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Evobrutinib is a highly selective, orally administered Bruton's tyrosine kinase (BTK) inhibitor. The objective of this phase II, multicenter, randomized, double-blind, placebo-controlled trial was to evaluate the efficacy and safety of evobrutinib in patients with active autoantibody-positive systemic lupus erythematosus (SLE).Patients were diagnosed with SLE by either the Systemic Lupus International Collaborating Clinics criteria or at least four American College of Rheumatology criteria 6 months or more prior to screening, had an SLE Disease Activity Index-2000 score of 6 or more, were autoantibody-positive and on standard-of-care therapy. Randomization was 1:1:1:1 to oral evobrutinib 25 mg once daily (QD), 75 mg QD, 50 mg twice daily, or placebo. Primary efficacy endpoints were SLE responder index (SRI)-4 response at week 52 and SRI-6 response at week 52 in the high disease activity subpopulation. Safety endpoints included treatment-emergent adverse events (TEAEs).A total of 469 patients were randomized and received at least one dose of evobrutinib or placebo at the time of primary analysis. Mean (SD) age at baseline was 40.7 (±12.3) years; 94.9% of patients were female. Neither primary efficacy endpoint was met. All doses of evobrutinib were well tolerated, and there was no clear dose effect on the incidence of reported TEAEs, or serious TEAEs, including severe infections.This phase II, dose-ranging trial in SLE failed to show a treatment effect of evobrutinib versus placebo at any dose. Evobrutinib was generally well tolerated, with no dose effect observed for TEAEs. These results suggest that BTK inhibition does not appear to be an effective therapeutic intervention for patients with SLE.

Individuals with potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections1–3. Here we hypothesize that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-2 (refs. 4–11), would expand in vivo to support rapid viral control, aborting infection. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication–transcription complex (RTC)12,13, in intensively monitored healthcare workers (HCWs) who tested repeatedly negative according to PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals from before the pandemic (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27, a robust early innate signature of SARS-CoV-2 (ref. 14), suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae., Seronegative healthcare workers with an innate signature of infection preferentially expand pre-existing T cells targeting the conserved replication transcription complex of SARS-CoV-2 in abortive infection.

Objectives To perform an exploratory study to identify a JDM serum metabolic profile that differs from healthy controls (HCs) and responds to immunosuppressive treatment. Methods Blood was collected from 9 HCs and 10 patients diagnosed with probable (n = 4) or definite (n = 6) JDM based on the criteria of Bohan and Peter for myositis, with 7 of the 10 providing longitudinal samples following initiation of treatment; these patients comprised the treatment-naïve cohort. Sera underwent mass spectroscopy–based measurements of targeted metabolic intermediates, including 15 amino acids, 45 acylcarnitines (ACs), 15 ceramides and 29 sphingomyelins. Principal components analysis reduced metabolites into smaller sets of factors each comprised of correlated metabolic intermediates. Factor scores and metabolite concentrations were compared with HCs using two-sample t-tests while treatment effects were evaluated using paired t-tests. Results Of eight principal components analysis–derived metabolite factors (one AC, two amino acids, three sphingosine and two ceramide), two were significantly associated with JDM: one AC factor containing mostly long-chain ACs (P = 0.049) and one ceramide factor (P Conclusion While additional validation is needed, these lipids have potential as JDM serum diagnostic and/or treatment biomarkers. Additionally, the significant association of long-chain ACs and ceramides with JDM offers insights regarding pathogenesis, implicating dysregulation of mitochondrial fatty acid β-oxidation.

Systemic lupus erythematous is a systemic autoimmune disease that can cause severe pain and impair quality of life. Pain in lupus can arise from a variety of mechanisms and is usually assessed in terms of activity and damage. In contrast, categorization of symptoms as type 1 and type 2 manifestations encompasses a broader array of symptoms, including widespread pain, fatigue, and depression that may track together. The categorization of symptoms as type 1 and type 2 manifestations can facilitate communication between patient and provider as well as provide a framework to address more fully the complex symptoms experienced by patients.

Objective The type 1 and type 2 systemic lupus erythematosus (SLE) categorization system was recently proposed to validate the patients' perspective of disease and to capture a more comprehensive spectrum of symptoms. The objective of this study was to characterize the clinical manifestations of SLE subtypes and to determine the correlation between the patient- and physician-reported measures used in the model. Methods This was a cross-sectional study of patients with SLE in a university clinic. Patients completed the Systemic Lupus Activity Questionnaire (SLAQ) and 2011 American College of Rheumatology fibromyalgia (FM) criteria. Active SLE was defined as Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score ≥6, clinical SLEDAI score ≥4, or active lupus nephritis. We identified 4 groups: type 1 SLE (active SLE without FM), type 2 SLE (inactive SLE with FM), mixed SLE (active SLE with FM), and minimal SLE (inactive SLE without FM). Results In this cohort of 212 patients (92% female, mean age 45 years), 30% had type 1 SLE, 8% had type 2 SLE, 13% had mixed SLE, and 49% had minimal SLE. Regardless of SLE disease activity, patients with FM (21%), reported higher SLAQ scores, patient global assessment scores, and self-reported lupus flare that resulted in discordance between patient- and physician-reported measures. Conclusion Fatigue, widespread pain, sleep dysfunction, and mood disorders are common symptoms in SLE. Identifying these symptoms as type 2 SLE may be a method to improve patient communication and understanding. The level of type 2 SLE impacts patients' perception of disease and self-reported symptoms. The SLAQ may need to be reinterpreted based on the FM severity scale.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus, a prototypic autoimmune disease. These antibodies bind to conserved sites on single-stranded and double-stranded DNA and display variable region somatic mutations consistent with antigen selection. Nevertheless, the interaction of anti-DNA with DNA has unconventional features. Anti-DNA antibodies bind by a mechanism called monogamous bivalency, in which stable interaction requires contact of both Fab sites with determinants on the same extended DNA molecule; the size of this DNA can be hundreds to thousands of bases, especially in solid phase assays. This binding also requires the presence of the Fc portion of IgG, a binding mechanism known as Fc-dependent monogamous bivalency. As shown by the effects of ionic strength in association and dissociation assays, anti-DNA binding is primarily electrostatic. Like anti-DNA autoantibodies, anti-DNA antibodies that bind specifically to non-conserved sites on bacterial DNA, a type of anti-DNA found in otherwise healthy individuals, also interact by monogamous bivalency. The unconventional features of anti-DNA antibodies may reflect the highly charged and polymeric nature of DNA and the need for molecular rearrangements to facilitate monogamous bivalency; the Fc portion contributes to binding in an as yet unknown way.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by antinuclear antibodies (ANAs) that form immune complexes that mediate pathogenesis by tissue deposition or cytokine induction. Some ANAs bind DNA or associated nucleosome proteins, whereas other ANAs bind protein components of complexes of RNA and RNA-binding proteins (RBPs). Levels of anti-DNA antibodies can fluctuate widely, unlike those of anti-RBP antibodies, which tend to be stable. Because anti-DNA antibody levels can reflect disease activity, repeat testing is common; by contrast, a single anti-RBP antibody determination is thought to suffice for clinical purposes. Experience from clinical trials of novel therapies has provided a new perspective on ANA expression during disease, as many patients with SLE are ANA negative at screening despite previously testing positive. Because trial results suggest that patients who are ANA negative might not respond to certain agents, screening strategies now involve ANA and anti-DNA antibody testing to identify patients with so-called ‘active, autoantibody-positive SLE’. Evidence suggests that ANA responses can decrease over time because of the natural history of disease or the effects of therapy. Together, these findings suggest that, during established disease, more regular serological testing could illuminate changes relevant to pathogenesis and disease status. Antinuclear antibodies (ANAs), characteristic features of systemic lupus erythematosus (SLE), are a requirement for disease classification and trial enrolment. In this Review, the authors re-examine the role of ANAs in SLE and discuss changing attitudes towards using ANAs as biomarkers.

Personalized medicine is an important goal for the treatment of rheumatic disease that seeks to improve outcomes by matching therapy more precisely with the underlying pathogenetic disturbances in the individual patient. Realization of this goal requires actionable biomarkers to identify these disturbances as well as pathways that can be targeted for novel therapy. Among advances in characterizing pathogenesis, Big Data provides an unprecedented picture of pathogenesis, with analysis of tissue lesions revealing disturbances that may not be apparent in blood. Big Data approaches include single cell RNAseq (scRNAseq) which can elucidate patterns of gene expression by individual cells. Galvanized by the Accelerating Medicines Partnership, a public-private initiative of the NIH, investigative teams have analyzed gene expression in cells in the synovium for rheumatoid arthritis and kidney for systemic lupus erythematosus. A review of basic and translational research for 2018-2019 provides the progress in these areas. Thus, the studies on rheumatoid arthritis have identified subpopulations of immune cells and fibroblasts implicated in synovitis. For lupus, transcriptomic studies have provided evidence for widespread effects of type 1 interferon. Studies in progressive sclerosis have demonstrated changes associated with stem cell therapy as well as potential new targets for anti-fibrotic agents. Other studies using molecular approaches have defined new mechanisms for vasculitis as well as the potential role of the microbiome in inflammatory arthritis and systemic lupus erythematosus. Future studies with Big Data will incorporate the spatial relationships of cells in inflammation as well as changes in gene expression over time.

Objective We developed a model that categorizes systemic lupus erythematosus (SLE) activity into two dimensions: Type 1 SLE consists of inflammatory activity, including arthritis, nephritis, and rashes; Type 2 SLE includes fatigue, myalgia, mood disturbance, and cognitive dysfunction. Patient-reported outcome (PRO) measures have received attention as a way to capture symptomatology of SLE. The objective of this study was to explore the use of existing PRO measures to classify Type 1 and 2 SLE activity. Methods Systemic lupus erythematosus patients completed three questionnaires: Systemic Lupus Activity Questionnaire (SLAQ), Polysymptomatic Distress Scale (PSD), and Patient Health Questionnaire (PHQ-2). SLE Disease Activity Index (SLEDAI) and physician global assessments (PGA; 0–3) for Type 1 and Type 2 activity were also recorded. High Type 1 SLE activity was defined as cSLEDAI ≥4 (scored without labs), SLEDAI ≥6, active nephritis, or Type 1 PGA ≥1.0. High Type 2 SLE activity was defined as Type 2 PGA ≥1.0. Patients with both high Type 1 and 2 activity were defined as Mixed SLE, and patients with low Type 1 and 2 activity were defined as Minimal SLE. Data were reduced with a factor analysis. Using a reduced set of 13 variables, multinomial logistic regression models estimated the probability of Minimal, Type 1, Type 2, and Mixed SLE classification. Results The study included 208 patients with SLE. The model accurately predicted the clinician-based Type 1 and 2 SLE classification in 63% of patients; 73% of patients had their Type 1 SLE activity accurately predicted; and 83% had their Type 2 SLE activity accurately predicted. Performance varied by group: 87% of Minimal patients were correctly predicted to be in the Minimal SLE group, yet only about one-third of patients in the Type 1 group were correctly predicted to be in the Type 1 group. Conclusions Our findings indicate Type 2 SLE activity can be identified by patient-reported data. The use of PROs was not as accurate at predicting Type 1 activity. These findings highlight the challenges of using PROs to categorize and classify SLE symptoms since some manifestations of Type 1 activity (e.g., nephritis) may be essentially clinically silent while other Type 1 manifestations may cause severe symptoms.

DNA is a polymeric macromolecule that can display a variety of backbone conformations. While the classical B-DNA is a right-handed double helix, Z-DNA is a left-handed helix with a zig-zag orientation. The Z conformation depends upon the base sequence, base modification and supercoiling and is considered to be transient. To determine whether the presence of Z-DNA can be detected immunochemically, the binding of monoclonal and polyclonal anti-Z-DNA antibodies to a panel of natural DNA antigens was assessed by an ELISA using brominated poly(dG-dC) as a control for Z-DNA. As these studies showed, among natural DNA tested (Micrococcus luteus, calf thymus, Escherichiacoli, salmon sperm, lambda phage), micrococcal (MC) DNA showed the highest binding with both anti-Z-DNA preparations, and E. coli DNA showed binding with the monoclonal anti-DNA preparation. The specificity for Z-DNA conformation in MC DNA was demonstrated by an inhibition binding assay. An algorithm to identify propensity to form Z-DNA indicated that DNA from Mycobacterium tuberculosis could form Z-DNA, a prediction confirmed by immunoassay. Together, these findings indicate that anti-Z-DNA antibodies can serve as probes for the presence of Z-DNA in DNA of various species origin and that the content of Z-DNA varies significantly among DNA sources.

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

BACKGROUND: Autoimmune diseases comprise a spectrum of illnesses and are on the rise worldwide. Although anti-nuclear antibodies (ANA) are detected in many autoimmune diseases, up to 20% of healthy women are ANA+ and most will never develop clinical symptoms. Further, disease transition is higher among ANA+ African Americans compared to European Americans. OBJECTIVE: To determine the immune features that might define and prevent transition to clinical autoimmunity in ANA+ healthy individuals. METHODS: We comprehensively phenotype immune profiles of African Americans and European Americans who are ANA- healthy, ANA+ healthy, or have systemic lupus erythematosus (SLE) using single cell mass cytometry, next-generation RNA sequencing, multiplex cytokine profiling, and phospho-signaling analyses. RESULTS: We found that SLE patients of both races displayed T cell expansion and elevated expression of Type I and II interferon pathways compared to both ANA- and ANA+ healthy individuals. We discovered a unique immune signature that suggests a suppressive immune phenotype and reduced CD11C+ autoimmunity-associated B cells in healthy ANA+ European Americans that is absent in their SLE or even healthy ANA- counterparts, or among African American cohorts. In contrast, ANA+ healthy African Americans exhibited elevated expression of T cell activation markers and higher plasma levels of IL-6 compared to healthy ANA+ European Americans. CONCLUSIONS: We propose that this novel immune signature identified in ANA+ healthy European Americans protects them from T cell expansion, heightened activation of interferon pathways, and disease transition.

Immune checkpoint inhibitors are novel biologic agents to treat cancer by inhibiting the regulatory interactions that limit T cell cytotoxicity to tumours. Current agents target either CTLA-4 or the PD-1/PD-L1 axis. Because checkpoints may also regulate autoreactivity, immune checkpoint inhibitor therapy is complicated by side effects known as immune-related adverse events (irAEs). The aim of this article is to review the mechanisms of these events. irAEs can involve different tissues and include arthritis and other rheumatic manifestations. The frequency of irAEs is related to the checkpoint inhibited, with the combination of agents more toxic. Because of their severity, irAEs can limit therapy and require immunosuppressive treatment. The mechanisms leading to irAEs are likely similar to those promoting anti-tumour responses and involve expansion of the T cell repertoire; furthermore, immune checkpoint inhibitors can affect B cell responses and induce autoantibody production. Better understanding of the mechanisms of irAEs will be important to improve patient outcome as well as quality of life during treatment.

Objective In the development of novel therapies for systemic lupus erythematosus, antinuclear antibody (ANA) positivity represents a criterion for trial eligibility. Since as many as 30% of patients enrolled in trials have been ANA negative, we evaluated the performance characteristics of immunofluorescence assays (IFAs) for ANA determinations for screening. Methods This study used 5 commercially available IFAs to assess the ANA status of 181 patients enrolled in a phase II clinical trial for an anti-interleukin-6 antibody. Enrollment included a detailed review of medical records to verify a historical ANA value. IFA results were related to various clinical and serologic features at enrollment. Results While the frequency of ANA negativity assessed by the central laboratory was 23.8% in a cohort of 181 patients, the evaluated IFA kits demonstrated frequencies of negativity from 0.6 to 27.6%. With 2 IFA kits showing a significant frequency of ANA negativity, positive and negative samples differed in levels of anti-double-stranded DNA, C3, and presence of other ANAs as well as the frequency of high interferon (IFN) expression. Conclusion These findings indicate that, when used for screening, IFAs can vary because of performance characteristics of kits and thus can affect determination of trial eligibility. With kits producing a significant frequency of ANA negativity, ANA status can be associated with other serologic measures as well as the presence of the IFN signature, potentially affecting responsiveness to a trial agent.

ObjectiveWe have developed a new conceptual model to characterise the signs and symptoms of SLE: the Type 1 and 2 SLE Model. Within the original model, Type 1 SLE consists of inflammatory manifestations like arthritis, nephritis and rashes; Type 2 SLE includes symptoms of fatigue, myalgia, mood disturbance and cognitive dysfunction. Through in-depth interviews, we explored how the Type 1 and 2 SLE Model fits within the lived experience of patients with SLE, with a focus on the connection between Type 1 and Type 2 SLE symptoms.MethodsSemistructured in-depth interviews were conducted among adult participants meeting 1997 American College of Rheumatology or Systemic Lupus International Collaborating Clinics criteria for SLE. Participants were purposefully selected for age, race, sex and nephritis history. All interviews were audio-recorded and transcribed. Data were analysed through episode profile and thematic analysis.ResultsThrough interviews with 42 patients with SLE, two patterns of Type 2 SLE emerged: Intermittent (n=18) and Persistent (n=24). Participants with Intermittent Type 2 SLE described feeling generally well when Type 1 is inactive; these participants were younger and had more internal SLE manifestations. Participants with Persistent Type 2 described always experiencing Type 2 symptoms despite inactive Type 1, although the severity may fluctuate. Participants with Persistent Type 2 SLE experienced traditional lupus symptoms of joint pain, hair loss and rash, but less often had severe organ system involvement.ConclusionsBy listening to the stories of our patients, we found two underlying patterns of Type 2 SLE: Intermittent Type 2 symptoms that resolve in synchrony with Type 1 inflammatory symptoms, and Persistent Type 2 symptoms that continue despite remission of Type 1 symptoms.

We have read with interest the reply by Klareskog et al , ‘ How to communicate in science ’,1 to our editorial, ‘ Pathogenic effector function of …

We have read with interest the comments by Dr Holmdahl1 on our editorial entitled ‘Pathogenic effector functions of ACPA: where do we stand?’2 and thank him for the additional information. We fully agree …

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus. Previous studies have indicated that the phosphodiester backbone is the main antigenic target, with electrostatic interactions important for high avidity. To define further these interactions, the effects of ionic strength on anti-DNA binding of SLE plasmas were assessed in association and dissociation assays by ELISA. As these studies demonstrated, increasing ionic strength to a concentration of 1000 mM NaCl reduced antibody binding although the extent of the reduction varied among samples. In dissociation assays, differences among plasmas were also observed. For one of the plasmas, binding to DNA displayed resistance to dissociation by increasing ionic strength even though these concentrations limited binding in association assays. Time course studies showed a gradual change in binding interactions. These studies indicate that anti-DNA binding can involve both electrostatic and non-electrostatic interactions, with binding in some plasmas showing evidence of hysteresis.

The past two decades have witnessed remarkable advances in the treatment of inflammatory arthritis that have made remission in previously untreatable conditions a realistic goal for many patients. These advances derive from new insights into disease mechanisms; the advent of the biologics and other new therapies; the development of robust measures of disease activity; coherent treatment strategies to guide therapy (ie, treat to target or T2T); and a sufficient supply of rheumatologists to implement the new approaches.1 The advances in rheumatology have been unprecedented and, arguably, the treatment of inflammatory arthritis has progressed faster and further than that of any other serious chronic disease in all of medicine. Indeed, a patient with rheumatoid arthritis (RA) today can lead an essentially normal life, whereas, a few decades ago, such a patient would have a restricted existence and be easily recognised by the appearance of grave illness, wasting and deformity. Along with better outcomes have come challenges in the utilisation of the current armamentarium of disease modifying antirheumatic drugs or DMARDs (table 1). These challenges are, of course, welcome since they signify progress. In the face of literally hundreds to thousands of ways to treat arthritis and the continuing influx of novel agents, they also demand guidance for both providers and patients to establish a treatment plan, recognising potential risks, benefits and costs. The publication of three articles on European League Against Rheumatism (EULAR) recommendations for the treatment of early inflammatory arthritis, RA and axial spondyloarthritis (axSpA) is therefore an important event and provides a much needed perspective and framework for the delivery of best care.2–4 View this table: Table 1 Disease-modifying antirheumatic drugs (DMARDs) Each article represents a remarkable effort by panels of rheumatologists, health professionals and patient representatives to encompass an ever-expanding literature and provide overarching principles as well as specific recommendations. The work …

'Hope’ by Dr Kushboo Sheth is a profound and moving depiction of the inner life of a rheumatologist confronting the COVID-19 viral pandemic. The feelings that Sheth describes are as varied as they are powerful and range from fear to rage to distress. Burdened by the weight of these feelings, she vows to adapt, persevere and trudge onward. Most of all, she strives to get beyond the pandemic, aspiring to be a better person whatever battering and damage may come along the way. The feelings that Sheth describes are inescapable at this time given the upheaval that the pandemic has caused in all aspects of life. Vulnerability is universal. Anyone can infect me and I can infect anyone. Those who can infect and be infected are the same: families, friends, patients, the checkout clerk at the grocery, a random passerby walking too close to you on the sidewalk. In this calculus, infection is a matter of bad luck or, to use a word that has fallen out of fashion, fate. Even if infection does not lead to hospitalisation, it can lead to confinement at home, an isolation tantamount to imprisonment. What I find striking about ‘Hope’ is not the range and depth of Sheth’s feelings but rather the identity of the person who is experiencing them. Sheth is a physician. Physicians by training should be accustomed …

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by antinuclear antibody (ANA) production. ANAs bind to DNA, RNA and complexes of proteins and nucleic acids and are important markers for diagnosis and activity. According to current models, ANAs originate from antigen-driven processes; nevertheless, antibody responses to both DNA and RNA binding proteins display features unexpected in terms of current paradigms for antigenicity. These differences may reflect disturbances in both B and T cells critical for autoreactivity. Clinically, ANA testing has new uses for determining classification as well as assessing eligibility for clinical trials. Studies of patients with established disease show frequent seronegativity. In this setting, seronegativity may indicate a stage of disease called post-autoimmunity in which the natural history of disease or effects of immunosuppressive therapies modifies responses. The new uses of ANA testing highlight the importance of understanding autoantigenicity and developing sensitive and informative assays for clinical assessments.

Immune checkpoint inhibitors (ICI) are a new class of biological agents that has revolutionized the treatment of cancer. Unlike conventional cytotoxic agents that are designed to kill cells, ICI block interactions that regulate the T cell response to cancer. At present, two classes of ICI are available to treat a wide variety of malignancies: antibodies to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and antibodies directed to the programmed death-1 (PD-1) and PD-1 ligand (PD-L1) axis, either to PD-1 or PD-L1. While both anti-CTLA-4 and antibodies to PD-1/PD-L1 are effective, these systems differ in the T cell populations affected, the location of these cells and the downstream signaling pathways involved. ICI can be used alone or in combination. Despite differences in mechanism of action, treatment with both types of ICI is associated with severe side effects that have been termed immune-related adverse reactions (irAE). irAE include, among other manifestations, dermatitis, colitis, pneumonitis, endocrinopathy (hypothyroidism, hypophysitis, adrenal insufficiency) and arthritis and related conditions. Because of the goal of ICI therapy is the induction or stimulation of cytotoxic T cells, the effect of these agents on B cells has received much less attention. In general, effects of ICI on tumors are considered the action of T cells. irAE, however, may result from B cell effects and the induction of autoantibodies. Thus, autoantibodies to thyroid antigens may lead to thyroiditis and subsequent hypothyroidism. Similarly, ICI can lead to the production of antibodies to islet antigens as well as glutamic acid decarboxylase 65 and induction of diabetes. On the other hand, while arthritis can be an irAE, affected patients usually do not show antibodies to citrullinated proteins (ACPA). Importantly, functional and phenotypic properties of B cells following ICI may help predict the emergence of irAE. Much of the data on this issue comes from a detailed analysis of patients with melanoma treated with anti-CTLA-4, anti-PD-1 or the combination. With this combination, B cells of treated patients showed characteristic changes that include a decrease in the number of circulating B cells in conjunction with an increase in plasmablasts and a population of B cells characterized by low expression of CD21. Furthermore, in treated patients, the CD21lo B cells population showed greater clonality as well as a higher frequency of clones in comparison to the CD21hi population. These changes, which may resemble those seen in patients haploinsufficent for CTLA-4, may predict the development of irAE. The CD21lo B cell population may have a particular role in the development of irAE since these cells appear to be recent emigrants from germinal centers and may undergo rapid activation. The effects of ICI on B cell populations is also relevant for ICI use in the setting of pre-existent inflammatory or autoimmune disease marked by autoantibody production. While ICI, either alone or in combination, can lead to the exacerbation of these conditions, the effect on autoantibody production has not yet been well studied. These exacerbations, however, can respond to agents such as glucocorticoids or TNF blockers whose B cell effects are not clear. Since B cells can express PD-1 as well as PD-L1, the effects of ICI may direct actions or the indirect effects on other cell populations. Future studies are needed to delineate more precisely the contribution of B cells to the response of cancer to ICI as well as the development of irAE. Disclosure of Interests: None declared

Background The physician global assessment (PGA) is a 3-point visual analog scale assessing lupus disease activity. We implemented a Type II PGA to assess what we have termed Type II SLE symptoms. Type II SLE symptoms encompass fatigue, widespread pain, depression, sleep and cognitive dysfunction, and differ from Type I SLE symptoms, such as nephritis and inflammatory arthritis, the classic manifestations of SLE inflammation. Methods This was a cross sectional study of SLE patients (ACR 1997 or SLICC 2012 criteria). All patients completed Systemic Lupus Activity Questionnaire (SLAQ), Patient Health Questionnaire-9 (PHQ9), and 2016 ACR Fibromyalgia criteria. The authors recorded PGA, SLEDAI and Type II SLE therapy. Patients were divided into 4 groups based on lupus disease activity. Type I SLE group was defined as SLEDAI 6, clinical SLEDAI 4, or active nephritis. Type II SLE group was defined as meeting fibromyalgia criteria. Mixed SLE group met criteria for Type I and Type II SLE. Low activity group did not meet criteria for either Type I or Type II SLE. Differences across groups were analyzed by Fishers exact test and ANOVA. A step-wise linear regression analysis examined predictors of Type II SLE treatment. Results The authors performed a Type II PGA on 197 patients (92% female, mean age 45.8) from July to November 2018. Type II PGA strongly correlated with patient reported fibromyalgia and depression symptoms. Type II PGA did not correlate with Type I PGA or SLEDAI. Type II PGA was significantly higher in the Type II SLE and mixed SLE groups. Type II symptoms were addressed (counseling or intervention) at 45% of visits. In a regression model predicting Type II management, factors associated with increased Type II treatment were Type II PGA (OR 2.37, CL: 1.37, 4.08) and fibromyalgia severity score (OR 1.41, CL: 1.15, 1.72). Type I PGA (OR 0.46; 95% CI: 0.24, 0.89) was associated with decreased Type II SLE management. Compared to a cohort of 212 SLE patient visits from January to May 2018, prior to implementing the Type II PGA score, the rate of documented Type II SLE management in the Type II SLE group increased from 53% to 89% (p=0.03). Conclusions A PGA specifically addressing Type II SLE symptoms can be implemented in practice and correlates with patient reported Type II SLE symptoms. Scoring a Type II PGA encourages the physician to address these important SLE symptoms. Further work is needed to validate the scoring. Funding Source(s): None

Background Patients with SLE have poor health related quality of life (HRQoL), however the predominant causes for decreased HRQoL in different subgroups of SLE are not well understood. Features of fibromyalgia, including fatigue, widespread pain, depression, sleep and cognitive dysfunction, are prevalent in SLE affecting 20%–65% of patients and often contribute to disability and poor health related quality of life (HRQoL). These aspects of fibromyalgia have not been well described in lupus nephritis (LN). We evaluated self-reported symptoms of fibromyalgia and mood disorders in patients with and without lupus nephritis Methods This was a cross sectional study of SLE patients (ACR 1997 or SLICC 2012 criteria) from July to November 2018. All patients completed Systemic Lupus Activity Questionnaire (SLAQ), Patient Health Questionnaire-9 (PHQ9), and 2016 ACR Fibromyalgia criteria. Active nephritis was defined as UPC>500 mg and/or active urinary sediment excluding other causes. Fibromyalgia was defined as 7 areas of pain with 5 symptoms severity score (SSS) or 4 areas of pain with 9 SSS. Differences across groups were analyzed by Fishers exact test and ANOVA. Results 205 patients completed patient reported outcome measures (92% female, mean age 45.1 years). In our cohort 17% had active nephritis, 16% were in nephritis remission and 67% had no history of nephritis. Of patients with nephritis, 28% had membranous class V, 27% had proliferative or proliferative/mixed LN, 3% class I/II, 0.5% post-transplant, 0.5% ESRD on dialysis, and class was unknown in 32%. The prevalence of fibromyalgia was 10.5% in those with nephritis compared to 23% of patients without nephritis (p=0.04). Patients with LN had statistically significantly lower fibromyalgia severity scores, symptom severity scores, areas of pain, fatigue, cognitive dysfunction, sleep disturbance, forgetfulness, muscle pain, and numbness. Patient-reported disease activity was significantly lower in patients with LN remission. Depression and anxiety were not significantly different between groups. Conclusions Patients with both active and inactive lupus nephritis have lower rates of fibromyalgia, fatigue, sleep and cognitive dysfunction compared to non-nephritis lupus patients. Depression and anxiety were pervasive among all lupus groups and persisted even after achieving lower disease activity. The drivers of low HRQoL may be distinct across SLE subgroups. Lupus nephritis patients have high rates of disability and poor HRQoL, however the mechanism underlying these outcomes in nephritis is less likely related to features of fibromyalgia Funding Source(s): None

Purpose The communication of cell injury and death is a critical element in host defense. Although immune cells can serve this function by elaborating cytokines and chemokines, somatic cells can repurpose nuclear macromolecules to function as damage-associated molecular patterns (DAMPs) or alarmins to exert similar activity. Among these molecules, DNA, high-mobility group box-1, and histone proteins can all act as DAMPs once they are in an extracellular location. This review describes current information on the role of the nuclear DAMPs, their translocation to the outside of cells, and pathways of activation after uptake into the inside of immune cells. Methods MEDLINE and PubMed databases were searched for citations (1990–2016) in English related to the following terms: DAMPs, high-mobility group box-1, DNA, histones, cell death, danger, and immune activation. Selected articles with the most relevant studies were included for a more detailed consideration. Findings Although nuclear molecules have important structural and genetic regulatory roles inside the cell nucleus, when released into the extracellular space during cell death, these molecules can acquire immune activity and serve as alarmins or DAMPs. Although apoptosis is generally considered the source of extracellular nuclear material, other cell death pathways such as necroptosis, NETosis, and pyroptosis can contribute to the release of nuclear molecules. Importantly, the release of nuclear DAMPs occurs with both soluble and particulate forms of these molecules. The activity of nuclear molecules may depend on posttranslational modifications, redox changes, and the binding of other molecules. Once in an extracellular location, nuclear DAMPs can engage the same pattern recognition receptors as do pathogen-associated molecular patterns. These interactions can activate immune cells and lead to cytokine and chemokine production. Among these receptors, internal receptors for DNA are key to the response to this molecule; the likely function of these internal sensors is the recognition of DNA from intracellular infection by bacteria or viruses. Activation of these receptors requires translocation of extracellular DNA into specialized compartments. In addition to nuclear DNA, mitochondrial DNA can also serve as a DAMP. Implications The communication of cell injury and death is a critical element in host defense and involves the repurposing of nuclear molecules as immune triggers. As such, the presence of extracellular nuclear material can serve as novel biomarkers for conditions involving cell injury and death. Targeting of these molecules may also represent an important new approach to therapy.

There has been much recent interest in the protein “corona,” the nonspecific adsorption of proteins on the surface of nanoparticles used in biological applications. This research investigates an analogous DNA corona. We find that particles (200 nm and 1 μm) incubated with DNA form a DNA corona, with a higher concentration of DNA adsorbed on the surface of cationic nanoparticles. With protein present, a combined DNA and protein corona is formed although DNA in solution displaces protein from the nanoparticle surface. Displacement of protein from the nanoparticle surface is dependent on the concentration of DNA in solution and was also observed for planar surfaces. Overall, we expect this investigation of the DNA corona to be important for nanomedicine applications, as well as disease states, especially systemic lupus erythematosus, in which biological particles with bound DNA are important mediators of inflammation and thrombosis.

We thank Dr Pacheco and colleagues for their comments1 on our article2 on the variation of testing for antinuclear antibodies (ANA) by indirect immunofluorescence (IIF) in the context of systemic lupus erythematosus (SLE). Along with findings described in other letters,3–8 the observations by Pacheco and colleagues on three different line immunoassays (LIA) and an ELISA indicate that, as in the case of other technologies, LIAs can show considerable discordance among different kits that are currently available. This study also shows that results by LIA can differ from those obtained by an ELISA. Differences among ANA tests can have a variety of causes including assay conditions as well as the origin and biochemical properties of test antigens. …

Background Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the expression of antibodies to extracellular vesicles (EVs). These vesicles can arise from dead and dying cells and display nuclear and cytoplasmic molecules. Since EV preparations contain mitochondria, we performed experiments to test directly the binding of SLE antibodies to mitochondria. Methods Mitochondria were prepared from mouse liver and immobilized on microtiter plates pre-coated with poly-l-lysine at 0.5 µg/ml. Bound antibodies were detected using a peroxidase-conjugated anti-IgG reagent. To determine whether DNA contributed to the antigenicity of the mitochondria, the ability of DNA to inhibit binding was tested. The binding of sera from 211 SLE patients who met 1982 ACR criteria for classification was determined by ELISA and results compared with an ELISA for M2 antimitochondrial antibodies (AMA). Results Using an ELISA assay with immobilized mitochondria, 60.2% of SLE sera showed positive responses defined as greater than two standard deviations above the mean of control sera. Samples were also analyzed using a commercial AMA ELISA (Euroimmun US, Morris Plains, New Jersey) for IgG antibodies to the M2 antigen. With this kit, 5.7% of the SLE samples tested positive. To determine the relationship of antibodies to anti-DNA, the ability of calf thymus (CT) DNA to inhibit binding to mitochondria was investigated for a subset of samples. In the ELISA, CT DNA at 50 µg/ml inhibited binding for 7 of 8 SLE plasmas, with inhibition ranging from 7.7%–59.3%. Conclusion These results indicate that blood of patients with SLE contain antibodies to mitochondria. Among these antibodies, some may react to DNA as shown by the ability of soluble DNA to inhibit ELISA binding. While binding mitochondria, these antibodies differ from AMA found in primary biliary cholangitis since few reacted in the ELISA for M2 AMA. Since studies using flow cytometry have demonstrated that IgG positive EVs in the blood of patients contain mitochondria as shown by MitoTracker Deep Red, these results suggest that, like nuclei, mitochondria may be a subcellular organelle that can display autoantigenic determinants to form immune complexes in SLE.

We very much appreciate the comments of Willems et al 1 on our article on the variability of antinuclear antibody (ANA) testing for patients with established systemic lupus erythematosus (SLE).2 Others have provided their perspective on test variability.3–6 In their letter, Willems et al discuss the importance of antibody titre especially in the context of patient classification. In the new classification criteria under development, a positive ANA at a titre of 1:80 or higher is required.7 In …

OBJECTIVE: We previously described the multiplex autoantibody SLE-key Rule-Out test, which detects a signature of autoantibody reactivity that distinguishes healthy subjects from SLE patients with 94% sensitivity, 75% specificity and 93% negative predictive value; thus, an individual manifesting a positive Rule-Out test score is unlikely to have SLE (e.g. lupus is excluded). The objective of this current study was to evaluate the stability of the lupus-associated signature over time. METHODS: We used banked serum samples from healthy subjects (n = 51) and lupus patients (n = 50 individual samples and n = 181 paired samples, for a total of n = 412 serum samples). The samples were drawn at different times after diagnosis to analyse the impact on the SLE-key Rule-Out test of time elapsed since diagnosis and any changes in disease activity (as reflected by the SLEDAI score). RESULTS: The SLE signature remains stable for the first 10 years after diagnosis; in this time frame