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Anti-dsDNA antibodies are pathogenically heterogeneous, implying distinct origins and antigenic properties. Unexpectedly, during the clinical and molecular characterization of autoantibodies to the endonuclease DNase1L3 in patients with systemic lupus erythematosus (SLE), we identified a subset of neutralizing anti-DNase1L3 antibodies previously catalogued as anti-dsDNA. Based on their variable heavy-chain (V H ) gene usage, these antibodies can be divided in two groups. One group is encoded by the inherently autoreactive V H 4-34 gene segment, derives from anti-DNase1L3 germline-encoded precursors, and gains cross-reactivity to dsDNA - and some additionally to cardiolipin - following somatic hypermutation. The second group, originally defined as nephritogenic anti-dsDNA antibodies, is encoded by diverse V H gene segments. Although affinity maturation results in dual reactivity to DNase1L3 and dsDNA, their binding efficiencies favor DNase1L3 as the primary antigen. Clinical, transcriptional and monoclonal antibody data support that cross-reactive anti-DNase1L3/dsDNA antibodies are more pathogenic than single reactive anti-dsDNA antibodies. These findings point to DNase1L3 as the primary target of a subset of antibodies classified as anti-dsDNA, shedding light on the origin and pathogenic heterogeneity of antibodies reactive to dsDNA in SLE., (© 2023. The Author(s).)

Introduction: It is documented that a series of autoantibodies can be detected with increased frequency in women with recurrent pregnancy loss (RPL) and they may impact the pregnancy prognosis negatively. It is unknown whether the autoantibodies per se or the basic immune disturbances underlying autoantibody production, are the reason for this association. Our group has previously found that some genetically determined immunological biomarkers are associated with RPL and the same biomarkers are also in various degrees known to predispose to autoantibody production. The aim of this study was to clarify whether the RPL-associated immunogenetic biomarkers are associated with positivity for three major classes of autoantibodies associated with RPL., Methods: In 663 patients with RPL in whom we had results for HLA-DRB1 typing and plasma mannose-binding lectin (p-MBL) measurement, it was investigated whether there is a correlation between positivity for the autoantibodies: anticardiolipin antibodies, β2 glycoprotein I antibodies, and lupus anticoagulant (jointly called antiphospholipid antibodies), thyroid-peroxidase antibodies, and antinuclear antibodies and each of the HLA-DRB1 alleles HLA-DRB1*03 or HLA-DRB1*07 either alone or in combination with low p-MBL defined as ≤500 µg/l., Results: Although slightly higher frequencies of positivity of two or more autoantibodies were seen in patients with either p-MBL ≤500 µg/l or being positive for HLA-DRB1*03, none were significantly associated. However, in patients with the combination of low p-MBL and HLA-DRB1*03, presence of at least one autoantibody was significantly more frequent than in patients with no such combination (OR= 2.4; 95% CI 1.2-5.0, p = 0.01). In an analysis of which autoantibodies were most strongly associated with the low p-MBL/HLA-DRB1*03 combination, antinuclear antibodies were significantly more frequent in these patients (OR 2.0; 95% CI 1.0-3.9, p=0.05) whereas the other autoantibodies were also positively but more weakly associated with this combination., Discussion: In conclusion, to clarify the pathogenetic background, underlying immunogenetic factors should be examined in autoantibody positive RPL patients (as well as other patients with autoimmune diseases) but the genetic background may be complex., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nørgaard-Pedersen, Steffensen, Kesmodel and Christiansen.)

Systemic lupus erythematosus is a chronic disease characterized by autoantibodies, renal and cutaneous disease, and immune complex formation. Emerging evidence suggests that aberrant DNA repair is an underlying mechanism of lupus development. We previously showed that the POLBY265C/C mutation, which results in development of an aberrant immune repertoire, leads to lupus-like disease in mice. To address whether the hematopoietic compartment is sufficient for lupus development, we transplanted bone marrow cells from POLBY265C/C and POLB+/+ into wild-type congenic mice. Only mice transplanted with the POLBY265C/C bone marrow develop high levels of antinuclear antibodies and renal disease. In conclusion, we show that the hematopoietic compartment harvested from the POLBY265C/C mice is sufficient for development of autoimmune disease., Competing Interests: The authors have declared that no competing interests exist.

The anti-La mab 312B, which was established by hybridoma technology from human-La transgenic mice after adoptive transfer of anti-human La T cells, immunoprecipitates both native eukaryotic human and murine La protein. Therefore, it represents a true anti-La autoantibody. During maturation, the anti-La mab 312B acquired somatic hypermutations (SHMs) which resulted in the replacement of four aa in the complementarity determining regions (CDR) and seven aa in the framework regions. The recombinant derivative of the anti-La mab 312B in which all the SHMs were corrected to the germline sequence failed to recognize the La antigen. We therefore wanted to learn which SHM(s) is (are) responsible for anti-La autoreactivity. Humanization of the 312B ab by grafting its CDR regions to a human Ig backbone confirms that the CDR sequences are mainly responsible for anti-La autoreactivity. Finally, we identified that a single amino acid replacement (D > Y) in the germline sequence of the CDR3 region of the heavy chain of the anti-La mab 312B is sufficient for anti-La autoreactivity.

Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from V H 10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (V H 10) or not (V H 4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. V H 10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the V H 4 segment did not. The CDR2 loop shuffling hampered V H 10 reactivity while displaying a gain-of-function in the nonbinding V H 4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the V H 10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.

Anti-DNA antibodies are usually produced against histone-DNA complexes appearing during cell apoptosis, while histones are known as damage-associated molecules. A myelin sheath of axons contains myelin basic protein (MBP) playing an important role in the pathogenesis of autoimmune diseases. Antibodies with enzymatic activities (abzymes) are distinctive features of some autoimmune and viral diseases. Abzymes against different proteins can usually only hydrolyze these specific proteins. Using sequential chromatographies of homogeneous IgG preparations from sera of HIV-infected patients on columns with immobilized MBP, H2a, and H2b histones, the anti-MBP, anti-H2a, and anti-H2b antibodies were obtained. It was first shown that IgGs against H2a and H2b effectively hydrolyze these histones and MBP, while anti-MBP split MBP, H2a, and H2b, but no other control proteins. Using the MALDI mass spectrometry, the cleavage sites of H2a, H2b, and MBP by abzymes against these three proteins were found. Among 14 sites of hydrolysis of H2a by IgGs against H2a and 10 sites by anti-MBP IgGs, only one site of hydrolysis was the same for these abzymes. Eleven cleavage sites of H2b with IgGs against H2b and 10 sites of its hydrolysis with antibodies against MBP were different. Anti-H2a, anti-H2b, and anti-MBP abzymes are unpredictable examples of IgGs possessing not only cross-complexation but also catalytic cross-reactivity, which may be a common phenomenon for such abzymes in patients with different autoimmune diseases. The existence of cross-reactivity of abzymes against H2a and H2b histones and MBP represent a great danger to humans since, in contrast with MBP, histones due to cell apoptosis constantly occur in human blood. Anti-H2a, anti-H2b, and anti-MBP can attack and hydrolyze myelin basic protein of the myelin sheath of axons and plays a negative role in the pathogenesis of several pathologies.

Background: Systemic lupus erythematosus (SLE) is a severe autoimmune disease in which immune tolerance defects drive production of pathogenic anti-nuclear autoantibodies. Anergic B cells are considered a potential source of these autoantibodies due to their autoreactivity and overrepresentation in SLE patients. Studies of lupus-prone mice have shown that genetic defects mediating autoimmunity can breach B cell anergy, but how this breach occurs with regards to endogenous nuclear antigen remains unclear. We investigated whether B and T cell defects in congenic mice (c1) derived from the lupus-prone New Zealand Black strain can breach tolerance to nuclear self-antigen in the presence of knock-in genes (Vκ8/3H9; dKI) that generate a ssDNA-reactive, anergic B cell population., Methods: Flow cytometry was used to assess splenic B and T cells from 8-month-old c1 dKI mice and serum autoantibodies were measured by ELISA. dKI B cells stimulated in vitro with anti-IgM were assessed for proliferation and activation by examining CFSE decay and CD86. Cytokine-producing T cells were identified by flow cytometry following culture of dKI splenocytes with PMA and ionomycin. dKI B cells from 6-8-week-old mice were adoptively transferred into 4-month-old wild type recipients and assessed after 7 days via flow cytometry and immunofluorescence microscopy., Results: c1 dKI mice exhibited B cell proliferation indicative of impaired anergy, but had attenuated autoantibodies and germinal centres compared to wild type littermates. This attenuation appeared to stem from a decrease in PD-1hi T helper cells in the dKI strains, as c1 dKI B cells were recruited to germinal centres when adoptively transferred into c1 wild type mice., Conclusion: Anergic, DNA-specific autoreactive B cells only seem to drive profound autoimmunity in the presence of concomitant defects in the T cell subsets that support high-affinity plasma cell production., Competing Interests: The authors have declared that no competing interests exist.

Phenotypic variation of quantitative traits is orchestrated by a complex interplay between the environment (e.g. diet) and genetics. However, the impact of gene-environment interactions on phenotypic traits mostly remains elusive. To address this, we feed 1154 mice of an autoimmunity-prone intercross line (AIL) three different diets. We find that diet substantially contributes to the variability of complex traits and unmasks additional genetic susceptibility quantitative trait loci (QTL). By performing whole-genome sequencing of the AIL founder strains, we resolve these QTLs to few or single candidate genes. To address whether diet can also modulate genetic predisposition towards a given trait, we set NZM2410/J mice on similar dietary regimens as AIL mice. Our data suggest that diet modifies genetic susceptibility to lupus and shifts intestinal bacterial and fungal community composition, which precedes clinical disease manifestation. Collectively, our study underlines the importance of including environmental factors in genetic association studies.

Anti-nuclear antibodies to speckled 100 kDa (sp100) and glycoprotein 210 (gp210) are specific serologic markers of primary biliary cholangitis (PBC) of uncertain/controversial clinical or prognostic significance. To study the genetic determinants associated with sp100 and gp210 autoantibody subphenotypes, we performed a genome-wide association analysis of 930 PBC cases based on their autoantibody status, followed by a replication study in 1,252 PBC cases. We confirmed single-nucleotide polymorphisms rs492899 (P = 3.27 × 10 -22 ; odds ratio [OR], 2.90; 95% confidence interval [CI], 2.34-3.66) and rs1794280 (P = 5.78 × 10 -28 ; OR, 3.89; 95% CI, 3.05-4.96) in the human major histocompatibility complex (MHC) region associated with the sp100 autoantibody. However, no genetic variant was identified as being associated with the gp210 autoantibody. To further define specific classical human leukocyte antigen (HLA) alleles or amino acids associated with the sp100 autoantibody, we imputed 922 PBC cases (211 anti-sp100-positive versus 711 negative cases) using a Han Chinese MHC reference database. Conditional analysis identified that HLA-DRβ1-Asn77/Arg74, DRβ1-Ser37, and DPβ1-Lys65 were major determinants for sp100 production. For the classical HLA alleles, the strongest association was with DRB1*03:01 (P = 1.51 × 10 -9 ; OR, 2.97; 95% CI, 2.06-4.29). Regression analysis with classical HLA alleles identified DRB1*03:01, DRB1*15:01, DRB1*01, and DPB1*03:01 alleles can explain most of the HLA association with sp100 autoantibody. Conclusion: This study indicated significant genetic predisposition to the sp100 autoantibody, but not the gp210 autoantibody, subphenotype in PBC patients. Additional studies will be necessary to determine if these findings have clinical significance to PBC pathogenesis and/or therapeutics., (© 2019 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

Juvenile-onset systemic sclerosis (jSSc) is a rare and severe autoimmune disease with associated life-threatening organ inflammation and evidence of fibrosis. The organ manifestations of jSSc resemble adult SSc, but with better outcomes and survival. The etiology of jSSc appears to reflect adult-onset SSc, with similar inflammatory mediators and autoantibodies, but with a significant population of children with uncharacterized anti-nuclear antibodies. The genetics of patients with jSSc differ from women with SSc, resembling instead the genes of adult males with SSc, with additional HLA genes uniquely associated with childhood-onset disease. Current treatments are aimed at inhibiting the inflammatory aspect of disease, but important mechanisms of fibrosis regulated by dermal white adipose tissue dendritic cells may provide an avenue for targeting and potentially reversing the fibrotic stage.

Anti-double-stranded (ds)DNA autoantibodies are prototypical serological markers of systemic lupus erythematosus (SLE), but little is known about their immunoglobulin variable (IgV) region composition at the level of the secreted (serum) proteome. Here, we use a novel proteomic workflow based on de novo mass spectrometric sequencing of anti-dsDNA precipitins to analyse IgV subfamily expression and mutational signatures of high-affinity, precipitating anti-dsDNA responses. Serum anti-dsDNA proteomes were oligoclonal with shared (public) expression of immunoglobulin (Ig)G heavy chain variable region (IGHV) and kappa chain variable region (IGKV) subfamilies. IgV peptide maps from eight subjects showed extensive public and random (private) amino acid replacement mutations with prominent arginine substitutions across heavy (H)- and light (L)-chains. Shared sets of L-chain complementarity determining region 3 (CDR3) peptides specified by arginine substitutions were sequenced from the dominantly expressed IGKV3-20 subfamily, with changes in expression levels of a clonal L-chain CDR3 peptide by quantitative multiple reaction monitoring (MRM) paralleling the rise and fall of anti-dsDNA levels by Farr radioimmunoassays (RIA). The heavily mutated IgV peptide signatures of precipitating anti-dsDNA autoantibody proteomes reflect the strong selective forces that shape humoral anti-dsDNA responses in germinal centres. Direct sequencing of agarose gel precipitins using microlitre volumes of stored sera streamlines the antibody sequencing workflow and is generalizable to other precipitating serum antibodies., (© 2018 British Society for Immunology.)

BACKGROUND MAD2 is the gene controlling mitosis. Many studies have assessed MAD2 in various types of carcinoma. Antinuclear mitotic spindle apparatus antibody (MSA) and anticentromere antibody (ACA) are related mitotic antibodies, playing roles in autoimmune diseases and carcinomas, but the expression of MAD2, MSA, and ACA in SCLC is unclear. MATERIAL AND METHODS We enrolled 70 SCLC patients, 72 non-small cell lung cancer (NSCLC) patients, and 65 pulmonary nodule (PN) patients. MAD2 expression was measured through agarose electrophoresis and qt-PCR. Antinuclear mitotic spindle apparatus antibody (MSA) and anticentromere antibody (ACA) were detected by indirect immunofluorescence (IIF). RESULTS MAD2 was found both in SCLC and NSCLC. Interestingly, there was a significant difference found between SCLC and NSCLC using qt-PCR (P<0.05). The area under the ROC curve of MAD2 expression was 0.799, with medium diagnostic value. MAD2 expression was related to age, lymphatic metastasis, and survival time, but not with sex. The positivity for MSA and ACA by IIF assay were 37.20% and 34.00%, respectively, in the SCLC group, which were higher than in the NSCLC and pulmonary nodule groups (P<0.05). The kappa values of MSA and ACA with MAD2 expression were 0.73 and 0.65, respectively, with moderate consistency. Combining MAD2 with MSA and ACA enhanced the sensitivity and specificity for diagnosing SCLC. CONCLUSIONS MAD2 expression was found to be involved in carcinogenesis and prognosis of SCLC. The combination of MAD2 with MSA and ACA is useful for early diagnosis and shows promise in treatment of SCLC.

Anti-DNA autoantibodies are a hallmark of systemic lupus erythematosus (SLE). A subset of anti-DNA IgG autoantibodies is cell-internalizable; thus they can enter living cells in the form of free IgG. However, the contribution made by the Fc region of internalized free-form IgG to the cytokine response has not been studied, despite the recent discovery of tripartite motif-containing 21 (TRIM21), a cytosolic Fc receptor involved in immune signaling. This study used an internalizable IgG anti-DNA antibody (3D8) to examine the cytokine responses of human monocytes to the Fc region of cytosolic free IgG. Internalization of 3D8 IgG and a 3D8 single-chain variable fragment-Fc (scFv-Fc) induced production of IL-8 and TNF-α via activation of NF-κB. By contrast, a 3D8 scFv (comprising variable domains alone) did not. This suggests Fc-dependent cytokine signaling. A 3D8 IgG-N434D mutant that is not recognized by TRIM21 induced greater production of cytokines than 3D8 IgG. Moreover the amounts of cytokines induced by 3D8 IgG in TRIM21-knockdown THP-1 cells were higher than those in control cells, indicating that cytokine signaling is not mediated by TRIM21. The results suggest the existence of a novel Fc-dependent signaling pathway that is activated upon internalization of IgG antibodies by human monocytes.

N 6 -Methyladenosine (m 6 A) is an abundant post-transcriptional RNA modification that influences multiple aspects of gene expression. In addition to recruiting proteins, m 6 A can modulate RNA function by destabilizing base pairing. Here, we show that when neighbored by a 5' bulge, m 6 A stabilizes m 6 A-U base pairs, and global RNA structure by ~1 kcal mol -1 . The bulge most likely provides the flexibility needed to allow optimal stacking between the methyl group and 3' neighbor through a conformation that is stabilized by Mg 2+ . A bias toward this motif can help explain the global impact of methylation on RNA structure in transcriptome-wide studies. While m 6 A embedded in duplex RNA is poorly recognized by the YTH domain reader protein and m 6 A antibodies, both readily recognize m 6 A in this newly identified motif. The results uncover potentially abundant and functional m 6 A motifs that can modulate the epitranscriptomic structure landscape with important implications for the interpretation of transcriptome-wide data.

A key challenge in the development of novel chemotherapeutics is the design of molecules capable of selective toxicity to cancer cells. Antibodies have greater target specificity compared to small molecule drugs, but most are unable to penetrate cells, and predominantly target extracellular antigens. A nuclear-penetrating anti-DNA autoantibody isolated from the MRL/lpr lupus mouse model, 3E10, preferentially localizes to tumors, inhibits DNA repair, and selectively kills cancer cells with defects in DNA repair. A murine divalent single chain variable fragment of 3E10 with mutations for improved DNA binding affinity, 3E10 (D31N) di-scFv, has previously been produced in P. pastoris and yielded promising pre-clinical findings, but is unsuitable for clinical testing. The present study reports the design, expression and testing of a panel of humanized 3E10 (D31N) di-scFvs, some of which contain CDR substitution. These variants were expressed in a modified CHO system and evaluated for their physicochemical attributes and ability to penetrate nuclei to selectively cause DNA damage accumulation in and kill cancer cells with DNA repair defects. Secondary structure was conserved and most variants retained the key characteristics of the murine 3E10 (D31N) di-scFv produced in P. pastoris. Moreover, several variants with CDR substitutions outperformed the murine prototype. In conclusion, we have designed several humanized variants of 3E10 (D31N) di-scFv that have potential for application as monotherapy or conjugates for targeted nuclear drug delivery., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The signature lesion of SSA/Ro autoantibody-associated congenital heart block (CHB) is fibrosis and a macrophage infiltrate, supporting an experimental focus on cues influencing the fibroblast component. The transcriptomes of human fetal cardiac fibroblasts were analyzed using two complementary approaches. Cardiac injury conditions were simulated in vitro by incubating human fetal cardiac fibroblasts with supernatants from macrophages transfected with the SSA/Ro-associated noncoding Y ssRNA. The top 10 upregulated transcripts in the stimulated fibroblasts reflected a type I interferon (IFN) response [e.g., IFN-induced protein 44-like (IFI44L), of MX dynamin-like GTPase (MX)1, MX2, and radical S -adenosyl methionine domain containing 2 (Rsad2)]. Within the fibrotic pathway, transcript levels of endothelin-1 (EDN1), phosphodiesterase (PDE)4D, chemokine (C-X-C motif) ligand (CXCL)2, and CXCL3 were upregulated, while others, including adenomedullin, RAP guanine nucleotide exchange factor 3 (RAPGEF3), tissue inhibitor of metalloproteinase (TIMP)1, TIMP3, and dual specificity phosphatase 1, were downregulated. Agnostic Database for Annotation, Visualization and Integrated Discovery analysis revealed a significant increase in inflammatory genes, including complement C3A receptor 1 (C3AR1), F2R-like thrombin/trypsin receptor 3, and neutrophil cytosolic factor 2. In addition, stimulated fibroblasts expressed high levels of phospho-MADS box transcription enhancer factor 2 [a substrate of MAPK5 (ERK5)], which was inhibited by BIX-02189, a specific inhibitor of ERK5. Translation to human disease leveraged an unprecedented opportunity to interrogate the transcriptome of fibroblasts freshly isolated and cell sorted without stimulation from a fetal heart with CHB and a matched healthy heart. Consistent with the in vitro data, five IFN response genes were among the top 10 most highly expressed transcripts in CHB fibroblasts. In addition, the expression of matrix-related genes reflected fibrosis. These data support the novel finding that cardiac injury in CHB may occur secondary to abnormal remodeling due in part to upregulation of type 1 IFN response genes. NEW & NOTEWORTHY Congenital heart block is a rare disease of the fetal heart associated with maternal anti-Ro autoantibodies which can result in death and for survivors, lifelong pacing. This study provides in vivo and in vitro transcriptome-support that injury may be mediated by an effect of Type I Interferon on fetal fibroblasts., (Copyright © 2017 the American Physiological Society.)

Targeting and modification of important intracellular proteins using efficient vehicles are invaluable in diagnostic and therapeutic fields. Cell-penetrating antibodies and their fragments can be utilized as vehicles for the delivery of modifiers into cells. In this study, we explored the applicability of variable heavy chain (VH) domain as delivery vehicles for mammalian cells. The characteristics of the recombinant VH domain produced from a cell-penetrating monoclonal anti-double stranded DNA antibody 2C10 were analyzed using flow cytometry, confocal microscopy, cell proliferation assay, and cell cycle analysis in various mammalian cell lines. The VH domain penetrated into various cell lines in a time- and dose-dependent manner, although the internalization efficiency varied. The domain was localized in the nuclei as well as the cytoplasm of living cells. It was also internalized into cells mainly through the clathrin-mediated endocytosis pathway. We tested further its efficiency in delivering specific biomolecule(s) using the conjugates of the single domain molecule and small interfering RNA (siRNA) for the testicular nuclear auto-antigenic sperm protein (tNASP). It was found that the siRNA was successfully delivered by the VH domain into cancer cells, and knockdown effects from the delivered tNASP-siRNA were observed. The levels of the RNA transcript and protein of tNASP were decreased and the down-regulated tNASP inhibited cell proliferation and caused G0G1 phase arrest of the cell cycle. These results indicate that the recombinant 2C10 VH domain could be applied as an efficient vehicle capable of delivering valuable biomolecule into the cytoplasm or cell nuclei for clinical uses.

Background Estrogens have a modulatory effect on several immune responses, many of which are correlated to autoimmune diseases. Estrogens act through binding to their receptors, and an overexpression of these receptors has been identified in patients with different autoimmune diseases. Here we analyzed the association of a putative functional genetic variant in the main estrogen receptor (ERα) gene ( ESR1), and the susceptibility to clinical findings and severity of SLE. Methods A total of 426 individuals (266 healthy controls and 160 SLE patients) were genotyped for the polymorphism rs2234693 in the ESR1 gene. Allele and genotype frequencies were calculated and analyzed between cases and controls using Unphased software. Results The SNP rs2234693 was not associated with SLE per se but the minor allele rs2234693-C was correlated with the presence of nephritis and discoid skin rash. On the other hand, the rs2234693-CC genotype was correlated with the absence of arthritis as well as anti-ANA and anti-RNP autoantibodies. The comprehensive clinical analysis of these patients revealed a more severe status of the disease, characterized by a younger age of onset and higher number of organs involved when compared to European populations. Conclusions Minor allele rs2234693-C was associated with renal and cutaneous involvement, as well as the absence of arthritis, anti-ANA and anti-RNP autoantibodies.

IgG anti-DNA antibodies are both diagnostic and pathogenic for systemic lupus erythematosus (SLE). They contribute to tissue inflammation through direct tissue binding and to systemic inflammation through activation of Toll-like receptors by nucleic acid-containing immune complexes. IgG DNA-reactive antibodies originate when B cell tolerance mechanisms are impaired. The heterogeneous immune perturbations in SLE lead to the survival and activation of DNA-reactive B cells in various B cell subsets at distinct stages of B cell maturation and differentiation. We propose that the spectrum of B cell alterations and failed tolerance mechanisms for DNA-reactive B cells in lupus patients is best understood by studying genetic risk alleles. This implies that the B cells producing IgG anti-DNA antibodies and the failed tolerance mechanisms(s) will differ across patients. A better understanding of these differences should lead to better patient stratification, improved outcomes of clinical trials, and the identification of novel therapeutic targets., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

MHC, especially HLA-DR3 and HLA-DR2, is one of the most important genetic susceptibility regions for systemic lupus erythematosus. Human studies to understand the role of specific HLA alleles in disease pathogenesis have been hampered by the presence of strong linkage disequilibrium in this region. To overcome this, we produced transgenic mice expressing HLA-DR3 (DRβ1*0301) and devoid of endogenous class II (both I-A and I-E genes, AE(0)) on a lupus-prone NZM2328 background (NZM2328.DR3(+)AE(0)). Both NZM2328 and NZM2328.DR3(+)AE(0) mice developed anti-dsDNA and glomerulonephritis, but anti-dsDNA titers were higher in the latter. Although kidney histological scores were similar in NZM2328 and NZM2328.DR3(+)AE(0) mice (7.2 ± 4.3 and 8.6 ± 5.7, respectively, p = 0.48), the onset of severe proteinuria occurred earlier in NZM2328.DR3(+)AE(0) mice compared with NZM2328 mice (median, 5 and 9 mo respectively, p < 0.001). Periarterial lymphoid aggregates, classic wire loop lesions, and occasional crescents were seen only in kidneys from NZM2328.DR3(+)AE(0) mice. Interestingly, NZM2328.DR3(+)AE(0) mice, but not NZM2328 mice, spontaneously developed anti-Smith (Sm) Abs. The anti-Sm Abs were seen in NZM2328.DR3(+)AE(0) mice that were completely devoid of endogenous class II (AE(-/) (-)) but not in mice homozygous (AE(+/+)) or heterozygous (AE(+/-)) for endogenous MHC class II. It appears that only HLA-DR3 molecules can preferentially select SmD-reactive CD4(+) T cells for generation of the spontaneous anti-Sm immune response. Thus, our mouse model unravels a critical role for HLA-DR3 in generating an autoimmune response to SmD and lupus nephritis in the NZM2328 background., Competing Interests: The authors have no financial conflicts of interest., (Copyright © 2015 by The American Association of Immunologists, Inc.)

We showed previously that C57BL/6 congenic mice with an introgressed homozygous 70 cM (125.6 Mb) to 100 cM (179.8 Mb) interval on c1 from the lupus-prone New Zealand Black (NZB) mouse develop high titers of antinuclear Abs and severe glomerulonephritis. Using subcongenic mice, we found that a genetic locus in the 88-96 cM region was associated with altered dendritic cell (DC) function and synergized with T cell functional defects to promote expansion of pathogenic proinflammatory T cell subsets. In this article, we show that the promoter region of the NZB gene encoding the SLAM signaling pathway adapter molecule EWS-activated transcript 2 (EAT-2) is polymorphic, which results in an ∼ 70% reduction in EAT-2 in DC. Silencing of the EAT-2 gene in DC that lacked this polymorphism led to increased production of IL-12 and enhanced differentiation of T cells to a Th1 phenotype in T cell-DC cocultures, reproducing the phenotype observed for DC from congenic mice with the NZB c1 70-100 cM interval. SLAM signaling was shown to inhibit production of IL-12 by CD40L-activated DCs. Consistent with a role for EAT-2 in this inhibition, knockdown of EAT-2 resulted in increased production of IL-12 by CD40-stimulated DC. Assessment of downstream signaling following CD40 cross-linking in the presence or absence of SLAM cross-linking revealed that SLAM coengagement blocked activation of p38 MAPK and JNK signaling pathways in DC, which was reversed in DC with the NZB EAT-2 allele. We conclude that EAT-2 negatively regulates cytokine production in DC downstream of SLAM engagement and that a genetic polymorphism that disturbs this process promotes the development of lupus., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Only 2% of mothers positive for anti-SSA/Ro (Ro) antibodies have children with congenital heart block (CHB). This study aimed to determine whether reactivity with p305, an epitope within the α1G T-type calcium channel, confers added risk over anti-Ro antibodies., Methods and Results: Using sera from anti-Ro-exposed pregnancies resulting in offspring with CHB, no disease but CHB-sibling, and no disease and no CHB-sibling, as well as disease (lupus without anti-Ro) and healthy controls, reactivities were determined for binding to Ro60, p305, and an epitope within Ro60, p133-Ro60, which shares structural properties with p305, including key amino acids and an α-helical structure. Candidate peptides were further evaluated in an in vitro model that assessed the binding of maternal antibodies to apoptotic cells. In anti-Ro-positive mothers, anti-p305 autoantibodies (>3 SD above healthy controls) were detected in 3/59 (5%) CHB pregnancies, 4/30 (13%) unaffected pregnancies with a CHB-sibling, and 0/42 (0%) of unaffected pregnancies with no CHB-sibling. For umbilical bloods (61 CHB, 41 healthy with CHB sibling), no association of anti-p305 with outcome was detected; however, overall levels of anti-p305 were elevated compared to mothers during pregnancy in all groups studied. For anti-p133-Ro60, reactivity paralleled that of anti-p305. In the screen employing apoptotic cells, p133-Ro60, but not p305, significantly attenuated the binding of immunoglobulin G isolated from a mother whose child had CHB (42.1% reduced to 13.9%, absence/presence of p133-Ro60, respectively, P<0.05)., Conclusions: These data suggest that anti-p305 is not a robust maternal marker for assessing increased risk of CHB during an anti-SSA/Ro pregnancy., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Random recombination of antibody heavy- and light-chain genes results in a diverse B-cell receptor (BCR) repertoire including self-reactive BCRs. However, tolerance mechanisms that prevent the development of self-reactive B cells remain incompletely understood. The absence of the surrogate light chain, which assembles with antibody heavy chain forming a pre-BCR, leads to production of antinuclear antibodies (ANAs). Here we show that the naive follicular B-cell pool is enriched for cells expressing prototypic ANA heavy chains in these mice in a non-autoimmune background with a broad antibody repertoire. This results in the spontaneous formation of T-cell-dependent germinal centres that are enriched with B cells expressing prototypic ANA heavy chains. However, peripheral tolerance appears maintained by selection thresholds on cells entering the memory B-cell and plasma cell pools, as exemplified by the exclusion of cells expressing the intrinsically self-reactive V(H)81X from both pools.

The autoimmune disease systemic lupus erythematosus has a complex environmental and multifactorial genetic basis. Genome-wide association studies have recently identified numerous disease-associated polymorphisms, but it remains unclear in which cells and during which step of pathogenesis specific polymorphisms interact to cause disease. Using a mouse model in which the same activating mutation (CD45E613R) causes distinct genetic background-dependent disease phenotypes, we performed a screen for genetic modifiers of autoreactivity between anti-nuclear Ab (ANA)-resistant CD45E613R.B6 and ANA-permissive CD45E613R.BALB/c mice. Within a novel autoreactivity-associated locus on chromosome 9, we identify a putative modifier, TLR9. Validating a role for TLR9 in modifying autoreactivity in the context of the CD45E613R mutation, manipulation of TLR9 gene dosage eliminates ANA in CD45E613R.BALB/c mice, but confoundingly permits ANA in CD45E613R.B6 mice. We demonstrate that sensitivity to ANA is modulated by strength of TLR9 signal, because stronger TLR9(B6) signals, but not weaker TLR9(BALB/c) signals, negatively regulate CD45E613R B cell development during competitive reconstitution at the central tolerance checkpoint. Our results identify a novel autoreactivity-associated locus and validate Tlr9 as a candidate gene within the locus. We further demonstrate a novel role for TLR9 signal strength in central tolerance, providing insight into the interplay of disease-associated polymorphisms at a discrete step of systemic lupus erythematosus pathogenesis., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Serum IgG anti-nuclear antibodies (ANA) directed to complexes of DNA and histones are a hallmark of systemic lupus erythematosus (SLE) and reflect a failure in lymphocyte self-tolerance. A prior study utilizing spontaneously autoimmune B6.Nba2 mice deficient in terminal deoxynucleotidyl transferase (TdT) and with heterozygous deficiencies in Jh and Igk loci underscored the importance of somatic hypermutation (SHM) as a major generator of SLE-associated ANA. This interpretation had to be qualified because of severely limited opportunities for receptor editing and restricted VHCDR3 diversity. Therefore, we performed the converse study using mice that carried functional Tdt genes and wild type Jh and Igk loci but that could not undergo SHM. Analyses of ANA and ANA-producing hybridomas from B6.Nba2 Aicda(-/-) mice revealed that few animals produced high titers of the prototypical ANA directed to complexes of histones and DNA, that this response was delayed and that those cells that did produce such antibody exhibited limited clonal expansion, unusual Jk use and only infrequent dual receptor expression. This, together with the additional finding of an intrinsic propensity for SHM to generate Arg codons selectively in CDRs, reinforce the view that most IgG autoimmune clones producing prototypical anti-nucleosome antibodies in wild type mice are created by SHM., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life., (© 2015 The Authors.)

Recent advances in mass spectrometry-based proteomic methods have allowed variable (V)-region peptide signatures to be derived from human autoantibodies present in complex serum mixtures. Here, we analysed the clonality and V-region composition of immunoglobulin (Ig) proteomes specific for the immunodominant SmD protein subunit of the lupus-specific Sm autoantigen. Precipitating SmD-specific IgGs were eluted from native SmD-coated ELISA plates preincubated with sera from six patients with systemic lupus erythematosus (SLE) positive for anti-Sm/RNP. Heavy (H)- and light (L)-chain clonality and V-region sequences were analysed by 2-dimensional gel electrophoresis and combined de novo database mass spectrometric sequencing. SmD autoantibody proteomes from all six patients with SLE expressed IgG1 kappa restricted clonotypes specified by IGHV3-7 and IGHV1-69 H-chains and IGKV3-20 and IGKV2-28 L-chains, with shared and individual V-region amino acid replacement mutations. Clonotypic sharing and restricted V-region diversity of systemic autoimmunity can now be extended from the Ro/La cluster to Sm autoantigen and implies a common pathway of anti-Sm autoantibody production in unrelated patients with SLE., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

LCN2 (Lipocalin 2) is a 25 KD secreted acute phase protein, reported to be a novel regulator of angiogenesis in breast cancer. Up regulation of LCN2 had been observed in multiple cancers including breast cancer, pancreatic cancer and ovarian cancer. However, the role of LCN2 promoter methylation in the formation of microvessels is poorly understood. The aim of this study was to analyze the association of LCN 2 promoter methylation with microvessel formation and tumor cell proliferation in breast cancer patients. The LCN2 promoter methylation status was studied in 64 breast cancer tumors by methylation specific PCR (MSP). Evaluation of microvessel density (MVD) and Ki67 cell proliferation index was achieved by immunohistochemical staining using CD34 and MIB-1 antibodies, respectively. LCN2 promoter unmethylation status was observed in 43 (67.2%) of breast cancer patients whereas LCN2 methylation status was seen in 21 (32.8%). Further, LCN2 promoter unmethylation status was associated with aggressive tumor phenotype and elevated mean MVD in breast cancer patients.

The patients with CD3γ deficiency can present with different clinical findings despite having the same homozygous mutation. We report three new CD3gamma-deficient siblings from a consanguineous family with a combined T-B+NK+ immunodeficiency and their variable clinical and cellular phenotypes despite the same homozygous mutation of the CD3G gene (c.80-1G>C). We also re-evaluate a previously reported non-consanguineous family with two CD3gamma-deficient siblings with the same mutation. The median age at diagnosis was 11 years (14 months-20 years). We found all five patients to display autoimmunity: autoimmune thyroiditis (n = 5), autoimmune haemolytic anaemia (n = 2), immune thrombocytopenia (n = 1), autoimmune hepatitis (n = 1), minimal change nephrotic syndrome (n = 1), vitiligo (n = 1) and positive antinuclear antibodies (n = 3) as well as high IgE (n = 2) and atopic eczema (n = 2). While CD3(+) TCRαβ+T cell percentages were low in all patients, only one had lymphopenia and 3 had CD3(+) T cell lymphopenia. Strikingly, we report frequent and multiple autoimmunity in tested heterozygous carriers in both families (n = 6; in 67%), and frequent autoimmunity in family members not available for testing (n = 5, in 80%). The results suggest that CD3G should be studied as a candidate gene for autoimmunity and that CD3gamma deficiency should be considered among other primary immunodeficiencies with predominantly autoimmune manifestations., (© 2014 John Wiley & Sons Ltd.)

A strong gender bias is seen in many autoimmune diseases including systemic lupus erythematosus (SLE). To investigate the basis for the female preponderance in SLE, we have been studying BALB/c mice in which B cells express the R4A heavy chain of an anti-DNA antibody in association with an endogenous light chain repertoire (R4Atg mice). In unmanipulated mice, approximately 5% of B cells express the R4A transgene. R4Atg mice do not spontaneously develop elevated serum titers of anti-DNA antibodies. Administration of either estradiol (E2) or prolactin (Pr) results in escape from tolerance of autoreactive B cells, expressed as an increase in transgene-expressing B cells and elevated serum titers of anti-DNA antibodies. We previously demonstrated that autoreactive B cells maturing in an estrogenic milieu develop as marginal zone (MZ) B cells; when these same B cells mature in the presence of increased prolactin, they develop as follicular (Fo) B cells. To determine the long term consequence of this differential maturation of DNA-reactive B cells, we treated R4Atg BALB/c mice with E2 or Pr for 6 weeks until serum titers of anti-DNA antibody were high, at which time hormonal exposure was discontinued. In E2-treated mice, the anti-DNA titers remained high even 3 months after discontinuation of hormone exposure. Nascent B cells underwent normal tolerance induction, but existing autoreactive MZ B cells persisted and continued to secrete autoantibody. In contrast, Pr caused only a short-term increase in anti-DNA antibody titers. By 3 months after cessation of hormone treatment, serum anti-DNA antibody titers and B cell subsets were indistinguishable from those in placebo (P) treated mice. These findings suggest that autoantibody responses are sustained for variable lengths of time depending on the B cell subset producing the autoantibodies. This observation may be relevant to understanding the heterogeneous presentation of patients with SLE and to the design of therapies targeting specific B-cell populations in autoimmune disease., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The hallmark of systemic lupus erythematosus (SLE) is the presence of high levels of anti-double-stranded DNA autoantibody (anti-dsDNA) in sera. In addition, pathogen infections coincide frequently with the occurrence of lupus. Our study was designed to investigate the contribution of anti-dsDNA, extracellular and intracellular Toll-like receptors (TLRs), a family of pattern-recognition receptors for sensing invading pathogens, in the pathogenesis of lupus. Although cell surface-expressed TLR4 may promote lupus progression, intracellular nucleic acid-sensing TLR9 plays either stimulatory or protective roles in different murine lupus models. To examine the role of TLR4, TLR9, and anti-dsDNA in SLE, we generated transgenic mice carrying anti-dsDNA antibody transgene and challenged the mice with TLR4- and TLR9-agonists, lipopolysaccharides (LPS), and CpG oligodeoxynucleotide (CpG ODN1826 and 2216), respectively. Splenocytes from these mice were found to secrete higher levels of interleukin-10 (IL-10) and anti-dsDNA when treated with a combination of TLR4 and TLR9 agonists (LPS + CpG). In addition, the transgenic mice were intraperitoneally administered with CpG or combined CpG and LPS to determine whether extracellular TLR4 and intracellular TLR9 activations could affect lupus progression in vivo. It was found that serum levels of anti-dsDNA antibodies and interferon-alpha were higher in CpG + LPS-treated transgenic mice than those in non-transgenic mice. Besides, elevated levels of proteinuria, blood urine nitrogen, and immune complex depositions in kidney were found in treated transgenic mice. Anti-dsDNA and simultaneous activation of surface-expressed TLR4 and endosomal TLR9 are crucial to promote the lupus progression.

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by autoantibodies targeting nucleic acid-associated Ags. The endosomal TLRs TLR7 and TLR9 are critical for generation of Abs targeting RNA- or DNA-associated Ags, respectively. In murine lupus models, deletion of TLR7 limits autoimmune inflammation, whereas deletion of TLR9 exacerbates disease. Whether B cell or myeloid TLR7/TLR9 signaling is responsible for these effects has not been fully addressed. In this study, we use a chimeric strategy to evaluate the effect of B cell-intrinsic deletion of TLR7 versus TLR9 in parallel lupus models. We demonstrate that B cell-intrinsic TLR7 deletion prevents RNA-associated Ab formation, decreases production of class-switched Abs targeting nonnuclear Ags, and limits systemic autoimmunity. In contrast, B cell-intrinsic TLR9 deletion results in decreased DNA-reactive Ab, but increased Abs targeting a broad range of systemic autoantigens. Further, we demonstrate that B cell-intrinsic TLR9 deletion results in increased systemic inflammation and immune complex glomerulonephritis, despite intact TLR signaling within the myeloid compartment. These data stress the critical importance of dysregulated B cell-intrinsic TLR signaling in the pathogenesis of systemic lupus erythematosus.

Purpose: Systemic lupus erythematosus (SLE) patients have anti-nuclear autoantibodies directed against dsDNA and RNA-associated antigens (extractable nuclear antigens; ENA). In this study, we investigated the differences in microRNA (miRNA) expression and its biological implications in SLE patients with distinct autoantibody specificities., Methods: The SLE patients were grouped into three subsets based on the type of autoantibodies present in their sera (anti- ENA+ group with autoantibodies against ENA alone; antidsDNA+ group having autoantibodies against dsDNA only, and anti-ENA+dsDNA+ group having autoantibodies to both dsDNA and ENA). Global miRNA expression profiling was done for each of these three groups using TaqMan® low density miRNA arrays., Results: We report that different sets of miRNAs are dysregulated in SLE patients with different autoantibody specificities. Further, Ingenuity pathway analysis (IPA) software revealed specific biological pathways that were targeted by miRNAs dysregulated in different SLE subsets. Molecules involved in cell cycle and cytoskeleton remodeling were the prime targets of miRNAs dysregulated in anti-ENA+ patients whereas miRNAs dysregulated in anti-dsDNA+ patients were found to be implicated in multiple cytokine signaling pathways. IPA analysis of gene targets of miRNAs commonly dysregulated in all three SLE subsets identified several metabolic-, hormone-, and interferon-related pathways to be affected., Conclusion: The differential miRNA expression in patients with distinct autoantibodies is suggestive of different regulatory mechanisms operating among them. Based on these observations, we are hopeful that this 'sub-grouping' approach could be used to identify other defective processes associated with varying disease manifestations in SLE and may be considered when designing therapeutic interventions.

Broadly HIV-1-neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1-infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1-infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient's plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.

The intracellular tyrosine kinase Lyn mediates inhibitory receptor function in B cells and myeloid cells, and Lyn(-/-) mice spontaneously develop an autoimmune and inflammatory disease that closely resembles human systemic lupus erythematosus. TLR-signaling pathways have been implicated in the production of anti-nuclear Abs in systemic lupus erythematosus and mouse models of it. We used a conditional allele of Myd88 to determine whether the autoimmunity of Lyn(-/-) mice is dependent on TLR/MyD88 signaling in B cells and/or in dendritic cells (DCs). The production of IgG anti-nuclear Abs, as well as the deposition of these Abs in the glomeruli of the kidneys, leading to glomerulonephritis in Lyn(-/-) mice, were completely abolished by selective deletion of Myd88 in B cells, and autoantibody production and glomerulonephritis were delayed or decreased by deletion of Myd88 in DCs. The reduced autoantibody production in mice lacking MyD88 in B cells or DCs was accompanied by a dramatic decrease in the spontaneous germinal center (GC) response, suggesting that autoantibodies in Lyn(-/-) mice may depend on GC responses. Consistent with this view, IgG anti-nuclear Abs were absent if T cells were deleted (TCRβ(-/-) TCRδ(-/-) mice) or if T cells were unable to contribute to GC responses as the result of mutation of the adaptor molecule SAP. Thus, the autoimmunity of Lyn(-/-) mice was dependent on T cells and on TLR/MyD88 signaling in B cells and in DCs, supporting a model in which DC hyperactivity combines with defects in tolerance in B cells to lead to a T cell-dependent systemic autoimmunity in Lyn(-/-) mice.

The Lyn tyrosine kinase regulates inhibitory signaling in B and myeloid cells: loss of Lyn results in a lupus-like autoimmune disease with hyperactive B cells and myeloproliferation. We have characterized the relative contribution of Lyn-regulated signaling pathways in B cells specifically to the development of autoimmunity by crossing the novel lyn(flox/flox) animals with mice carrying the Cre recombinase under the control of the Cd79a promoter, resulting in deletion of Lyn in B cells. The specific deletion of Lyn in B cells is sufficient for the development of immune complex-mediated glomerulonephritis. The B cell-specific Lyn-deficient mice have no defects in early bone marrow B cell development but have reduced numbers of mature B cells with poor germinal centers, as well as increased numbers of plasma and B1a cells, similar to the lyn(-/-) animals. Within 8 mo of life, B cell-specific Lyn mutant mice develop high titers of IgG anti-Smith Ag ribonucleoprotein and anti-dsDNA autoantibodies, which deposit in their kidneys, resulting in glomerulonephritis. B cell-specific Lyn mutant mice also develop myeloproliferation, similar to the lyn(-/-) animals. The additional deletion of MyD88 in B cells, achieved by crossing lyn(flox/flox)Cd79a-cre mice with myd88(flox/flox) animals, reversed the autoimmune phenotype observed in B cell-specific Lyn-deficient mice by blocking production of class-switched pathogenic IgG autoantibodies. Our results demonstrate that B cell-intrinsic Lyn-dependent signaling pathways regulate B cell homeostasis and activation, which in concert with B cell-specific MyD88 signaling pathways can drive the development of autoimmune disease.

Aim: To assess TLR9 expression in systemic lupus erythematosus (SLE) patients, its correlation with disease activity, and impact of TLR9 expression on the response to oral glucocorticoids., Methods: Twenty-five active SLE, 15 inactive, and 15 control subjects were included. Anti-DNA, ANA, C3, C4, and TLR9 mRNA expressions were assessed. Active SLE patients only received oral steroid for 6 weeks. Post therapy, they were classified into steroid sensitive and steroid resistant. Data were reassessed after treatment., Results: SLEDAI, anti-DNA, ANA, and TLR9 expressions were significantly higher in active SLE patients. Based on retrograde analysis, TLR9 expression was significantly higher in steroid-resistant versus steroid-sensitive group before treatment, with no significant difference between them after treatment. There was a significant positive correlation between TLR9 expression and SLEDAI score and anti-DNA and negative correlation with C3 and C4 in all patients., Conclusion: TLR9 may play a role in the pathogenesis of SLE and correlates with the disease activity. Corticosteroids have no effect on TLR9 expression, explaining lack of corticosteroid response in some SLE patients. TLR 9 expression can be used in predicting glucocorticoid response in active SLE patients. New treatment modalities targeting TLR9 expression may be of value in steroid-resistant patients.

Glycosylphosphatidylinositols (GPI) are complex glycolipids that are covalently linked to the C terminus of proteins as a post-translational modification and tether proteins to the plasma membrane. One of the most striking features of GPI-anchored proteins (APs) is their enrichment in lipid rafts. The biosynthesis of GPI and its attachment to proteins occur in the endoplasmic reticulum. In the Golgi, GPI-APs are subjected to fatty acid remodeling, which replaces an unsaturated fatty acid at the sn-2 position of the phosphatidylinositol moiety with a saturated fatty acid. We previously reported that fatty acid remodeling is critical for the enrichment of GPI-APs in lipid rafts. To investigate the biological significance of GPI-AP enrichment in lipid rafts, we generated a PGAP3 knock-out mouse (PGAP3(-/-)) in which fatty acid remodeling of GPI-APs does not occur. We report here that a significant number of aged PGAP3(-/-) mice developed autoimmune-like symptoms, such as increased anti-DNA antibodies, spontaneous germinal center formation, and enlarged renal glomeruli with deposition of immune complexes and matrix expansion. A possible cause for this was the impaired engulfment of apoptotic cells by resident peritoneal macrophages in PGAP3(-/-) mice. Mice with conditional targeting of PGAP3 in either B or T cells did not develop such autoimmune-like symptoms. In addition, PGAP3(-/-) mice exhibited the tendency of Th2 polarization. These data demonstrate that PGAP3-dependent fatty acid remodeling of GPI-APs has a significant role in the control of autoimmunity, possibly by the regulation of apoptotic cell clearance and Th1/Th2 balance.

First homogenous immunoassay for sequence-specific nucleic acid detection is developed. The assay bases on our finding that a fluorophore inserted into a DNA probe instead of one of the internal nucleotides may get protected from fluorescence quenching caused by an anti-fluorophore antibody, if the probe is hybridized with the target sequence. This ensures a positive signal in the antibody presence. The assay enables quantitative detection and may have potential for development of homogenous high-throughput platforms., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: The significance of non-rheumatoid arthritis (RA) autoantibodies in patients with RA is unclear. The aim of this study was to assess associations of autoantibodies with autoimmune risk alleles and with clinical diagnoses from the electronic medical records (EMRs) among RA cases and non-RA controls., Methods: Data on 1,290 RA cases and 1,236 non-RA controls of European genetic ancestry were obtained from the EMRs of 2 large academic centers. The levels of anti-citrullinated protein antibodies (ACPAs), antinuclear antibodies (ANAs), anti-tissue transglutaminase antibodies (AGTAs), and anti-thyroid peroxidase (anti-TPO) antibodies were measured. All subjects were genotyped for autoimmune risk alleles, and the association between number of autoimmune risk alleles present and number of types of autoantibodies present was studied. A phenome-wide association study (PheWAS) was conducted to study potential associations between autoantibodies and clinical diagnoses among RA cases and non-RA controls., Results: The mean ages were 60.7 years in RA cases and 64.6 years in non-RA controls. The proportion of female subjects was 79% in each group. The prevalence of ACPAs and ANAs was higher in RA cases compared to controls (each P < 0.0001); there were no differences in the prevalence of anti-TPO antibodies and AGTAs. Carriage of higher numbers of autoimmune risk alleles was associated with increasing numbers of autoantibody types in RA cases (P = 2.1 × 10(-5)) and non-RA controls (P = 5.0 × 10(-3)). From the PheWAS, the presence of ANAs was significantly associated with a diagnosis of Sjögren's/sicca syndrome in RA cases., Conclusion: The increased frequency of autoantibodies in RA cases and non-RA controls was associated with the number of autoimmune risk alleles carried by an individual. PheWAS of EMR data, with linkage to laboratory data obtained from blood samples, provide a novel method to test for the clinical significance of biomarkers in disease., (Copyright © 2013 by the American College of Rheumatology.)

The formation of antibodies against double-stranded (ds)DNA is considered to be the serologic hallmark of systemic lupus erythematosus (SLE) and anti-dsDNA antibodies play an important role in the pathogenesis of the disease. Anti-dsDNA antibodies from lupus mice and SLE patients arise by somatic mutation. Importantly, autoantibodies in which somatic mutations have been reverted to germ-line V regions did not show any measurable autoreactivity, suggesting that anti-dsDNA autoantibodies develop from non-autoreactive B-cells by somatic hypermutation, presumably during the germinal center reaction. As the random nature of somatic hypermutation will inevitably create autoreactive B cells, self-tolerance checkpoints at the postmutational stage of B cell differentiation have to exist that normally prevent the induction of pathogenic anti-dsDNA specificities. This review summarizes the proposed mechanisms for the generation of anti-dsDNA in murine lupus and in SLE patients.

Slipped-strand DNAs, formed by out-of-register mispairing of repeat units on complementary strands, were proposed over 55 years ago as transient intermediates in repeat length mutations, hypothesized to cause at least 40 neurodegenerative diseases. While slipped-DNAs have been characterized in vitro, evidence of slipped-DNAs at an endogenous locus in biologically relevant tissues, where instability varies widely, is lacking. Here, using an anti-DNA junction antibody and immunoprecipitation, we identify slipped-DNAs at the unstable trinucleotide repeats (CTG)n•(CAG)n of the myotonic dystrophy disease locus in patient brain, heart, muscle and other tissues, where the largest expansions arise in non-mitotic tissues such as cortex and heart, and are smallest in the cerebellum. Slipped-DNAs are shown to be present on the expanded allele and in chromatinized DNA. Slipped-DNAs are present as clusters of slip-outs along a DNA, with each slip-out having 1-100 extrahelical repeats. The allelic levels of slipped-DNA containing molecules were significantly greater in the heart over the cerebellum (relative to genomic equivalents of pre-IP input DNA) of a DM1 individual; an enrichment consistent with increased allelic levels of slipped-DNA structures in tissues having greater levels of CTG instability. Surprisingly, this supports the formation of slipped-DNAs as persistent mutation products of repeat instability, and not merely as transient mutagenic intermediates. These findings further our understanding of the processes of mutation and genetic variation., Competing Interests: The authors have declared that no competing interests exist.

Cross-presentation is important for initiating CTL responses against tumors. Delivery of exogenous Ags to the cross-presentation pathway in dendritic cells (DCs), using a number of different carriers, has been attempted to further understand the mechanisms underlying cross-presentation and to develop therapeutic tumor vaccines. The present study reports a new antigenic carrier molecule: a single-chain V region fragment (scFv) of a nucleic acid-hydrolyzing Ab, 3D8. A fusion protein comprising 3D8 scFv and the CTL epitope OVA(250-264) (chicken OVA aa 250-264) was internalized by DC2.4 DCs and processed via a proteasome-dependent, brefeldin- and cycloheximide-sensitive, chloroquine- and primaquine-insensitive pathway, resulting in loading of the CTL epitope onto H-2K(b). In vivo cross-presentation and cross-priming were efficient, even without adjuvant; injection of mice with 3D8 scFv-OVA(250-264) induced cross-presentation of the CTL epitope by draining lymph node CD11c(+) B7.1(+) MHC class II(high) DCs, elicited a CTL response, and suppressed the growth of tumors expressing the OVA epitope. This report shows that an anti-nucleic acid Ab is used to deliver exogenous Ag to the cross-presentation pathway and inhibit in vivo tumor growth.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the production of autoantibodies against nuclear components. Lyn-deficient mice are an excellent animal model of SLE manifesting clinical, pathological and biochemical features seen in the human disease. They develop autoreactive antibodies, glomerulonephritis and show generalized inflammation, and their B cells have a hyperactive phenotype. Since loss of Lyn confers hyper-activation of phosphoinositide 3-kinase (PI3K) signaling, we studied the effect of down-modulating PI3K in Lyn-deficient mice. We found that heterozygous inactivation of the p110δ isoform of PI3K was sufficient to restrain disease in Lyn-deficient mice, leading to significantly decreased autoantibody development and autoimmune-mediated kidney pathology, and improved survival. Intriguingly, haploinsufficiency of p110δ did not dampen signaling in Lyn-deficient B cells. However, plasma cell numbers, serum immunoglobulin titers, inflammation and T cell signaling and activation were significantly moderated in Lyn(-/-)p110δ(+/KD) mice. Importantly, we have shown that haploinsufficiency of p110δ has minor effects on the B cell compartment per se but leads to significant defects in T cell activation and B cell class-switching. These studies suggest that agents targeting p110δ PI3K need not achieve full blockade of the enzyme to be of great benefit in the treatment of SLE., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: This study was undertaken to determine the molecular characteristics of clonotypic autoantibodies in the sera of patients with primary Sjögren's syndrome (SS). This characterization is hampered by the presence of mixed anti-Ro/La specificities that may conceal clonotypic species. In order to narrow clonotypic diversity, a positive selection step was performed on a peg-like determinant of Ro 60 (termed Ro 60-peg) prior to analysis of the autoantibody proteome., Methods: Monospecific anti-Ro 60-peg IgG were isolated by affinity purification from the sera of 7 patients with primary SS and anti-Ro/La and subjected to 2-dimensional gel electrophoresis and high-resolution orbitrap mass spectrometric sequencing. V regions of heavy and light chains were analyzed by combined database and de novo amino acid sequencing., Results: Proteomic analysis revealed a Ro 60-peg-specific IgG1κ-restricted monoclonal autoantibody that was present in the sera of all patients and specified by a V(H) 3-23 heavy chain paired with a V(κ) 3-20 light chain. The public anti-Ro 60-peg clonotype was specified further by common mutations in the heavy-chain and light-chain complementarity-determining regions. Titers and relative affinities of clonotypic IgG did not vary over the course of the disease., Conclusion: The expression of a Ro 60-reactive public B cell clonotype in a subset of patients with primary SS as a long-lived, class-switched circulating autoantibody implies a common breach of B cell tolerance checkpoints in these patients. The unique heavy chain/light chain signature opens the possibility of tracking the development of a "forbidden" clone against a bona fide systemic autoantigen in human disease., (Copyright © 2011 by the American College of Rheumatology.)

Objective: Anti-U3-RNP, or anti-fibrillarin antibodies (AFA), are detected more frequently among African American (AA) patients with systemic sclerosis (SSc) compared to other ethnic groups and are associated with distinct clinical features. We examined the immunogenetic, clinical, and survival correlates of AFA in a large group of AA patients with SSc., Methods: Overall, 278 AA patients with SSc and 328 unaffected AA controls were enrolled from 3 North American cohorts. Clinical features, autoantibody profile, and HLA class II genotyping were determined. To compare clinical manifestations, relevant clinical features were adjusted for disease duration. Cox proportional hazards regression was used to determine the effect of AFA on survival., Results: Fifty (18.5%) AA patients had AFA. After Bonferroni correction, HLA-DRB1*08:04 was associated with AFA, compared to unaffected AA controls (OR 11.5, p < 0.0001) and AFA-negative SSc patients (OR 5.2, p = 0.0002). AFA-positive AA patients had younger age of disease onset, higher frequency of digital ulcers, diarrhea, pericarditis, higher Medsger perivascular and lower Medsger lung severity indices (p = 0.004, p = 0.014, p = 0.019, p = 0.092, p = 0.006, and p = 0.016, respectively). After adjustment for age at enrollment, AFA-positive patients did not have different survival compared to patients without AFA (p = 0.493)., Conclusion: Our findings demonstrate strong association between AFA and HLA-DRB1*08:04 allele in AA patients with SSc. AA SSc patients with AFA had younger age of onset, higher frequency of digital ulcers, pericarditis and severe lower gastrointestinal involvement, but less severe lung involvement compared to AA patients without AFA. Presence of AFA did not change survival.

Impaired immune functions leading to primary immunodeficiencies often correlate with paradoxical autoimmune complications; patients with hyper-IgM syndromes who are deficient in activation-induced cytidine deaminase (AID), which is required for class-switch recombination and somatic hypermutation, are prone to develop autoimmune diseases. To investigate the impact of AID-deficiency on early B-cell tolerance checkpoints in humans, we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from AID-deficient patients. New emigrant/transitional and mature naive B cells from AID-deficient patients express an abnormal Ig repertoire and high frequencies of autoreactive antibodies, demonstrating that AID is required for the establishment of both central and peripheral B-cell tolerance. In addition, B-cell tolerance was further breached in AID-deficient patients as illustrated by the detection of anti-nuclear IgM antibodies in the serum of all patients. Thus, we identified a major and previously unsuspected role for AID in the removal of developing autoreactive B cells in humans.

Increasing evidence demonstrates that pathological B cells play an essential role in the triggering and development of human systemic lupus erythematosus (SLE). A rational strategy for treating SLE might be to delete B cells thereby suppressing autoimmunity. Commercial monoclonal anti-CD20 antibody is widely used for treatment of B cell-related autoimmune disorders. However its long term use is limited by several factors including short half-life, high cost, and possible side effects of antibody protein therapy. Therefore, we constructed a recombinant adenovirus encoding the murine anti-CD20 antibody gene, and used it to immunize lupus-prone (BWF1) mice. Our data demonstrated that administration of adenovirus encoding the murine anti-CD20 antibody gene generated murine anti-CD20 antibody, which resulted in elimination of B cells in BWF1 mice. In addition, the anti-CD20 reduced serum anti-dsDNA antibody levels, impeded the development of proteinuria and improved the survival of BWF1 mice. These findings suggested that the adenovirus encoding murine anti-CD20 antibody gene might provide an alternative strategy for B cell-mediated diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Pathogenic anti-DNA antibodies expressed in systemic lupus erythematosis bind DNA mainly through electrostatic interactions between the positively charged Arg residues of the antibody complementarity determining region (CDR) and the negatively charged phosphate groups of DNA. The importance of Arg in CDR3 for DNA binding has been shown in mice with transgenes coding for anti-DNA V(H) regions; there is also a close correlation between arginines in CDR3 of antibodies and DNA binding. Codons for Arg can readily be formed by V(D)J rearrangement; thereby, antibodies that bind DNA are part of the preimmune repertoire. Anti-DNAs in healthy mice are regulated by receptor editing, a mechanism that replaces κ light (L) chains compatible with DNA binding with κ L chains that harbor aspartic residues. This negatively charged amino acid is thought to neutralize Arg sites in the V(H). Editing by replacement is allowed at the κ locus, because the rearranged VJ is nested between unrearranged Vs and Js. However, neither λ nor heavy (H) chain loci are organized so as to allow such second rearrangements. In this study, we analyze regulation of anti-DNA H chains in mice that lack the κ locus, κ-/κ- mice. These mice show that the endogenous preimmune repertoire does indeed include a high frequency of antibodies with Arg in their CDR3s (putative anti-DNAs) and they are associated mainly with the editor L chain λx. The editing mechanisms in the case of λ-expressing B cells include L chain allelic inclusion and V(H) replacement.