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Objective: Our objective was to evaluate the development of a systemic autoimmune rheumatic disease (SARD) in undifferentiated and asymptomatic individuals with antinuclear antibodies (ANAs). We comparatively evaluated those who did and did not develop a SARD and fulfillment of classification criteria., Methods: We conducted a cohort study of undifferentiated and asymptomatic patients with ANAs who were assessed for the development of a SARD. The primary outcome was a diagnosis of a SARD over a two-year period. We assessed fulfillment of classification criteria. Risk ratios (RRs) were used to evaluate differences among those who did and did not progress to a SARD., Results: We evaluated 207 asymptomatic ANA-positive or undifferentiated patients, of whom 23 (11%) progressed to a SARD, whereas 187 (89%) did not progress. Progressors developed systemic lupus erythematosus (SLE) (n = 11 [48%]), Sjögren disease (n = 5 [22%]), systemic sclerosis (n = 3 [13%]), rheumatoid arthritis (n = 1 [4%]), and from ANA-positive to undifferentiated connective tissue disease (n = 3 [13%]). Fever (RR 0.89, 95% confidence interval [CI] 0.8-0.93) and antiphospholipid antibodies (RR 0.89, 95% CI 0.87-0.93) occurred less frequently, whereas arthritis (RR 1.74, 95% CI 1.20-2.55) occurred more frequently in progressors. Progressors to SLE had arthritis (91%), whereas none developed delirium, psychosis, or nephritis. Among patients with SLE, 100% fulfilled the EULAR/American College of Rheumatology (ACR) SLE criteria (sensitivity 91.7%, specificity 100%), whereas 73% fulfilled the 1997 ACR SLE criteria (sensitivity 81.8%, specificity 98.9%)., Conclusion: Most undifferentiated/asymptomatic individuals with ANA do not progress to a SARD over a two-year period. SLE progressors appear to have mild disease in the short term. The EULAR/ACR SLE criteria have improved ability to identify those who develop SLE., (© 2024 The Author(s). ACR Open Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Objective: The aim of this study was to determine the immunologic profile associated with disease flares in patients with systemic lupus erythematosus (SLE) and to investigate the clinical significance of any differences observed between patients during and following a flare., Methods: Multiparameter flow cytometry was used to examine 47 immune populations within the peripheral blood of 16 healthy controls, 25 patients with clinically quiescent SLE, and 46 patients with SLE experiencing a flare at baseline and at 6- and 12-month follow-up visits. Unsupervised clustering was used to identify patients with similar immune profiles and to track changes over time. Parametric or nonparametric statistics were used when appropriate to assess the association of cellular phenotypes with clinical and laboratory parameters., Results: Five clusters of patients were identified that variably contained patients with active and quiescent SLE, and that had distinct clinical phenotypes. Patients characterized by increased T peripheral helper, activated B, and age-associated B cells were the most likely to be flaring at baseline, as well as the most likely to remain active or flare over the subsequent year if they acquired or retained this phenotype at follow-up. In contrast, patients who had increased T helper (T h ) cells in the absence of B cell changes, or who had increased T h 1 cells and innate immune populations, mostly developed quiescent SLE on follow-up. A significant proportion of patients with SLE had depletion of many immune populations at flare and only showed increases in these populations post-flare., Conclusion: Cellular phenotyping of patients with SLE reveals several distinct immunologic profiles that may help to stratify patients with regard to prognosis and treatment., (© 2023 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Objective: Elevated levels of serum interferon-α (IFNα) and the disruption of B cell tolerance are central to systemic lupus erythematosus (SLE) immunopathogenesis; however, the relationship between these 2 processes remains unclear. The purpose of this study was to investigate the impact of elevated IFNα levels on B cell tolerance mechanisms in vivo and determine whether any changes observed were due to the direct effect of IFNα on B cells., Methods: Two classical mouse models of B cell tolerance were used in conjunction with an adenoviral vector encoding IFNα to mimic the sustained elevations of IFNα seen in SLE. The role of B cell IFNα signaling, T cells, and Myd88 signaling was determined using B cell-specific IFNα receptor-knockout, CD4+ T cell-depleted, or Myd88-knockout mice, respectively. Flow cytometry, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and cell cultures were used to study the effects of elevated IFNα on the immunologic phenotype., Results: Elevation of serum IFNα disrupts multiple B cell tolerance mechanisms and leads to autoantibody production. This disruption was dependent upon B cell expression of IFNα receptor. Many of the IFNα-mediated alterations also required the presence of CD4+ T cells as well as Myd88, suggesting that IFNα acts directly on B cells to modify their response to Myd88 signaling and their ability to interact with T cells., Conclusion: The results provide evidence that elevated IFNα levels act directly on B cells to facilitate autoantibody production and further highlight the importance of IFN signaling as a potential therapeutic target in SLE., (© 2023 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Objectives: Cognitive dysfunction (CD) is a common manifestation of SLE that can have detrimental consequences for those affected. To date, no treatments have been approved for SLE-CD. This study aims to assess the association of azathioprine (AZA) and mycophenolate (MMF) use with SLE-CD, given that these medications have demonstrated neuroprotective qualities in prior studies., Methods: Consecutive adult SLE patients presenting to a single healthcare center were considered for participation. The ACR neuropsychological battery for SLE was administered to consenting patients at 0, 6 and 12 months. Scores were compared with age- and sex-matched controls. Primary outcome was CD, defined as a z-score ≤-1.5 in two or more cognitive domains. Mixed-effects logistic regression models were constructed to estimate the odds of CD with respect to AZA and MMF use., Results: A total of 300 participants representing 676 patient visits completed the study; 114 (38%) met criteria for CD at baseline. The cumulative AZA dose (g/kg) was associated with reduced odds of CD [odds ratio (OR) 0.76 (95% CI 0.58, 0.98), P = 0.04]. Years of AZA treatment was also associated with reduced odds of CD [OR 0.72 (95% CI 0.54, 0.97), P = 0.03]. MMF use was not associated with CD., Conclusion: AZA use was associated with significantly lower odds of SLE-CD, while MMF use was not. Additional studies are warranted to further investigate the relationship of AZA and SLE-CD., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: Screening for cognitive impairment (CI) in systemic lupus erythematosus (SLE) relies on the American College of Rheumatology (ACR) neuropsychological battery (NB). By studying the concurrent criterion validity, our goal was to assess the Montreal Cognitive Assessment (MoCA) as a screening tool for CI compared to the ACR-NB and to evaluate the added value of the MoCA to the Automated Neuropsychological Assessment Metrics (ANAM)., Methods: A total of 285 adult SLE patients were administered the ACR-NB, MoCA, and ANAM. For the ACR-NB, patients were classified as having CI if there was a Z score of ≤-1.5 in ≥2 domains. The area under the curve (AUC) and sensitivities/specificities were determined. A discriminant function analysis was applied to assess the ability of the MoCA to differentiate between CI, undetermined CI, and non-CI patients., Results: CI was not accurately identified by the MoCA compared to the ACR-NB (AUC of 0.66). Sensitivity and specificity were poor at 50% and 69%, respectively, for the cutoff of 26, and 80% and 45%, respectively, for the cutoff of 28. The MoCA had a low ability to identify CI status. The addition of the MoCA to the ANAM led to improvement on the AUC by only 2.5%., Conclusion: The MoCA does not have adequate concurrent criterion validity to accurately identify CI in patients with SLE. The low specificity of the MoCA may lead to overdiagnosis and concern among patients. Adding the MoCA to the ANAM does not substantially improve the accuracy of the ANAM. These results do not support using the MoCA as a screening tool for CI in patients with SLE., (© 2022 American College of Rheumatology.)

Systemic Autoimmune Rheumatic Diseases (SARDs) are characterized by the production of anti-nuclear antibodies (ANAs). ANAs are also seen in healthy individuals and can be detected years before disease onset in SARD. Both the immunological changes that promote development of clinical symptoms in SARD and those that prevent autoimmunity in asymptomatic ANA + individuals (ANA + NS) remain largely unexplored. To address this question, we used flow cytometry to examine peripheral blood immune populations in ANA + individuals, with and without SARD, including 20 individuals who subsequently demonstrated symptom progression. Several immune populations were expanded in ANA + individuals with and without SARD, as compared with ANA - healthy controls, particularly follicular and peripheral T helper, and antibody-producing B cell subsets. In ANA + NS individuals, there were significant increases in T regulatory subsets and TGF-ß1 that normalized in SARD patients, whereas in SARD patients there were increases in Th2 and Th17 helper cell levels as compared with ANA + NS individuals, resulting in a shift in the balance between inflammatory and regulatory T cell subsets. Patients with SARD also had increases in the proportion of pro-inflammatory innate immune cell populations, such as CD14 + myeloid dendritic cells, and intermediate and non-classical monocytes, as compared to ANA + NS individuals. When comparing ANA + individuals without SARD who progressed clinically over the subsequent 2 years with those who did not, we found that progressors had significantly increased T and B cell activation, as well as increased levels of LAG3 + T regulatory cells and TGF-ß1. Collectively, our findings suggest that active immunoregulation prevents clinical autoimmunity in ANA + NS and that this becomes impaired in patients who progress to SARD, resulting in an imbalance favoring inflammation., 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 © 2022 Gupta, Vanlieshout, Manion, Bonilla, Kim, Muñoz-Grajales, Nassar, Johnson, Hiraki, Ahmad, Touma, Bookman and Wither.)

Objective: We previously demonstrated the utility of the Automated Neuropsychological Assessment Metrics (ANAM) for screening cognitive impairment (CI) in patients with systemic lupus erythematosus (SLE) and developed composite indices for interpreting ANAM results. Our objectives here were to provide further support for the ANAM's concurrent criterion validity against the American College of Rheumatology neuropsychological battery (ACR-NB), identify the most discriminatory subtests and scores of the ANAM for predicting CI, and provide a new approach to interpret ANAM results using Classification and Regression Tree (CART) analysis., Methods: 300 adult SLE patients completed an adapted ACR-NB and ANAM on the same day. As per objectives, six models were built using combinations of ANAM subtests and scores and submitted to CART analysis. Area under the curve (AUC) was calculated to evaluate the ANAM's criterion validity compared to the adapted ACR-NB; the most discriminatory ANAM subtests and scores in each model were selected, and performance of models with the highest AUCs were compared to our previous composite indices; decision trees were generated for models with the highest AUCs., Results: Two models had excellent AUCs of 86 and 89%. Eight most discriminatory ANAM subtests and scores were identified. Both models demonstrated higher AUCs against our previous composite indices. An adapted decision tree was created to simplify the interpretation of ANAM results., Conclusion: We provide further validity evidence for the ANAM as a valid CI screening tool in SLE. The decision tree improves interpretation of ANAM results, enhancing clinical utility.

Objectives: To examine i) the relationship between neuropsychological performance and depression and anxiety over time, and ii) the overlap between classification of cognitive dysfunction, anxiety, and depression in SLE., Methods: 301 patients with SLE were included. Cognition was measured using a modified version of the ACR neuropsychological battery; cognitive dysfunction was defined as z-scores ≤-1.5 on ≥2 domains. Depression and anxiety were measured using the Beck Depression Inventory-II and the Beck Anxiety Inventory, respectively. All measures were assessed at baseline, 6, and 12 months. Their relationships were analyzed using Multiple Factor Analysis (MFA)., Results: Anxiety and depression and neuropsychological performance were stable across time. Factor analysis identified two dimensions explaining 42.2% of the variance in neuropsychological performance. The first dimension (33.1% of the variance) included primarily complex cognitive tests measuring executive function; verbal, visual, and working memory; and complex processing speed. The second dimension (9.1% of the variance) included primarily measures of simple information processing speed or motor dexterity. Anxiety and depression scores were consistently related to the first cognitive dimension. There was substantial overlap in participants classified with cognitive dysfunction and anxiety and depression., Conclusions: Depression and anxiety symptoms in SLE patients are related to a cognitive dimension incorporating memory, executive function and complex processing speed in a stable manner across one year. Many patients with cognitive dysfunction exhibit clinically significant anxiety and depression. Further research should examine whether cognition improves when anxiety and depression are treated and mechanistic links between anxiety and depression and cognitive dysfunction in SLE., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: To review: 1) degree of conformity to the American College of Rheumatology neuropsychological battery (ACR-NB) among studies that used a NB, 2) review definitions of cognitive impairment (CI) from studies that used a NB, and 3) characterize measurement tools used to assess CI in systemic lupus erythematosus (SLE)., Methods: The literature search was conducted in Ovid Medline, Embase, and PsycINFO for articles on CI in adult SLE patients. We reviewed studies that used a NB and compared their tests to the ACR-NB to assess the degree of conformity. Definitions of CI from studies that used a NB were reviewed when sufficient information was available. We reviewed and categorized CI measurement tools into four broad categories: NB, screening, incomplete/mixed batteries, and computerized batteries., Results: Of 8727 references, 118 were selected for detailed review and 97 were included in the final analysis. Of 43 studies that used a NB, none of the studies used the ACR-NB exactly as published. Many studies supplemented with other tests. Overall, there was inconsistent use of ACR-NB tests. Definitions for CI varied, with cut-offs ranging from 1 to 3 standard deviations below normative values on domains/tests varying in type and number. The most frequently used measurement tool for assessing CI in SLE was a NB. Use of screening tests and computerized batteries have also increased over the last decade., Conclusion: The assessment and definition of CI in SLE remains heterogeneous. A consensus meeting to address existing inconsistencies should be considered to harmonize the field of CI in SLE., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Screening for cognitive impairment in systemic lupus erythematosus (SLE) conventionally relies on the American College of Rheumatology (ACR) neuropsychologic battery (NB), which is not universally available. To develop a more accessible screening approach, we assessed validity of the Automated Neuropsychological Assessment Metrics (ANAM). Using the ACR NB as the gold standard for cognitive impairment classification, the objectives were 1) to measure overall discriminative validity of the ANAM for cognitive impairment versus no cognitive impairment, 2) to identify ANAM subtests and scores that best differentiate patients with cognitive impairment from those with no cognitive impairment, and 3) to derive ANAM composite indices and cutoffs., Methods: A total of 211 consecutive adult patients, female and male, with SLE were administered the ANAM and ACR NB. 1) For overall discriminative validity of the ANAM, we compared patients with cognitive impairment versus those with no cognitive impairment on 4 scores. 2) Six ANAM models using different scores were developed, and the most discriminatory subtests were selected using logistic regression analyses. The area under the receiver operating characteristic curve (AUC) was calculated to establish ANAM validity against the ACR NB. 3) ANAM composite indices and cutoffs were derived for the best models, and sensitivities and specificities were calculated., Results: Patients with no cognitive impairment performed better on most ANAM subtests, supporting ANAM's discriminative validity. Cognitive impairment could be accurately identified by selected ANAM subtests with top models, demonstrating excellent AUCs of 81% and 84%. Derived composite indices and cutoffs demonstrated sensitivity of 78-80% and specificity of 70%., Conclusion: This study provides support for ANAM's discriminative validity for cognitive impairment and utility for cognitive screening in adult SLE. Derived composite indices and cutoffs enhance clinical applicability., (© 2020, American College of Rheumatology.)

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.

Objectives: To systematically review and synthesize literature on 1) the overall prevalence of depression and anxiety in SLE patients in identified studies, and 2) the pooled prevalence per metrics of depression and anxiety in adult SLE patients., Methods: This review used (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) PRISMA guidelines and in-depth searches in four databases (1954-2016; Ovid-based Medline, Embase, PsycINFO and CINAHL) to identify articles on the prevalence of depression and/or anxiety in adult SLE patients. Included studies were critically appraised and analyzed. The prevalence of depression and anxiety was studied for all included studies, and whenever possible, pooled prevalence (PP) was determined for more commonly used metrics. Statistical and publication bias was assessed using funnel plots., Result: A total of 3103 references were identified, 226 were selected for detailed review and 72 were included in the final analysis., Overall Prevalence: The depression PP, obtained from 69 studies representing 23,386 SLE patients, was 35.0% (95% CI: 29.9%-40.3%). The anxiety PP, obtained from 38 studies representing 4439 SLE patients, was 25.8% (95% CI: 19.2%-32.9%)., Prevalence per Metrics Used: The more commonly used instruments included the Centre for Epidemiological Studies - Depression (CES-D), Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), Hospital Anxiety and Depression Scales (HADS-A/D), and Hamilton Rating Scales for Depression/Anxiety (HAM-D/A)]. The CES-D was utilized in 13 studies including 1856 SLE patients; depression PP was 41.5% (95% CI: 35.1%-48.1%). The BDI was utilized in 14 studies including 1355 SLE patients and the BAI in 3 studies including 489 patients; depression PP was 39.9% (95% CI: 31.1%-49.1) and anxiety PP was 38.4% (95% CI: 34.2%-42.8%). The HADS-D was utilized in 14 studies including 1238 SLE patients and the HADS-A in 12 studies including 1099 patients respectively; its depression PP was 24.4% (95% CI: 19.1%-30.1%) and anxiety PP was 38.3% (95% CI: 29.1%-47.9%). The HAM-D was utilized in 4 studies including 267 SLE patients and the HAM-A in 4 studies including 213 patients respectively; its depression PP was 40.0% (95% CI: 23.0%-59.0%) and anxiety PP was 39.0% (95% CI: 32.0%-45.0%)., Conclusion: There was high variability in the prevalence of depression and anxiety, ranging from 8.7%-78.6% and 1.1%-71.4%, respectively. This could be attributed to the lack of consistency in the metrics used and its definition for depression and anxiety in SLE. Studies that used a specific metric, such as the CES-D, BDI or HAM-D, yielded similar depression prevalence. The HADS-D had the lowest prevalence. All metrics of anxiety yielded similar anxiety prevalence., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: LN is one of the most common and severe manifestations of SLE. Our aim was to test the association of SLE risk loci with LN risk in childhood-onset SLE (cSLE) and adult-onset SLE (aSLE)., Methods: Two Toronto-based tertiary care SLE cohorts included cSLE (diagnosed <18 years) and aSLE patients (diagnosed ⩾18 years). Patients met ACR and/or SLICC SLE criteria and were genotyped on the Illumina Multi-Ethnic Global Array or Omni1-Quad arrays. We identified those with and without biopsy-confirmed LN. HLA and non-HLA additive SLE risk-weighted genetic risk scores (GRSs) were tested for association with LN risk in logistic models, stratified by cSLE/aSLE and ancestry. Stratified effect estimates were meta-analysed., Results: Of 1237 participants, 572 had cSLE (41% with LN) and 665 had aSLE (30% with LN). Increasing non-HLA GRS was significantly associated with increased LN risk [odds ratio (OR) = 1.26; 95% CI 1.09, 1.46; P = 0.0006], as was increasing HLA GRS in Europeans (OR = 1.55; 95% CI 1.07, 2.25; P = 0.03). There was a trend for stronger associations between both GRSs and LN risk in Europeans with cSLE compared with aSLE. When restricting cases to proliferative LN, the magnitude of these associations increased for both the non-HLA (OR = 1.30; 95% CI 1.10, 1.52; P = 0.002) and HLA GRS (OR = 1.99; 95% CI 1.29, 3.08; P = 0.002)., Conclusion: We observed an association between known SLE risk loci and LN risk in children and adults with SLE, with the strongest effect observed among Europeans with cSLE. Future studies will include SLE-risk single nucleotide polymorphisms specific to non-European ancestral groups and validate findings in an independent cohort., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Memory B cells and plasma cells are antigen-experienced cells tasked with the maintenance of humoral protection. Despite these prominent functions, definitive cell surface markers have not been identified for these cells. We report here the isolation and characterization of the monoclonal variable lymphocyte receptor B (VLRB) N8 antibody from the evolutionarily distant sea lamprey that specifically recognizes memory B cells and plasma cells in humans. Unexpectedly, we determined that VLRB N8 recognizes the human leukocyte antigen-I (HLA-I) antigen in a tyrosine sulfation-dependent manner. Furthermore, we observed increased binding of VLRB N8 to memory B cells in individuals with autoimmune disorders multiple sclerosis and systemic lupus erythematosus. Our study indicates that lamprey VLR antibodies uniquely recognize a memory B cell- and plasma cell-specific posttranslational modification of HLA-I, the expression of which is up-regulated during B cell activation.

Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Both a lack of biomarkers and relatively ineffective treatments constitute impediments to management of lupus nephritis (LN). Here we used gene expression microarrays to contrast the transcriptomic profiles of active SLE patients with and without LN to identify potential biomarkers for this condition. RNA isolated from whole peripheral blood of active SLE patients was used for transcriptomic profiling and the data analyzed by linear modeling, with corrections for multiple testing. Results were validated in a second cohort of SLE patients, using NanoString technology. The majority of genes demonstrating altered transcript abundance between patients with and without LN were neutrophil-related. Findings in the validation cohort confirmed this observation and showed that levels of RNA abundance in renal remission were similar to active patients without LN. In secondary analyses, RNA abundance correlated with disease activity, hematuria and proteinuria, but not renal biopsy changes. As abundance levels of the individual transcripts correlated strongly with each other, a composite neutrophil score was generated by summing all levels before examining additional correlations. There was a modest correlation between the neutrophil score and the blood neutrophil count, which was largely driven by the dose of glucocorticosteroids and not the proportion of low density and/or activated neutrophils. Analysis of longitudinal data revealed no correlation between baseline neutrophil score or changes over the first year of follow-up with subsequent renal flare or treatment outcomes, respectively. The findings argue that although the neutrophil score is associated with LN, its clinical utility as a biomarker may be limited.

Systemic lupus erythematosus (SLE) is an autoimmune disease with marked gender and ethnic disparities. We report a large transancestral association study of SLE using Immunochip genotype data from 27,574 individuals of European (EA), African (AA) and Hispanic Amerindian (HA) ancestry. We identify 58 distinct non-HLA regions in EA, 9 in AA and 16 in HA (∼50% of these regions have multiple independent associations); these include 24 novel SLE regions (P<5 × 10 -8 ), refined association signals in established regions, extended associations to additional ancestries, and a disentangled complex HLA multigenic effect. The risk allele count (genetic load) exhibits an accelerating pattern of SLE risk, leading us to posit a cumulative hit hypothesis for autoimmune disease. Comparing results across the three ancestries identifies both ancestry-dependent and ancestry-independent contributions to SLE risk. Our results are consistent with the unique and complex histories of the populations sampled, and collectively help clarify the genetic architecture and ethnic disparities in SLE.

Lupus is characterized by a loss of B cell tolerance leading to autoantibody production. In this study, we explored the mechanisms underlying this loss of tolerance using B6 congenic mice with an interval from New Zealand Black chromosome 1 (denoted c1(96-100)) sufficient for anti-nuclear antibody production. Transgenes for soluble hen egg white lysozyme (sHEL) and anti-HEL immunoglobulin were crossed onto this background and various tolerance mechanisms examined. We found that c1(96-100) mice produced increased levels of IgM and IgG anti-HEL antibodies compared to B6 mice and had higher proportions of germinal center B cells and long-lived plasma cells, suggesting a germinal center-dependent breach of B cell anergy. Consistent with impaired anergy induction, c1(96-100) double transgenic B cells showed enhanced survival and CD86 upregulation. Hematopoietic chimeric sHEL mice with a mixture of B6 and c1(96-100) HEL transgenic B cells recapitulated these results, suggesting the presence of a B cell autonomous defect. Surprisingly, however, there was equivalent recruitment of B6 and c1(96-100) B cells into germinal centers and differentiation to splenic plasmablasts in these mice. In contrast, there were increased proportions of c1(96-100) T follicular helper cells and long-lived plasma cells as compared to their B6 counterparts, suggesting that both B and T cell defects are required to breach germinal center tolerance in this model. This possibility was further supported by experiments showing an enhanced breach of anergy in double transgenic mice with a longer chromosome 1 interval with additional T cell defects.

Invariant NKT (iNKT) cells are innate lymphocytes that respond to glycolipids presented by the MHC class Ib molecule CD1d and are rapidly activated to produce large quantities of cytokines and chemokines. iNKT cell development uniquely depends on interactions between double-positive thymocytes that provide key homotypic interactions between signaling lymphocyte activation molecule (SLAM) family members. However, the role of SLAM receptors in the differentiation of iNKT cell effector subsets and activation has not been explored. In this article, we show that C57BL/6 mice containing the New Zealand Black Slam locus have profound alterations in Ly108, CD150, and Ly9 expression that is associated with iNKT cell hyporesponsiveness. This loss of function was only apparent when dendritic cells and iNKT cells had a loss of SLAM receptor expression. Using small interfering RNA knockdowns and peptide-blocking strategies, we demonstrated that trans -Ly108 interactions between dendritic cells and iNKT cells are critical for robust activation. LY108 costimulation similarly increased human iNKT cell activation. Thus, in addition to its established role in iNKT cell ontogeny, Ly108 regulates iNKT cell function in mice and humans., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Objective: Case ascertainment through self-report is a convenient but often inaccurate method to collect information. The purposes of this study were to develop, assess the sensibility, and validate a tool to identify cases of systemic autoimmune rheumatic diseases (SARD) in the outpatient setting., Methods: The SARD tool was administered to subjects sampled from specialty clinics. Determinants of sensibility - comprehensibility, feasibility, validity, and acceptability - were evaluated using a numeric rating scale from 1-7. Comprehensibility was evaluated using the Flesch Reading Ease and the Flesch-Kincaid Grade Level. Self-reported diagnoses were validated against medical records using Cohen's κ statistic., Results: There were 141 participants [systemic lupus erythematosus (SLE), systemic sclerosis (SSc), rheumatoid arthritis, Sjögren syndrome (SS), inflammatory myositis (polymyositis/dermatomyositis; PM/DM), and controls] who completed the questionnaire. The Flesch Reading Ease score was 77.1 and the Flesch-Kincaid Grade Level was 4.4. Respondents endorsed (mean ± SD) comprehensibility (6.12 ± 0.92), feasibility (5.94 ± 0.81), validity (5.35 ± 1.10), and acceptability (3.10 ± 2.03). The SARD tool had a sensitivity of 0.91 (95% CI 0.88-0.94) and a specificity of 0.99 (95% CI 0.96-1.00). The agreement between the SARD tool and medical record was κ = 0.82 (95% CI 0.77-0.88). Subgroup analysis by SARD found κ coefficients for SLE to be κ = 0.88 (95% CI 0.79-0.97), SSc κ = 1.0 (95% CI 1.0-1.0), PM/DM κ = 0.72 (95% CI 0.49-0.95), and SS κ = 0.85 (95% CI 0.71-0.99). The screening questions had sensitivity ranging from 0.96 to 1.0 and specificity ranging from 0.88 to 1.0., Conclusion: This SARD case ascertainment tool has demonstrable sensibility and validity. The use of both screening and confirmatory questions confers added accuracy.

The development and progression of systemic lupus erythematosus is mediated by the complex interaction of genetic and environmental factors. To decipher the genetics that contribute to pathogenesis and the production of pathogenic autoantibodies, our lab has focused on the generation of congenic lupus-prone mice derived from the New Zealand Black (NZB) strain. Previous work has shown that an NZB-derived chromosome 4 interval spanning 32 to 151 Mb led to expansion of CD5+ B and Natural Killer T (NKT) cells, and could suppress autoimmunity when crossed with a lupus-prone mouse strain. Subsequently, it was shown that CD5+ B cells but not NKT cells derived from these mice could suppress the development of pro-inflammatory T cells. In this paper, we aimed to further resolve the genetics that leads to expansion of these two innate-like populations through the creation of additional sub-congenic mice and to characterize the role of IL-10 in the suppression of autoimmunity through the generation of IL-10 knockout mice. We show that expansion of CD5+ B cells and NKT cells localizes to a chromosome 4 interval spanning 91 to 123 Mb, which is distinct from the region that mediates the majority of the suppressive phenotype. We also demonstrate that IL-10 is critical to restraining autoantibody production and surprisingly plays a vital role in supporting the expansion of innate-like populations.

Systemic lupus erythematosus (SLE) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies (GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry, constituting a new GWAS, a meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including ten new associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis-acting eQTL effects in a range of ex vivo immune cells. We found an over-representation (n = 16) of transcription factors among SLE susceptibility genes. This finding supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE.

Objective: Serologically active clinically quiescent (SACQ) patients with systemic lupus erythematosus (SLE) remain clinically quiescent for prolonged periods despite anti-dsDNA antibodies and/or low complements, indicating the presence of immune complexes. The immune mechanisms leading to this quiescence are unknown. However, in addition to activating complement, immune complex uptake by various cells leads to the production of interferon (IFN)-α and other proinflammatory factors that are also involved in tissue damage. Here we investigate whether production of these factors is reduced in SACQ patients., Methods: The levels of 5 IFN-induced genes and 19 cyto/chemokines were measured in SACQ patients and were compared with those in serologically and clinically active (SACA) and serologically and clinically quiescent (SQCQ) patients. SACQ and SQCQ were defined as ≥ 2 years without clinical activity, with/without persistent serologic activity, respectively, and off corticosteroids/immunosuppressives. SACA was defined as disease activity compelling immunosuppression. Levels of OAS1, IFIT1, MX1, LY6E, and ISG15 were measured by quantitative real-time polymerase chain reaction (PCR) and a composite score (IFN-5) derived from this. Plasma cyto/chemokines were measured by Luminex assay. Nonparametric univariate and logistic regression analyses were conducted., Results: There were no differences in gene expression or cyto/chemokine levels between SACQ and SQCQ patients. The SACQ IFN-5 score was significantly lower than that of SACA (p = 0.003) and was driven by SACQ status, not by autoantibody profile or disease duration. Levels of granulocyte-macrophage colony-stimulating factor, interleukin (IL) 6, IL-10, IFN-γ-inducible protein 10, monocyte chemoattractant protein 1, and tumor necrosis factor-α were significantly lower in SACQ than SACA., Conclusion: The levels of proinflammatory factors in SACQ mirror those of SQCQ patients, indicating reduced production of these factors despite the presence of immune complexes.

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.)

Systemic lupus erythematosus is a complex autoimmune disorder characterized by the production of pathogenic anti-nuclear antibodies. Previous work from our laboratory has shown that the introgression of a New Zealand Black-derived chromosome 4 interval onto a lupus-prone background suppresses the disease. Interestingly, the same genetic interval promoted the expansion of both Natural Killer T- and CD5(+) B cells in suppressed mice. In this study, we show that ablation of NKT cells with a CD1d knockout had no impact on either the suppression of lupus or the expansion of CD5(+) B cells. On the other hand, suppressed mice had an expanded population of IL-10-producing B cells that predominantly localized to the CD5(+)CD1d(low) compartment. The expansion of CD5(+) B cells negatively correlated with the frequency of pro-inflammatory IL-17 A-producing T-cells and kidney damage. Adoptive transfer with a single injection of total B cells with an enriched CD5(+) compartment reduced the frequency of memory/activated, IFNγ-producing, and IL-17 A-producing CD4 T-cells but did not significantly reduce autoantibody levels. Taken together, these data suggest that the expansion of CD5(+) IL-10-producing B cells and not NKT cells protects against lupus in these mice, by limiting the expansion of pro-inflammatory IL-17 A- and IFNγ-producing CD4 T-cells.

Previous studies suggest that the B cells of patients with Systemic Lupus Erythematosus (SLE) are hyper-responsive to BCR crosslinking; however, it has been unclear whether this is the result of altered B cell signaling or differences in various B cell subpopulations in SLE patients as compared to healthy controls. Here we have developed a novel Phosflow technique that permits examination of cell signaling in distinct B cell subpopulations stratified based upon developmental stage and cell surface IgM levels, which we use to show that the naïve B cells of SLE patients are hyper-responsive to IgM receptor crosslinking, resulting in increased SYK phosphorylation. We further demonstrate that this hyper-responsiveness is most marked in the transitional B cell subset and that it is associated with altered function, resulting in decreased apoptosis and increased proliferation of these cells. Examination of repeated samples from the same patients revealed that the hyper-responsiveness fluctuated over time, suggesting that it may be mediated by pro-inflammatory factors rather than genetic variations between patients. In support of this concept, incubation of healthy control B cells with IFN-α or SLE plasma induced the hyper-responsive phenotype, which was blocked by anti-IFN-α antibody. Furthermore, no obvious correlation was seen between genetic variants that are proposed to alter BCR signaling and the increased SYK phosphorylation. The findings suggest that pro-inflammatory factors, in particular Type I IFNs, modulate B cell function in SLE in a way that could contribute to the breach of tolerance in this condition., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: The occurrence of monoclonal gammopathy of undetermined significance (MGUS) is common in chronic immune mediated disorders. This increased monoclonal antibody production could result from chronic stimulation of lymphocytes, with the immunoglobulin G (IgG) subtype accounting for the majority of cases in psoriatic arthritis (PsA). We aimed to identify IgG subclass profiles in patients with PsA and to determine association with specific disease characteristics., Methods: Serum samples from 221 patients with PsA from a single cohort were analyzed for their serum IgG subclass levels. All patients fulfilled the ClASsification for Psoriatic ARthritis (CASPAR) criteria and were followed at 6-month to 12-month intervals according to a standard protocol. MGUS was defined as the occurrence of a discrete band in the gammaglobulin region on at least 2 separate serum protein electrophoresis tests performed 6 months apart. Patients with high abnormal IgG subclass levels were compared to patients with normal levels using descriptive tests., Results: Elevations of IgG1-4 were common in PsA, with ∼20%-49% of patients having elevations of each subclass, IgG2 being the most common subclass abnormality. However, no clinical-serological correlation was found in the group with abnormal IgG2 levels. Of the 38 patients with MGUS, elevations in IgG1 were most common. Patients with an abnormal IgG1 subclass level were more likely to have a discrete band in the gammaglobulin region, higher prevalence of MGUS, and abnormal erythrocyte sedimentation rate or C-reactive protein levels., Conclusion: Determination of the IgG subclass concentration in PsA did not seem to add any significant value in identifying specific disease manifestations. However, this study provides insight into the pathological process leading to MGUS in PsA.

Autoimmune disease is a critical health concern, whose etiology remains enigmatic. We hypothesized that immune responses to somatically mutated self proteins could have a role in the development of autoimmune disease. IFN-γ secretion by T cells stimulated with mitochondrial peptides encoded by published mitochondrial DNA was monitored to test the hypothesis. Human peripheral blood mononuclear cells (PBMCs) of healthy controls and autoimmune patients were assessed for their responses to the self peptides and mutated-self peptides differing from self by one amino acid. None of the self peptides but some of the mutated-self peptides elicited an immune response in healthy controls. In some autoimmune patients, PBMCs responded not only to some of the mutated-self peptides, but also to some of the self peptides, suggesting that there is a breach of self-tolerance in these patients. Although PBMCs from healthy controls failed to respond to self peptides when stimulated with self, the mutated-self peptide could elicit a response to the self peptide upon re-stimulation in vitro, suggesting that priming with mutated-self peptides elicits a cross-reactive response with self. The data raise the possibility that DNA somatic mutations are one of the events that trigger and/or sustain T cell responses in autoimmune diseases., (Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

We have previously shown that B6 congenic mice with a New Zealand Black chromosome 1 (c1) 96-100 cM interval produce anti-nuclear Abs and that at least two additional genetic loci are required to convert this subclinical disease to fatal glomerulonephritis in mice with a c1 70-100 cM interval (c1(70-100)). Here we show that the number of T follicular helper and IL-21-, IFN-γ-, and IL-17-secreting CD4(+) T cells parallels disease severity and the number of susceptibility loci in these mice. Immunization of pre-autoimmune mice with OVA recapitulated these differences. Differentiation of naïve T cells in-vitro under polarizing conditions and in-vivo following adoptive transfer of OVA-specific TCR transgenic cells into c1(70-100) or B6 recipient mice, revealed T cell functional defects leading to increased differentiation of IFN-γ- and IL-17-producing cells in the 96-100 cM and 88-96 cM intervals, respectively. However, in-vivo enhanced differentiation of pro-inflammatory T cell subsets was predominantly restricted to c1(70-100) recipient mice, which demonstrated altered dendritic cell function, with increased production of IL-6 and IL-12. The data provide support for the role of pro-inflammatory T cells in the conversion of subclinical disease to fatal autoimmunity and highlight the importance of synergistic interactions between individual susceptibility loci in this process.

Systemic lupus erythematosus is a chronic multi-organ autoimmune disease marked mainly by the production of anti-nuclear antibodies. Nuclear antigens become accessible to the immune system following apoptosis and defective clearance of apoptotic debris has been shown in several knockout mouse models to promote lupus. However, genetic loci associated with defective clearance are not well defined in spontaneously arising lupus models. We previously showed that introgression of the chromosome 13 interval from lupus-prone New Zealand Black (NZB) mice onto a non-autoimmune B6 genetic background (B6.NZBc13) recapitulated many of the NZB autoimmune phenotypes. Here, we show that B6.NZBc13 mice have impaired clearance of apoptotic debris by peritoneal and tingible-body macrophages and have narrowed down the chromosomal interval of this defect using subcongenic mice with truncated NZB chromosome 13 intervals. This chromosomal region (81-94 Mb) is sufficient to produce polyclonal B- and T-cell activation, and expansion of dendritic cells. To fully recapitulate the autoimmune phenotypes seen in B6.NZBc13 mice, at least one additional locus located in the centromeric portion of the interval is required. Thus, we have identified a novel lupus susceptibility locus on NZB chromosome 13 that is associated with impaired clearance of apoptotic debris.

Genetic loci on New Zealand Black (NZB) chromosomes 1 and 13 play a significant role in the development of lupus-like autoimmune disease. We have previously shown that C57BL/6 (B6) congenic mice with homozygous NZB chromosome 1 (B6.NZBc1) or 13 (B6.NZBc13) intervals develop anti-nuclear antibodies and mild glomerulonephritis (GN), together with increased T and B cell activation. Here, we produced B6.NZBc1c13 bicongenic mice with both intervals, and demonstrate several novel phenotypes including: marked plasmacytoid and myeloid dendritic cell expansion, and elevated IgA production. Despite these changes, only minor increases in anti-nuclear antibody production were seen, and the severity of GN was reduced as compared to B6.NZBc1 mice. Although bicongenic mice had increased levels of baff and tnf-α mRNA in their spleens, the levels of IFN-α-induced gene expression were reduced. Splenocytes from bicongenic mice also demonstrated reduced secretion of IFN-α following TLR stimulation in vitro. This reduction was not due to inhibition by TNF-α and IL-10, or regulation by other cellular populations. Because pDC in bicongenic mice are chronically exposed to nuclear antigen-containing immune complexes in vivo, we examined whether repeated stimulation of mouse pDC with TLR ligands leads to impaired IFN-α production, a phenomenon termed TLR tolerance. Bone marrow pDC from both B6 and bicongenic mice demonstrated markedly inhibited secretion of IFN-α following repeated stimulation with a TLR9 ligand. Our findings suggest that the expansion of pDC and production of anti-nuclear antibodies need not be associated with increased IFN-α production and severe kidney disease, revealing additional complexity in the regulation of autoimmunity in systemic lupus erythematosus.

Lupus susceptibility loci on chromosome 1 have an important role in the development of autoimmunity in the New Zealand Black (NZB) mouse. We have previously shown that C57BL/6 congenic mice with an introgressed homozygous NZB chromosome 1 interval extending from ∼35 to 106 cM develop anti-nuclear antibodies and mild glomerulonephritis. In this study, we produced subcongenic mouse strains to localize the susceptibility loci in this interval and investigate how they promote autoimmunity. Our results indicate at least four susceptibility alleles and a suppressor allele. One allele is located in the 96-100 cM region and is sufficient to breach tolerance to chromatin. Addition of a second locus in the 88-96 cM interval enhances anti-dsDNA antibody production and promotes renal disease, which together with a third susceptibility allele in the 70-88 interval results in significant mortality. We further demonstrate the presence of a suppressor locus in the 35-70 or 100-102 cM interval that abrogates these phenotypes and an additional susceptibility allele in the 102-106 cM interval that restores a milder autoimmune phenotype. Several of these loci alter T-cell function. Thus, there is substantial genetic complexity in the NZB 35-106 cM interval, with disease reflecting a balance between susceptibility and suppressor loci.

Numerous mapping studies have implicated genetic intervals from lupus-prone New Zealand Black (NZB) chromosomes 1 and 4 as contributing to lupus pathogenesis. By introgressing NZB chromosomal intervals onto a non-lupus-prone B6 background, we determined that: NZB chromosome 1 congenic mice (denoted B6.NZBc1) developed fatal autoimmune-mediated kidney disease, and NZB chromosome 4 congenic mice (denoted B6.NZBc4) exhibited a marked expansion of B1a and NKT cells in the surprising absence of autoimmunity. In this study, we sought to examine whether epistatic interactions between these two loci would affect lupus autoimmunity by generating bicongenic mice that carry both NZB chromosomal intervals. Compared with B6.NZBc1 mice, bicongenic mice demonstrated significantly decreased mortality, kidney disease, Th1-biased IgG autoantibody isotypes, and differentiation of IFN-γ-producing T cells. Furthermore, a subset of bicongenic mice exhibited a paucity of CD21(+)CD1d(+) B cells and an altered NKT cell activation profile that correlated with greater disease inhibition. Thus, NZBc4 contains suppressive epistatic modifiers that appear to inhibit the development of fatal NZBc1 autoimmunity by promoting a shift away from a proinflammatory cytokine profile, which in some mice may involve NKT cells.

New Zealand Black (NZB) mice spontaneously develop a lupus-like autoimmune disease. Since CD40-CD40L interactions are important for B cell class-switch recombination and germinal center formation, we sought to understand the impact of these interactions on the immune abnormalities in NZB CD40L gene-deleted (CD40L(-/-)) mice in vivo. NZB.CD40L(-/-) mice demonstrated abrogation of all IgG autoantibodies tested and attenuated kidney disease. However, polyclonal B cell activation in vivo and B cell proliferation and class-switching in response to TLR ligands in vitro were preserved in the absence of CD40L in NZB mice. Although, plasmacytoid dendritic cell expansion and elevated BAFF production were unaffected by the absence of CD40L, there was some evidence that IFN-α-induced gene expression was reduced in the bone marrow of NZB.CD40L(-/-) mice. Our results suggest that CD40-CD40L interactions play an important role in promoting pathogenic IgG autoantibody production and kidney disease in NZB mice., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To compare the healthcare cost and loss of productivity in patients with systemic lupus erythematosus (SLE) with (LN) and without lupus nephritis (lupus nephritis-negative, LNN)., Method: Patients were classified into those with active (ALN and ALNN) and inactive disease (ILN and ILNN). Patients reported on visits to healthcare professionals and use of diagnostic tests, medications, assistive devices, alternative treatments, hospital emergency visits, surgical procedures, and hospitalizations as well as loss of productivity in the 4 weeks preceding enrollment., Results: Enrollment was 141 patients, 79 with LN and 62 LNN. Patients with LN were more likely to visit rheumatologists and nephrologists, undergo diagnostic tests, and had higher costs for medications than patients who were LNN. The annual healthcare cost averaged $CAN 12,597 ± 9946 for patients with LN and $10,585 ± 13,149 for patients who were LNN, a difference of $2012 (95% CI -$2075, $6100). Patients with ALN had more diagnostic tests and surgical procedures, contributing to a significantly higher annual direct cost ($14,224 ± 10,265) compared to patients with ILN ($9142 ± 8419) and a difference of $5082 (95% CI $591, $9573). The healthcare cost was not different between patients with ALNN and patients with ILNN. In patients with LN and patients who were LNN, < 50% were employed and on average missed 6.5-9 days of work per month. The loss of productivity was significantly higher for caregivers of patients with LN than caregivers of patients who were LNN., Conclusion: Healthcare cost and loss of productivity were similar between patients with LN and patients who were LNN; the loss of productivity for caregivers is higher for patients with LN; and the healthcare cost is greater in ALN than in ILN.

Introgression of a New Zealand Black (NZB) chromosome 13 interval onto a C57BL/6 (B6) background (B6.NZBc13) is sufficient to produce many hallmarks of lupus, including high-titre anti-chromatin antibody production, abnormal B- and T-cell activation, and renal disease. In this study we sought to characterize the immune defects leading to these abnormalities. By generating hematopoietic chimeras and BCR transgenic mice, we show that the congenic autoimmune phenotype can be transferred by BM cells and requires the presence of autoreactive B cells. Using the hen egg white lysozyme immunoglobulin transgenic mouse model, we demonstrate that B-cell anergy, deletion, and receptor editing are intact. Nevertheless, congenic B cells exhibit altered peripheral B-cell selection, as demonstrated by enhanced survival and activation of endogenous B cells with autoreactivity to chromatin and Sm/ribonucleoprotein. Given the autoantibody specificities to nuclear antigens, TLR signalling was assessed. B6.NZBc13 B cells were hyper-responsive to poly(I:C), a TLR3 ligand, demonstrating enhanced proliferation and survival as compared to B6 B cells. Our findings indicate the presence of an intrinsic B-cell defect on NZB chromosome 13 that results in hyper-responsiveness to a dsRNA analogue and implicates its potential supporting role in the generation of autoimmunity in B6.NZBc13 mice., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Studies of human inflammatory arthritis would be significantly aided by the development of better animal models. Our hypothesis is that it is possible to develop humanized arthritis models through novel techniques of hematopoietic stem and progenitor cell (HSPC) delivery., Methods: Bone marrow was obtained from patients with osteoarthritis who were undergoing total hip replacement. HSPC were enriched by negative selection and injected into the femur of irradiated anti-CD122 treated nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Human cell engraftment was analyzed by flow cytometry. Arthritis was induced by an intraarticular injection of Chlamydia trachomatis and injected knee joints were examined 5 days later by histology and immunohistochemistry., Results: Human bone marrow HSPC successfully engrafted NOD/SCID mice, with some mice showing up to 90% engrafted human cells. Human B lymphoid and myeloid cells were detected in the bone marrow and spleen 6 weeks following transfer of HSPC, and engrafted recipient mice remained healthy up to 12 weeks postinjection. Chlamydia-injected mice that had been repopulated with HSPC had synovial inflammation, consisting of human neutrophils and macrophages., Conclusion: Bone marrow-derived human HSPC engraft NOD/SCID mice and traffic appropriately to an inflammatory stimulus in the joint, thus offering the potential for direct studies on the immunopathogenesis and treatment of human arthritis.

We used a candidate gene approach to identify a set of SNPs, located in a predicted regulatory region on chromosome 1q44 downstream of NLRP3 (previously known as CIAS1 and NALP3) that are associated with Crohn's disease. The associations were consistently replicated in four sample sets from individuals of European descent. In the combined analysis of all samples (710 father-mother-child trios, 239 cases and 107 controls), these SNPs were strongly associated with risk of Crohn's disease (P(combined) = 3.49 x 10(-9), odds ratio = 1.78, confidence interval = 1.47-2.16 for rs10733113), reaching a level consistent with the stringent significance thresholds imposed by whole-genome association studies. In addition, we observed significant associations between SNPs in the associated regions and NLRP3 expression and IL-1beta production. Mutations in NLRP3 are known to be responsible for three rare autoinflammatory disorders. These results suggest that the NLRP3 region is also implicated in the susceptibility of more common inflammatory diseases such as Crohn's disease.

Systemic lupus erythematosus (SLE) is an autoimmune disease influenced by genetic and environmental factors. We carried out a genome-wide association scan and replication study and found an association between SLE and a variant in TNFAIP3 (rs5029939, meta-analysis P = 2.89 x 10(-12), OR = 2.29). We also found evidence of two independent signals near TNFAIP3 associated with SLE, including one previously associated with rheumatoid arthritis (RA). These results establish that variants near TNFAIP3 contribute to differential risk of SLE and RA.

Polyclonal B cell activation is a well-described feature of systemic lupus erythematosus (SLE), but the immune mechanisms leading to this activation are unclear. To gain insight into these processes, we extensively characterized the activated peripheral blood B cell populations in SLE. PBMC from lupus patients and healthy controls were stained with various combinations of conjugated Ab to identify distinct peripheral B cell subsets, and activation was assessed by measurement of forward scatter and CD80 or CD86 expression using flow cytometry. SLE patients had altered proportions of several B cell subsets, many of which demonstrated increased activation as assessed by forward scatter. This activation occurred at an early developmental stage, as B cells in the transitional (T2) stage were already significantly larger than those seen in controls. Increased proportions of CD80- or CD86-expressing cells were also seen in multiple B cell subsets, with the most striking differences observed in the naive CD27-CD23+ population. Within the CD23+ subset, increased costimulatory molecule expression was most pronounced in an IgD+IgMlow population, suggesting that activation follows Ag engagement. Although controls also had IgD+IgMlowCD23+ cells, they were reduced in number and not activated. Thus, there is an altered response to Ig receptor engagement with self-Ags in lupus.

Polyclonal B cell activation is a hallmark of the immune dysregulation in New Zealand Black (NZB) mice. We have previously shown that the splenic B cell activation is associated with increased CD80 expression. Here we show that abnormal expansions of CD80-expressing GC, CD5(+), marginal zone (MZ) precursor and MZ B cells produce this increase. To investigate the role of BCR engagement in the generation and activation of these populations, a non-self-reactive Ig Tg was introduced onto the NZB background. NZB Ig-Tg mice lacked Tg CD5(+) and peanut agglutinin(+) B cells, confirming the role of endogenous Ag in their selection. Although the increased proportion of MZ B cells was retained in NZB Ig-Tg mice, CD80 expression on these cells was reduced as compared to non-Tg NZB mice, suggesting a role for BCR engagement with endogenous Ag in their activation. Examination of CD40L-knockout NZB mice showed no difference in the abnormal activation or selection of the B cell populations, with the exception of GC cells, as compared to wild-type NZB mice. Thus, polyclonal B cell activation in NZB mice does not require CD40 engagement, but results, in part, from dysregulated BCR-specific mechanisms.

Previous mapping studies have linked New Zealand Black (NZB) chromosome 4 to several lupus traits, including autoantibody production, splenomegaly, and glomerulonephritis. To confirm the presence of these traits, our laboratory introgressed homozygous NZB chromosome 4 intervals extending from either 114 to 149 Mb or 32 to 149 Mb onto the lupus-resistant C57BL/6 background (denoted B6.NZBc4S and B6.NZBc4L, respectively). Characterization of aged cohorts revealed that B6.NZBc4L mice exhibited a striking increase in splenic B1a and NKT cells in the absence of high titer autoantibody production and significant renal disease. Tissue-specific expansion of these subsets was also seen in the peritoneum and liver for B1a cells and in the bone marrow for NKT cells. Staining with CD1d tetramers loaded with an alpha-galactosylceramide analog (PBS57) demonstrated that the expanded NKT cell population was mainly CD1d-dependent NKT cells. The lack of both cellular phenotypes in B6.NZBc4S mice demonstrates that the genetic polymorphism(s) that result in these phenotypes are on the proximal region of NZB chromosome 4. This study confirms the presence of a locus that promotes the expansion of B1a cells and newly identifies a region that promotes CD1d-restricted NKT cell expansion on NZB chromosome 4. Taken together, the data indicate that neither an expansion of B1a cells and/nor NKT cells is sufficient to promote autoantibody production and ultimately, renal disease.

Genetic loci on New Zealand Black (NZB) chromosome 1 play an important role in the development of lupus-like autoimmune disease. We have shown previously that C57BL/6 mice with an introgressed NZB chromosome 1 interval extending from approximately 35 to 106 cM have significantly more severe autoimmunity than mice with a shorter interval extending from approximately 82 to 106 cM. Comparison of the cellular phenotype in these mice revealed that both mouse strains had evidence of increased T cell activation; however, activation was more pronounced in mice with the longer interval. Mice with the longer interval also had increased B cell activation, leading us to hypothesize that there were at least two independent lupus susceptibility loci on chromosome 1. In this study, we have used mixed hemopoietic radiation chimeras to demonstrate that autoimmunity in these mice arises from intrinsic B and T cell functional defects. We further show that a T cell defect, localized to the shorter interval, leads to spontaneous activation of T cells specific for nucleosome histone components. Despite activation of self-reactive T cells in mixed chimeric mice, only chromosome 1 congenic B cells produce anti-nuclear Abs and undergo class switching, indicating impaired B cell tolerance mechanisms. In mice with the longer chromosome 1 interval, an additional susceptibility locus exacerbates autoimmune disease by producing a positive feedback loop between T and B cell activation. Thus, T and B cell defects act in concert to produce and amplify the autoimmune phenotype.

Polyclonal B cell activation is a prominent feature of the lupus-prone New Zealand Black (NZB) mouse strain. We have previously demonstrated linkage between a region on NZB chromosome 13 and increased costimulatory molecule expression on B cells. In this study we have produced C57BL/6 congenic mice with an introgressed homozygous NZB interval extending from approximately 24 to 73 cM on chromosome 13 (denoted B6.NZBc13). We show that B6.NZBc13 female mice not only have enhanced B cell activation but also share many other B cell phenotypic characteristics with NZB mice, including expansion of marginal zone and CD5+ B cell populations, increased numbers of IgM ELISPOTs, and increased serum levels of total IgM and IgM autoantibodies. In addition these mice have increased T cell activation, increased numbers of germinal centers, mild glomerulonephritis, and produce high-titer IgM and IgG anti-chromatin Abs. Male B6.NZBc13 mice have a less pronounced cellular phenotype, lacking expansion of the marginal zone B cell population and IgG anti-chromatin Ab production, indicating the presence of gender dimorphism for this locus. Thus, we have identified a genetic locus that recapitulates with fidelity the B cell phenotypic abnormalities in NZB mice, and we demonstrate that this locus is sufficient to induce an autoimmune phenotype. The data provide further support to the contention that immune abnormalities leading to altered B cell activation and selection contribute to the development of autoimmunity in NZB mice.

The TCR alpha/beta chains recognize antigen peptides bound to the groove of the MHC class II molecule. The crystal structure analyses of the TCR/peptide/MHC class II complexes have revealed that the Valpha chains play a significant role in antigen recognition. However, molecular details which amino acid residues of the Valpha chain are able to contribute to fine antigen specificity are not clearly understood. Previously, we have classified a panel of T hybrids specific for insulin isotypes from different species of animals into four groups based on response profiles to these antigens. In particular, the group III (pork insulin > or = beef insulin hierarchy of responsiveness) and IV (pork insulin >> beef insulin hierarchy of responsiveness) T hybrids are interesting, since these TCR alpha/beta chains with marked different antigen specificities demonstrate identical gene usages and very similar sequences. To specifically address the molecular requirements for insulin recognition by TCR, the TCR alpha and beta chain genes from these group III and IV T hybrids were transfected into 58 alpha-beta- T hybrid. The experiments suggested that CDR3alpha dictates the fine antigen specificity. Then, we have introduced a series of mutations into position 95 of CDR3alpha. The mutation experiments clearly indicated that position 95alpha determines the antigen specificity of the group III and IV T hybrids.

To identify defects in B cell tolerance that may contribute to the production of autoantibodies in New Zealand Black (NZB) mice, we crossed soluble hen egg white lysozyme (sHEL) and anti-HEL Ig transgenes (Ig Tg) onto the NZB background. In this study, we have examined one of the first checkpoints involved in maintenance of peripheral B cell tolerance, follicular exclusion and elimination of self-reactive B cells in the absence of T cell help. Freshly isolated anti-HEL Ig Tg B cells were labeled with CFSE, adoptively transferred into sHEL recipients, and the fate of self-reactive anti-HEL Ig Tg B cells was followed using flow cytometry and immunofluorescence microscopy. Although anti-HEL Ig Tg B cells from NZB mice are appropriately excluded from B cell follicles in NZB sHEL recipient mice, they demonstrate aberrant survival, proliferation, and generation of anti-HEL Ab-producing cells. This abnormal response results from an intrinsic defect in NZB B cells, requires the presence of CD4(+) T cells, and is facilitated by the splenic environment in NZB mice. Thus, NZB mice have immune defects that interact synergistically to allow autoreactive B cells to become activated despite the presence of tolerizing autoantigens.

In previous work, we demonstrated linkage between a broad region on New Zealand Black (NZB) chromosome 1 and increased costimulatory molecule expression on B cells and autoantibody production. In this study, we produced C57BL/6 congenic mice with homozygous NZB chromosome 1 intervals of differing lengths. We show that both B6.NZBc1(35-106) (numbers denote chromosomal interval length) and B6.NZBc1(85-106) mice produce IgG anti-nuclear autoantibodies, but B6.NZBc1(35-106) mice develop significantly higher titers of autoantibodies and more severe renal disease than B6.NZBc1(85-106) mice. Cellular analysis of B6.NZBc1(85-106) mice revealed splenomegaly and increased numbers of memory T cells. In addition to these features, B6.NZBc1(35-106) mice had altered B and T cell activation with increased expression of CD69, and for B cells, costimulatory molecules and MHC. Introduction of an anti-hen egg white lysozyme Ig transgene, as a representative nonself-reactive Ig receptor, onto the B6.NZBc1(35-106) background corrected the B cell activation phenotype and led to dramatic normalization of splenomegaly and T cell activation, but had little impact on the increased proportion of memory T cells. These findings indicate that there are multiple lupus susceptibility genes on NZB chromosome 1, and that although B cell defects play an important role in lupus pathogenesis in these mice, they act in concert with T cell activation defects.