Different Immunologic Profiles Are Associated With Distinct Clinical Phenotypes in Longitudinally Observed Patients With Systemic Lupus Erythematosus.

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 (Th ) cells in the absence of B cell changes, or who had increased Th 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.

Elevated Levels of Interferon-α Act Directly on B Cells to Breach Multiple Tolerance Mechanisms Promoting Autoantibody Production.

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.

Assessing the Utility of the Montreal Cognitive Assessment in Screening for Cognitive Impairment in Patients With Systemic Lupus Erythematosus.

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.

Association of mycophenolate and azathioprine use with cognitive function in systemic lupus.

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.

Altered Balance of Pro-Inflammatory Immune Cells to T Regulatory Cells Differentiates Symptomatic From Asymptomatic Individuals With Anti-Nuclear Antibodies.

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.

Validation of the automated neuropsychological assessment metrics for assessing cognitive impairment in systemic lupus erythematosus.

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.

Longitudinal relationships between cognitive domains and depression and anxiety symptoms in systemic lupus erythematosus.

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.

Metrics and definitions used in the assessment of cognitive impairment in systemic lupus erythematosus: A systematic review.

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.

Impaired B cell anergy is not sufficient to breach tolerance to nuclear antigen in Vκ8/3H9 lupus-prone mice.

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.

Association of systemic lupus erythematosus (SLE) genetic susceptibility loci with lupus nephritis in childhood-onset and adult-onset SLE.

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.

Prevalence and metric of depression and anxiety in systemic lupus erythematosus: A systematic review and meta-analysis.

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.

Validity Evidence for the Use of Automated Neuropsychologic Assessment Metrics As a Screening Tool for Cognitive Impairment in Systemic Lupus Erythematosus.

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.

A tyrosine sulfation-dependent HLA-I modification identifies memory B cells and plasma cells.

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.

Identification of a neutrophil-related gene expression signature that is enriched in adult systemic lupus erythematosus patients with active nephritis: Clinical/pathologic associations and etiologic mechanisms.

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.

Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins.

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.

Multiple tolerance defects contribute to the breach of B cell tolerance in New Zealand Black chromosome 1 congenic mice.

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 Cell Activation Is Potentiated by Homotypic trans-Ly108 Interactions.

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.

Development, Sensibility, and Validity of a Systemic Autoimmune Rheumatic Disease Case Ascertainment Tool.

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.

IL-10 Production Is Critical for Sustaining the Expansion of CD5+ B and NKT Cells and Restraining Autoantibody Production in Congenic Lupus-Prone Mice.

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.

Lack of Interferon and Proinflammatory Cyto/chemokines in Serologically Active Clinically Quiescent Systemic Lupus Erythematosus.

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.