.95 months, 17.7% (n = 20) presented a renal flare after immunosuppressive drugs withdrawal. Time from LN to relapse was 89.05±39.87 months, and time from immunosuppression discontinuation to relapse was 37.75±32.13 months. There were no differences in baseline characteristics between patients who relapsed and patients who did not, neither in histologic characteristics at the time of LN diagnostic biopsy except for glomerulosclerosis, that was less frequent in the relapse group (20% vs 34.41%, p = 0.0435). However, a tendency to a shorter time under immunosuppression in patients who relapsed was observed (73.55±79.06 vs 110.79±206.87 months). Data about treatment withdrawal in each group are summarized in Figure 1. Interestingly, there were no differences between both groups in steroids withdrawal (75% vs 78.49%,p = 0.7327) and hydroxychloroquine withdrawal (33.33% vs 21.74%, p = 0.3393). At the time of immunosuppression discontinuation, patients who relapsed presented lower serum C3 and C4 levels (78.79±24.89 vs 112.26±35.52, p = 0.0080 and 13.82±6.07 vs 23.09±12.56, p = 0.0333), but there were no differences in terms of renal function at this moment. At the end of follow up, patients who relapsed presented more albuminuria (227.87±259.79 vs 78.65±24.77, p = 0.0289) but creatinine levels were similar in both groups (0.84±0.30 vs 0.84±0.30, 0.5105). Only one patient died and two patients required renal replacement therapy, all in the no relapse group. Conclusion In a cohort of 113 patients with biopsy proven LN who were treated con immunosuppression and subsequently discontinued, 17.7% presented renal relapse at a mean time of 37 months after withdrawal. There was a tendency to a short time on immunosuppressive regimens in patients who relapsed. Also, lower serum C3 and C4 levels at the time of immunosuppression withdrawal were associated to renal relapse. At the end of a follow up of 14 years, patients who relapsed those who did not showed a similar maintained renal function but those who relapsed presented lower albuminuria.

A case of refractory systemic lupus erythematosus with monocytosis exhibiting somatic KRAS mutation

BackgroundSystemic lupus erythematosus (SLE), an autoimmune disorder that damages various organ systems, is caused by a combination of genetic and environmental factors. Although germline mutations of several genes are known to cause juvenile SLE, most of the susceptibility genetic variants of adult SLE are common variants of the population, somatic mutations that cause or exacerbate SLE have not been reported. We hereby report a refractory SLE case with monocytosis accompanying somatic KRAS mutation that have been shown to cause lupus-like symptoms.Case presentationA 60-year-old female patient who had been diagnosed with SLE was admitted to our hospital. Although prednisolone and tacrolimus treatments had kept her thrombocytopenia and anti-DNA Ab level at bay for more than 4 years, a diagnosis of transverse myelitis was made when she became acutely ill with pleocytosis. Elevated cells (predominately monocytes), protein, IgG, and IL-6 levels were also found in the cerebrospinal fluid (CSF) of the patient. Standard pulse treatments of methylprednisolone, high-dose of prednisolone, and intravenous cyclophosphamide in combination with plasma exchange could not alleviate the refractory neural and autoimmune manifestation. Monocytosis of peripheral blood was also noted. Flow cytometric analysis revealed elevated ratio of CD14+CD16+ atypical monocytes, which excluded the possibility of chronic myelomonocytic leukemia. Lupus-like symptoms with monocytosis reminded us of Ras-associated autoimmune leukoproliferative disorder, and Sanger sequencing of KRAS and NRAS genes from the patients’ peripheral blood mononuclear cells (PBMC), sorted CD3+ lymphocytes and CD14+ monocytes, and cerebrospinal fluid were performed. An activating KRAS somatic mutation was found in the patients’ DNA at the time of encephalomyelitis diagnosis.ConclusionSomatic mutations of some genes including KRAS may cause the refractoriness of SLE.

The Binding Mechanisms of Antibodies to DNA from Healthy Subjects and Patients with Systemic Lupus Erythematosus: The Role of Monogamous Bivalency and Fc Dependence.

Abs to DNA (anti-DNA) are a unique population of Abs that bind structural determinants on the DNA molecule. In systemic lupus erythematosus (SLE), anti-DNA Abs bind to conserved antigenic determinants, with the phosphodiester backbone being the most likely. In contrast, otherwise healthy subjects (HS) express anti-DNA that bind selectively to nonconserved sites on certain bacterial and viral DNA. As shown previously, SLE anti-DNA bind by a mechanism termed Fc-dependent monogamous bivalency. In this mechanism, both Fab sites interact with determinants on the same extended DNA molecule, reflecting the low affinity of each Fab site; the requirement for the Fc region suggests some contribution of the C region to increase avidity. In this study, we investigated whether anti-DNA from HS also bind to bacterial DNA by Fc-dependent monogamous bivalency. For this purpose, we compared the activity of intact IgG with Fab and F(ab')2 fragments prepared from the plasmas of SLE patients and HS using ELISAs with DNA from calf thymus or Micrococcus luteus These studies showed that Fab fragments from all plasmas tested, both SLE and HS, failed to bind significantly to DNA compared with intact IgG. By contrast, some, but not all, F(ab')2 preparations from both SLE patients and HS showed binding to M. luteus DNA; F(ab')2 fragments from SLE plasmas, however, did not bind significantly to calf thymus DNA. Together, these findings suggest that although anti-DNA Abs, whether from SLE or HS, bind by monogamous bivalency, binding to bacterial DNA does not require the Fc region.

Effects of Peptide-Induced Immune Tolerance on Murine Lupus.

The regulation of autoimmunity and the molecular mechanisms by which different immune cells, including T cells, polymorphonuclear leukocytes (PMN-granulocytes), and B cells suppress autoimmune diseases is complex. We have shown previously that BWF1 lupus mice are protected from autoimmunity after i.v. injection or oral administration of tolerogenic doses of pCons, an artificial synthetic peptide based on sequences containing MHC class I and MHC class II determinants in the VH region of a J558-encoded BWF1 anti-DNA Ab. Several T cell subsets can transfer this tolerance. In this study, we determined the potential roles of granulocytes, B cells and regulatory T cells altered by pCons treatment in the BWF1 (NZB/NZW) mouse model of lupus. Immunophenotyping studies indicated that pCons treatment of BWF1 mice significantly increased CD4+FoxP3+ T cells, reduced the percent of B cells expressing CD19+CD5+ but increased the percent of CD19+CD1d+ regulatory B cells and increased the ability of the whole B cell population to suppress IgG anti-DNA production in vitro. pCons treatment significantly decreased the expression of CTLA-4 (cytotoxic T-lymphocyte-associated protein-4) in CD8+ T cells. In addition, peptide administration modified granulocytes so they became suppressive. We co-cultured sorted naïve B cells from mice making anti-DNA Ab (supported by addition of sorted naive CD4+ and CD8+ T cells from young auto-antibody-negative BWF1 mice) with sorted B cells or granulocytes from tolerized mice. Both tolerized granulocytes and tolerized B cells significantly suppressed the production of anti-DNA in vitro. In granulocytes from tolerized mice compared to saline-treated littermate controls, real-time PCR analysis indicated that expression of interferon-induced TNFAIP2 increased more than 2-fold while Ptdss2 and GATA1 mRNA were up-regulated more than 10-fold. In contrast, expression of these genes was significantly down-regulated in tolerized B cells. Further, another IFN-induced protein, Bcl2, was reduced in tolerized B cells as determined by Western blot analyses. In contrast, expression of FoxP3 was significantly increased in tolerized B cells. Together, these data suggest that B cells and granulocytes are altered toward suppressive functions by in vivo tolerization of BWF1 mice with pCons and it is possible these cell types participate in the clinical benefits seen in vivo.

Type II NKT cells directed immune regulatory mechanism(s) control spontaneous lupus nephritis in mice

Abstract NKT cells recognize lipid antigens presented by CD1d molecules and can be categorized as iNKT cells and type II NKT cells. Type II NKT cells recognize sulfatide or lysophosphatidylcholine (LPC), and their activation results in cross-regulation of iNKT cells, tolerization of DC and control of inflammatory diseases. iNKT cells have been shown to play a pathogenic role in (NZB x NZW)F1 or BWF1 mice, a prototype for human SLE. Interestingly, anti-glycosphingolipid antibody responses have been found in lupus patients. Since sulfatide is enriched in kidney glomeruli, we have examined the role of type II NKT cells in the spontaneous development of lupus in BWF1 mice. We found that both iNKT (αGalCer/CD1d-tetramer+) and type II (sulfatide/CD1d-tetramer+) NKT cells accumulate in kidney tissues with progression of disease in BWF1 mice. Notably, iNKT cells are also activated in SLE patients and secrete the pro-inflammatory cytokine IFNg. Sulfatide-mediated activation of type II NKT cells leads to a significant inhibition of nephritis in BWF1 mice. Importantly, a clinically relevant structural analog of LPC, Miltefosine, also binds to CD1d, activates type II NKT cells and upon oral administration induces inhibition of proteinuria, anti-DNA Abs as well as infiltration of CD4+/CD8+T cells and B cells into kidney of BWF1 mice. Interestingly, the phenotype and frequency of type I interferon-secreting pDC is also altered in kidney of mice treated with Miltefosine. These studies suggest a key role for a type II NKT cell-based immune regulatory mechanism in the control of lupus and, since the CD1d-dependent pathway is highly conserved from mice to human, they have important implications for repurposing a drug for immunotherapeutic for lupus.

Anti-DNA antibodies in the blood of patients with schizophrenia possess DNA-hydrolyzing activity

IntroductionAutoantibodies (Abs) to different neuronal receptors and DNA were detected in the blood of patients with schizophrenia. Abs hydrolyzing DNA were detected in pool of polyclonal autoantibodies in autoimmune and infectious diseases, such catalytic Abs were named abzymes.ObjectivesTo investigate the level of anti-DNA antibodies and DNA-hydrolyzing activity of IgG from the serum of patients with schizophrenia depending on leading clinical symptoms.Aims– To measure the concentration of anti-DNA Abs in serum of patients with leading positive and negative symptoms;– to determine DNA-hydrolyzing activity of IgG.MethodsIn our study, 51 patients were included. The levels of antiDNA Abs were determined using ELISA. DNA-hydrolyzing activity was detected as the level(%) of supercoiled pBluescript DNA transition in circular and linear forms. Statistical analysis was performed in “Statistica 9.0”.ResultsAnti-DNA Abs of patients with schizophrenia not only bind DNA, but quite efficiently hydrolyze the substrate. IgG of patient with schizophrenia were shown to possess DNA hydrolyzing activity. It should be noted that DNAase activity of IgG in patients with schizophrenia with a negative symptoms was significantly higher, than in patients with positive symptoms (Table 1).ConclusionsThe data show a correlation with the level of DNase activity and leading symptoms of patients with schizophrenia.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease, and disease activity is associated with serum IFN-α level. Plasmacytoid dendritic cells (pDCs) sense microbial as well as self-nucleic acids by TLRs 7 and 9 and produce a large amount of IFN-α. Here, we show that heat shock protein 90 (Hsp90) associates with and delivers TLR7/9 from the ER to early endosomes for ligand recognition. Inhibition of Hsp90 by various approaches including the use of Hsp90 inhibitor, a geldanamycin derivative, suppressed the Hsp90 association with TLR7/9, which resulted in inhibition of IFN-α production, leading to improvement of SLE symptoms in mice. Notably, we observed that serum Hsp90 is clearly increased in patients with active SLE compared with that in patients with inactive disease. Furthermore, we demonstrated that serum Hsp90 detected in SLE patients binds to self-DNA and/or anti-DNA Ab, thus leading to stimulation of pDCs to produce IFN-α. Our data demonstrate that Hsp90 plays a crucial role in the pathogenesis of SLE and that an Hsp90 inhibitor will therefore provide a new therapeutic approach to SLE and other nucleic acid-related autoimmune diseases.

The lupus susceptibility locus Sle1 facilitates the peripheral development and selection of anti-DNA B cells through impaired receptor editing.

Systemic lupus erythematosus is characterized by the spontaneous production of IgG autoantibodies in patients and lupus-prone mice. In this study, we investigated the effect of the Sle1 lupus susceptibility locus on the peripheral development of 56R(+) anti-DNA transgenic B cells by tracking 56R(+) B cells in mice without (B6.56R) or with (B6.Sle1.56R) the Sle1 locus. Compared with B6.56R mice, B6.Sle1.56R mice exhibited increased class-switched IgG2a anti-DNA Abs in their serum, encoded by the transgene. Interestingly, within the spleen, Sle1 facilitated the development of these cells into clusters of IgG2a class-switched B cells juxtaposed to CD4(+) T cells within extrafollicular sites. Through sequence analysis of B cell hybridomas, we also found that B cells from B6.Sle1.56R mice are inefficient at Ig H and L chain editing. Thus, the Ig H chains in Sle1.56R(+) B cells are partnered more often with cationic L chains that facilitate DNA binding. Taken together, these findings indicate that the Sle1 lupus-susceptibility locus may facilitate the emergence of anti-DNA B cells by subduing BCR revision and possibly by shaping the extrafollicular development of effector B cells, although the precise molecular mechanisms await further study.

FRI0288 Immunogenicity is Associated with Lupus-Like Autoimmunity in Rheumatoid Arthritis Patients Treated with Infliximab

BackgroundImmunogenicity, a formation of anti-drug antibodies (ADA) against a biologic agent, has also been reported in patients with rheumatoid arthritis (RA) receiving anti-TNF antibody (Ab) treatment. In case of infliximab...

The properties of microparticles from RAW 264.7 macrophage cells undergoing in vitro activation or apoptosis.

Microparticles (MPs) are small, membrane-bound vesicles that arise from dead and dying cells, and display pro-inflammatory and pro-thrombotic activity. As shown previously, the RAW 264.7 murine macrophage cell line can release MPs following stimulation with LPS or polyinosinic:polycytidylic acid [poly (I:C)], ligands of TLR4 and TLR3 respectively. To determine the relationship of these MPs to those released during apoptosis, the nucleic acid content of MPs from cultures stimulated with LPS or poly (I:C) was compared with the nucleic acid content of MPs from untreated cells or cells induced to undergo apoptosis by treatment with etoposide or staurosporine (STS). As results of these studies showed, MPs from activated, apoptotic and untreated cells had features in common, as demonstrated by binding of the nucleic acid dyes SYTO 13 and propidium iodide; molecular mass of DNA; and binding of monoclonal anti-DNA and anti-nucleosome Abs. While MPs from the different culture conditions all contained ribosomal RNA, ribosomal RNA from MPs from STS-treated cells showed cleavage and degradation. Taken together, these studies indicate that the nucleic acid content of MPs from activated and apoptotic cells have important similarities, suggesting that events during TLR activation may lead to apoptosis and subsequent MP release.

SAT0236 Role of serological markers at baseline and follow-up in predicting biopsy proven lupus nephritis

BackgroundStudies have shown that 30-50% of SLE patients (pts) develop lupus nephritis (LN). Patients with high affinity anti-dsDNA antibodies (abs) are more likely to develop LN. The Farr radioimmunoassay is...

The constant region affects antigen binding of anti-DNA antibodies by altering secondary structure (P4002)

Abstract We previously demonstrated an important role of the constant (C) region in the pathogenicity of anti-DNA antibodies (Ab) through modulation of immunoglobulin affinity and specificity. However, the mechanisms by which the C region affects the binding of autoantibodies to nuclear antigens (Ag) have yet to be elucidated. From a parent IgG3 anti-nuclear Ab, PL9-11, we generated IgG1, IgG2b, and IgG2a variants that share identical variable regions but that differ in their C region. The affinity of these Abs for histone antigens was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine the wavelength shift of the Ab panel upon the addition of DNA and histone Ags. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure of the PL9-11 panel corresponding to Ab-Ag interactions. SPR analysis revealed significant differences of histone 2A/2B binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the Ab isotype, while increasing salt concentrations lowered KD values. Circular dichroism analysis determined that changes in Ab secondary structure content differed between the IgG isotypes upon Ag binding. In conclusion, the Ag-binding affinity of anti-nuclear Abs is dependent on the particular C region. Ag binding effects on Trp fluorescence, as well as secondary structure shifts, were also C region dependent. Alteration of secondary structures influenced by C regions may explain differences in fine specificity of anti-DNA Abs between Abs with similar variable regions, as well as cross-reactivity of anti-DNA Abs with non-DNA Ags.

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.

Naturally occurring V region connected antibodies inhibit anti-dsDNA antibody reactivity with dsDNA

The production of autoantibodies against a vast array of self antigens, most notably double stranded (ds) DNA, characterized systemic lupus erythematosus (SLE). The purpose of this work is to study specific Ig fractions isolated from normal human serum (NHS) and their effect on the binding of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies (Abs) to dsDNA. A fraction named immunoglobulin G (IgG)-reactive IgG was purified from total NHS IgG by absorption onto (CNBr)-activated Sepharose 4B linked to intact IgG molecules (IgG-Sepharose column). IgG-reactive IgG was co-incubated with systemic lupus erythematosus (SLE) patient's serum and binding of the anti-dsDNA Abs to dsDNA was measured by enzyme-linked immunosorbent assay (ELISA). Co-incubation of SLE patient's serum with IgG-reactive IgG resulted in a dose-dependent reduction in binding of anti-dsDNA Abs to dsDNA. A reduction greater than 70% was observed at a concentration of 300μg of IgG-reactive IgG per mL of a 400-fold diluted SLE patient's serum whereas total NHS IgG, at the same concentration, resulted in a 10% reduction in binding. The purification process used to isolate IgG-reactive IgG was based on interactions between intact Ig rather than on interactions between F(ab’)2 portions. IgG2 is the predominant immunoglobulin (Ig) subclass in IgG-reactive IgG. Thus, IgG2 might have an important role in the connectivity characteristics of NHS IgG. The capacity of IgG-reactive IgG to inhibit anti-DNA Ab binding to dsDNA may have potential application in the treatment of SLE. This targeted biological approach may provide an alternative strategy to immunosuppressants.

Antigen Is Required for Maturation and Activation of Pathogenic Anti-DNA Antibodies and Systemic Inflammation

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies and systemic inflammation that results in part from dendritic cell activation by nucleic acid containing immune complexes. There are many mouse models of lupus, some spontaneous and some induced. We have been interested in an induced model in which estrogen is the trigger for development of a lupus-like serology. The R4A transgenic mouse expresses a transgene-encoded H chain of an anti-DNA Ab. This mouse maintains normal B cell tolerance with deletion of high-affinity DNA-reactive B cells and maturation to immunocompetence of B cells making nonglomerulotropic, low-affinity DNA-reactive Abs. When this mouse is given estradiol, normal tolerance mechanisms are altered; high-affinity DNA-reactive B cells mature to a marginal zone phenotype, and the mice are induced to make high titers of anti-DNA Abs. We now show that estradiol administration also leads to systemic inflammation with increased B cell-activating factor and IFN levels and induction of an IFN signature. DNA must be accessible to B cells for both the production of high-affinity anti-DNA Abs and the generation of the proinflammatory milieu. When DNase is delivered to the mice at the same time as estradiol, there is no evidence for an abrogation of tolerance, no increased B cell-activating factor and IFN, and no IFN signature. Thus, the presence of autoantigen is required for positive selection of autoreactive B cells and for the subsequent positive feedback loop that occurs secondary to dendritic cell activation by DNA-containing immune complexes.

Invariant NKT Cells Inhibit Autoreactive B Cells in a Contact- and CD1d-Dependent Manner

Autoantibody production is a hallmark of autoimmune diseases, such as lupus and rheumatoid arthritis. Accumulating evidence suggests a role of invariant NKT (iNKT) cells in their pathogenesis. Mechanisms underlying the role of iNKT cells in these diseases, however, remain unclear. In this study, we show that iNKT cells suppress IgG anti-DNA Ab and rheumatoid factor production and reduce IL-10-secreting B cells in a contact-dependent manner, but increase total IgG production and enhance activation markers on B cells via soluble factors. In vivo reconstitution with iNKT cells also reduces autoantibody production in iNKT-deficient mice and in SCID mice implanted with B cells. Using an anti-DNA transgenic model, we found that autoreactive B cells spontaneously produce IL-10 and are activated in vivo. In the presence of activated iNKT cells, these autoreactive B cells are selectively reduced, whereas nonautoreactive B cells are markedly activated. Because iNKTs recognize CD1d, we reasoned that CD1d might play a role in the differential regulation of autoreactive versus nonautoreactive B cells by iNKT cells. Indeed, autoreactive B cells express more CD1d than nonautoreactive B cells, and CD1d deficiency in lupus mice exacerbates autoantibody production and enhances Ab response to a self-peptide but not to a foreign peptide. Importantly, iNKT cells fail to inhibit autoantibody production by CD1d-deficient B cells. Thus, iNKT cells inhibit autoreactive B cells in a contact- and CD1d-dependent manner but activate nonautoreactive B cells via cytokines. Such ability of iNKTs to suppress autoantibody production, without causing global suppression of B cells, has important implications for the development of iNKT-based therapy for autoimmune diseases.

Autoreactive marginal zone B cells enter the follicles and interact with CD4+ T cells in lupus-prone mice

BackgoundMarginal zone B cells have been implicated in the production of autoantibodies in murine models of lupus. It has been suggested that they contribute to lupus immunopathogenesis through their enhanced effector functions and their repertoire that is biased toward autoreactive specificities. In the B6.NZM2410.Sle.Sle2.Sle3 (B6.TC) model of lupus, the majority of marginal zone B cells are located outside the marginal zone and inside the follicles. Genetic alterations of this strain have shown a correlation between autoimmune pathogenesis and the presence of intrafollicular marginal zone B cells. This study was designed first to strengthen our original observations and to determine how the marginal zone B cells from the lupus-prone mice respond to stimulations and interact with T cells.ResultsThe intrafollicular location of B6.TC MZB cells starts before disease manifestations and puts MZB cells in direct contact with CD4+ T cells. Two different autoreactive B cell receptor (BCR) transgenic models showed that the expression of the Sle susceptibility loci enhances the presence of MZB cells inside the follicles. In vitro, B6.TC MZB cells were better effectors than B6 MZB cells with enhanced proliferation and antibody (Ab) production, including anti-DNA Ab, in response to stimulation with TLR ligands, immune complexes or anti-CD40. Furthermore, B6.TC MZB and CD4+ T cells showed a reciprocally enhanced activation, which indicated that their contacts inside B6.TC follicles have functional consequences that suggest an amplification loop between these two cell types.ConclusionsThese results showed that the NZM2410 susceptibility loci induce MZB cells to locate into the follicles, and that this breach of follicular exclusion occurs early in the development of the autoimmune pathogenesis. The enhanced responses to stimulation and increased effector functions of MZB cells from lupus-prone mice as compare to non-autoimmune MZB cells provide a mechanism by which the failure of MZB cell follicular exclusion contributes to the autoimmune process.

Cutting Edge: IL-23 Receptor Deficiency Prevents the Development of Lupus Nephritis in C57BL/6–lpr/lpr Mice

IL-17-producing T cells infiltrate kidneys of patients with lupus nephritis, and IL-23-treated lymph node cells from lupus-prone mice may transfer disease to Rag1-deficient mice. In this study, we show that IL-23R-deficient lupus-prone C57BL/6-lpr/lpr mice display decreased numbers of CD3(+)CD4(-)CD8(-) cells and IL-17A-producing cells in the lymph nodes and produce less anti-DNA Abs. In addition, clinical and pathology measures of lupus nephritis are abrogated. The presented experiments document the importance of IL-23R-mediated signaling in the development of lupus nephritis and urge the consideration of proper biologics for the treatment of the disease.