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OBJECTIVE To evaluate the clinical efficacy and safety of baminercept, a lymphotoxin β receptor IgG fusion protein (LTβR-Ig), for the treatment of primary Sjogren's syndrome (SS), and to explore the possible mechanisms of action of this treatment. METHODS In this multicenter trial, 52 patients with primary SS were randomized in a 2:1 ratio to receive subcutaneous injections of 100 mg of baminercept every week for 24 weeks or matching placebo. The primary end point was the change between screening and week 24 in the stimulated whole salivary flow (SWSF) rate. Secondary end points included the European League Against Rheumatism Sjogren's Syndrome Disease Activity Index (ESSDAI), as well as measurements of select chemokines and cytokines and enumeration of peripheral blood B and T cell subsets. RESULTS The change from baseline to week 24 in the SWSF rate was not significantly different between the baminercept and placebo treatment groups (baseline-adjusted mean change -0.01 versus 0.07 ml/minute; P = 0.332). The change in the ESSDAI during treatment was also not significantly different between the treatment groups (baseline-adjusted mean change -1.23 versus -0.15; P = 0.104). Although the incidence of adverse events was similar between the treatment groups, baminercept therapy was associated with a higher incidence of liver toxicity, including 2 serious adverse events. Baminercept also produced a significant decrease in plasma levels of CXCL13 and significant changes in the number of circulating B and T cells, consistent with its known inhibitory effects on LTβR signaling. CONCLUSION In this trial, treatment with baminercept failed to significantly improve glandular and extraglandular disease in patients with primary SS, despite evidence from mechanistic studies showing that it blocks LTβR signaling.

Genetic variation within the major histocompatibility complex (MHC) contributes substantial risk for systemic lupus erythematosus, but high gene density, extreme polymorphism and extensive linkage disequilibrium (LD) have made fine mapping challenging. To address the problem, we compared two association techniques in two ancestrally diverse populations, African Americans (AAs) and Europeans (EURs). We observed a greater number of Human Leucocyte Antigen (HLA) alleles in AA consistent with the elevated level of recombination in this population. In EUR we observed 50 different A—C—B—DRB1—DQA—DQB multilocus haplotype sequences per hundred individuals; in the AA sample, these multilocus haplotypes were twice as common compared to Europeans. We also observed a strong narrow class II signal in AA as opposed to the long-range LD observed in EUR that includes class I alleles. We performed a Bayesian model choice of the classical HLA alleles and a frequentist analysis that combined both single nucleotide polymorphisms (SNPs) and classical HLA alleles. Both analyses converged on a similar subset of risk HLA alleles: in EUR HLA– B*08:01 + B*18:01 + (DRB1*15:01 frequentist only) + DQA*01:02 + DQB*02:01 + DRB3*02 and in AA HLA–C*17:01 + B*08:01 + DRB1*15:03 + (DQA*01:02 frequentist only) + DQA*02:01 + DQA*05:01+ DQA*05:05 + DQB*03:19 + DQB*02:02. We observed two additional independent SNP associations in both populations: EUR rs146903072 and rs501480; AA rs389883 and rs114118665. The DR2 serotype was best explained by DRB1*15:03 + DQA*01:02 in AA and by DRB1*15:01 + DQA*01:02 in EUR. The DR3 serotype was best explained by DQA*05:01 in AA and by DQB*02:01 in EUR. Despite some differences in underlying HLA allele risk models in EUR and AA, SNP signals across the extended MHC showed remarkable similarity and significant concordance in direction of effect for risk-associated variants.

Background The role of sleep in the etiology of systemic lupus erythematosus (SLE) has not been well studied. We examined whether sleep duration was associated with subsequent transitioning to SLE in individuals at risk for SLE. Methods Four hundred and thirty-six relatives of SLE patients who did not have SLE themselves at baseline were evaluated again an average of 6.3 (± 3.9) years later. Fifty-six individuals transitioned to SLE (≥ 4 cumulative American College of Rheumatology (ACR) criteria). Sleep duration, medication use and medical history were assessed by questionnaire; ACR criteria were confirmed by medical record review. Vitamin D was measured by ELISA. Generalized estimating equations, accounting for correlation within families, assessed associations between baseline sleep and the outcome of transitioning to SLE. Results Reporting sleeping less than 7 hours per night at baseline was more common in those who subsequently transitioned than those who did not transition to SLE (55% versus 32%, p = 0.0005; OR: 2.8, 95% CI 1.6–4.9). Those who transitioned to SLE were more likely to sleep less than 7 hours per night than those who did not transition to SLE adjusting for age, sex and race (OR: 2.8, 95% CI 1.6–5.1). This association remained after individual adjustment for conditions and early symptoms that could affect sleep, including prednisone use, vitamin D deficiency and number of ACR criteria (OR: 2.0, 95% CI 1.1–4.2). Conclusion Lack of sleep may be associated with transitioning to SLE, independent of early clinical manifestations of SLE that may influence sleep duration. Further evaluation of sleeping patterns and biomarkers in at-risk individuals is warranted.

Objective To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjogren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. Methods A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. Results V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. Conclusion These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Objective Sjogren's Syndrome (SS) patients are prone to develop malignant lymphomas and a correlation has been established between lymphoproliferations and the presence in patients’ blood of an unusual B cell population that downregulated complement receptor 2/CD21. We sought to determine from which B cell compartment lymphoproliferations in SS patients emerge and what are the mechanisms that promote these clonal B cell expansions. Methods Using a PCR-based approach that allows us to clone and express, in vitro, recombinant antibodies produced by single B cells, we tested the reactivity of antibodies expressed by CD19+CD10-CD27-IgM+CD21-/low cells from SS patients. Results We identified clonal expansions in CD21-/low B cells isolated from the blood of three SS patients. All three lymphoproliferations expressed B cell receptors (BCRs) that displayed somatic hypermutation lineage trees characteristic of strong selection by antigens, one of which was identified as a ribosomal self-antigen. When mutated BCR sequences expressed by SS expanded clones were reverted, in vitro, to their germline counterparts, one remained autoreactive. Conclusion Clonal lymphoproliferations in SS patients preferentially accumulate in the autoreactive CD21-/low B cell compartment, which is often amplified in these subjects, and (self)-antigen recognition may drive expansion while further refining BCR (self)-reactivity. This article is protected by copyright. All rights reserved.

Objective Incomplete lupus erythematosus (ILE) involves clinical and/or serologic manifestations consistent with but insufficient for systemic lupus erythematosus (SLE) classification. Because the nature of ILE is poorly understood and no treatment recommendations exist, we examined the clinical manifestations, medication history, and immunologic features in a diverse collection of ILE and SLE patients. Methods Medical records of subjects enrolled in the Lupus Family Registry and Repository were reviewed for medication history and American College of Rheumatology (ACR) classification criteria to identify ILE patients (3 ACR criteria; n = 440) and SLE patients (≥4 ACR criteria; n = 3,397). Participants completed the Connective Tissue Disease Screening Questionnaire. Anticardiolipin and plasma B lymphocyte stimulator (BLyS) were measured by enzyme-linked immunosorbent assay, antinuclear antibodies (ANAs) by indirect immunofluorescence, and 13 autoantibodies by bead-based assays. Results On average, ILE patients were older than SLE patients (46.2 years versus 42.0 years; P < 0.0001), and fewer ILE patients were African American (23.9% versus 32.2%; P < 0.001). ILE patients exhibited fewer autoantibody specificities than SLE patients (1.3 versus 2.6; P < 0.0001) and were less likely to have ANA titers ≥1:1,080 (10.5% versus 19.5%; P < 0.0001). BLyS levels were intermediate in ILE patients (controls < ILE; P = 0.016; ILE < SLE; P = 0.008). Pericarditis, renal, or neurologic manifestations occurred in 12.5% of ILE patients and were associated with non–European American race/ethnicity (P = 0.012). Hydroxychloroquine use increased over time, but was less frequent in ILE than SLE patients (65.2% versus 83.1%; P < 0.0001). Conclusion Although usually characterized by milder symptoms, ILE manifestations may require immunomodulatory treatments. Longitudinal studies are necessary to understand how ILE affects organ damage and future SLE risk, and to delineate molecular pathways unique to ILE.

Anthrax vaccine adsorbed (AVA) is a significant line of defense against bioterrorist attack from Bacillus anthracis spores. However, in a subset of individuals, this vaccine may produce a suboptimal quantity of anti-protective antigen (PA), antibodies that are poorly neutralizing, and/or antibody titers that wane over time, necessitating annual boosters. To study individuals with such poor responses, we examine the properties of anti-PA in a subset of vaccinated individuals that make significant quantities of antibody but are still unable to neutralize toxin. In this cohort, characterized by poorly neutralizing antibody, we find that increased IgG4 to IgG1 subclass ratios, low antibody avidity, and insufficient antibody targeting domain 4 associate with improper neutralization. Thus, future vaccines and vaccination schedules should be formulated to improve these deficiencies.

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by overproduction of autoantibodies by B cells and the loss of tolerance to nucleic acids. Our previous studies show that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein, is more abundant in SLE B cells than in healthy control B cells. Increased numbers of ARID3a+ B cells in SLE correlated with increased disease activity in patients. ARID3a expression in SLE patient blood occurred in naïve B cell subsets which do not express ARID3a in healthy controls. Studies by the Sanz laboratory revealed that activated naïve B cells and may be precursors for the double negative (DN) B cells linked to pathogenic responses in SLE. We hypothesize that ARID3a expression in naïve B cells also defines precursors of pathogenic B cells. In individual patients, ARID3a is bimodally expressed, such that only a fraction of the naïve B cells express ARID3a at any given time. We took advantage of this bimodal expression using single cell technology to perform RNA-seq analyses of ARID3a+ and ARID3a− SLE B cells to identify differentially expressed genes associated with ARID3a. PCA analyses of scRNA seq data show that ARID3a+ naïve B cells cluster together and away from ARID3a− cells. This allows for analyses within and among individual samples by binning cells based on ARID3a expression. Preliminary data indicate that ARID3a expression is correlated with innate immune response genes, transcription factors, and epigenetic regulators. These studies will be informative regarding how ARID3a expression contributes to disease pathogenesis in patients with SLE and will provide novel information defining a new subset of naive B cells that may be directly linked to breaches in tolerance.

Objective:To determine whether Epstein-Barr virus (EBV) or cytomegalovirus (CMV) seropositivity is associated with multiple sclerosis (MS) in blacks and Hispanics and to what extent measures of the hygiene hypothesis or breastfeeding could explain these findings. EBV and CMV have been associated with MS risk in whites, and the timing and frequency of both viruses vary by factors implicated in the hygiene hypothesis.Methods:Incident cases of MS or its precursor, clinically isolated syndrome (CIS), and matched controls (blacks, 111 cases/128 controls; Hispanics, 173/187; whites, 235/256) were recruited from the membership of Kaiser Permanente Southern California. Logistic regression models accounted for HLA-DRB1*1501 status, smoking, socioeconomic status, age, sex, genetic ancestry, and country of birth.Results:Epstein-Barr nuclear antigen-1 (EBNA-1) seropositivity was independently associated with an increased odds of MS/CIS in all 3 racial/ethnic groups (p < 0.001 for blacks and whites, p = 0.02 for Hispanics). In contrast, CMV seropositivity was associated with a lower risk of MS/CIS in Hispanics (p = 0.004) but not in blacks (p = 0.95) or whites (p = 0.96). Being born in a low/middle-income country was associated with a lower risk of MS in Hispanics (p = 0.02) but not after accounting for EBNA-1 seropositivity. Accounting for breastfeeding did not diminish the association between CMV and MS in Hispanics.Conclusions:The consistency of EBNA-1 seropositivity with MS across racial/ethnic groups and between studies points to a strong biological link between EBV infection and MS risk. The association between past CMV infection and MS risk supports the broader hygiene hypothesis, but the inconsistency of this association across racial/ethnic groups implies noncausal associations.

Objective Systemic lupus erythematosus (SLE) and other autoimmune diseases cause significant morbidity. Identifying populations at risk of developing SLE is essential for curtailing irreversible inflammatory damage. The aim of this study was to identify factors associated with transition to classified disease that would inform our understanding of the risk of SLE. Methods Previously identified blood relatives of patients with SLE, who had

Background:We recently reported Phase 2 SLE trial results of obexelimab, an FcγRIIb agonist (suppressor of B cell activation). Obexelimab did not meet the primary endpoint (% of patients without flare at Day 225) (p=0.183) but other endpoints such as time to flare (p=0.025) were met.Objectives:1. To assign SLE patients to phenotypic subsets based on patterns of gene expression in immune-related pathways.12. To explore the association of immune patterns and clinical response to obexelimab.Methods:This analysis included 71 of the 104 participants in the obexelimab study, those who either completed the protocol or terminated for disease flare, if there were adequate blood samples (biomarker subset). At screening, patients were assigned to clusters based on 41 SLE co-expression signature modules from the Human Immune Phenotyping Consortium via unsupervised random forest and K-means clustering.2Other markers of SLE disease were also examined. TheBOLD3study design mandated withdrawal of background immunosuppressants, supporting less ambiguous pharmacodynamic analysis as the trial progressed.Results:Immune pathway expression patterns of 7 patient clusters (Fig 1a) confirmed our prior characterization of 200 non-overlapping SLE patients.2The biomarker subset retained a trend of longer time to first flare in patients receiving obexelimab (n=38) vs placebo (n=33) (Fig1b, HR 0.61, p=0.11). A smaller set of only Clusters 3 and 6 demonstrated marked increased time to flare in the obexelimab group (n=13) compared to placebo (n=14) (Fig 1c, HR 0.22, p=0.025). Obexelimab had no effect on other clusters (Fig 1d). The responder clusters shared low expression of inflammation modules (p < 0.001) compared to other clusters and high expression of T Cell, immune response, cell cycle, mitochondrial modules (all p < 0.001) and B Cell modules (p=0.006). We therefore sorted patients by these specific features regardless of cluster assignment. Figure 2 shows significant impact of obexelimab on time to flare in 64 patients with B Cell pathway activation (HR 0.5, p=0.038), although less robust by itself than found in Clusters 3 and 6. In a group with high B or plasma cell modules but low inflammation (n=46), treatment effect increased (HR 0.35, p=0.019). Between Screening and Baseline, as brief steroids were given and background treatments withdrawn, expression of B Cell and Plasma Cell pathways increased. Both then decreased after treatment with obexelimab but not placebo (p< 0.0001 and p< 0.001 respectively), an effect not seen with other immune pathway modules.Conclusion:Precision medicine for SLE has been hampered by heterogenous immune signals with variable expression. Clustering of patients by gene co-expression pathways enabled an efficient, hierarchical array that reduplicated results of a prior SLE cohort, suggesting these are not random phenotypes. Of these 7 reproducible SLE subsets, the combination of clusters 3 and 6 distinguished an obexelimab responder population of 27 out of 71 subjects (38%) with high expression of B and T Cell modules and cell activation pathways. Focus on the defining features shared by these clusters revealed specific factors associated with response, enabling inclusion of some patients from other clusters in an optimized responder population of 46/71 (65% of subjects). B Cell and Plasma Cell pathways demonstrated mechanism-related pharmacodynamic effects of obexelimab. Lack of responders with high expression of inflammation modules could implicate inhibitory factors to obexelimab within inflammatory pathways, potentially targetable by complementary treatments.References:[1]Banchereau Cell 165:1548 2016[2]Lu ACR Abstract #2977 2017[3]Merrill Arthritis Rheumatol 69: 1257 2017Disclosure of Interests: :Joan T Merrill Grant/research support from: Xencor, Bristol Myers Squibb, Glaxo Smith Kline, Consultant of: Xencor, Abbvie, UCB, Glaxo Smith Kline, EMD Serono, Astellas, Remegen, Celgene/Bristol Myers Squibb, Exagen, Astra Zeneca, Amgen, Jannsen, Servier, ILTOO, Daitchi Sankyo, Lilly, Paid instructor for: Abbvie, Bristol Myers Squibb, Joel Guthridge Grant/research support from: Xencor, Bristol Myers Squibb, DXterity, Debra Zack Shareholder of: Xencor, Employee of: Xencor, Paul Foster Shareholder of: Xencor Inc, Employee of: Xencor Inc, Bart Burington Shareholder of: Xencor Inc, Employee of: Xencor Inc, Ly Tran: None declared, Miles Smith: None declared, Judith A. James Grant/research support from: Progentec Diagnostics, Inc, Consultant of: Abbvie, Novartis, Jannsen

Background:Trials and clinical observations have demonstrated the efficacy of mycophenolate mofetil (MMF) for SLE treatment. Long-term use of MMF is associated with adverse events, pregnancy risks, drug monitoring, and increased cost. Current management continues therapy indefinitely. Whether immunosuppression may be safely withdrawn or whether risks of withdrawal outweigh the benefits of continuation is unknown.Objectives:To compare rates of clinically significant disease reactivation (CSDR), major flares, and all flares in patients with quiescent SLE on stable MMF randomized to maintain or withdraw MMF. The goal is to provide guidance for clinicians and patients on the risks of MMF withdrawal.Methods:Adults with quiescent SLE (SELENA-SLEDAI without serologies Results:102 subjects were randomized (50 MA, 52 WA); 1 subject in each arm was ineligible and 10 terminated early (7 MA, 3 WA). Mean disease duration was 13 years; 76% had a history of nephritis; mean baseline SLEDAI was 2.2. 5 MA subjects (10%) had CSDR, compared to 9 WA (17%). Median time to CDSR was 38 weeks in both arms. BILAG A flares occurred in 1MA subject (pancreatitis) vs. 4 WA (cranial neuropathy, panniculitis, 2 nephritis). Kaplan-Meier curves overlapped for CDSR, BILAG A flares, and all SLEDAI flares (Figure). Based on these data, we are 86% confident that the increased risk of CDSR with MMF withdrawal is less than 15% over 60 weeks. AEs were similar between groups; infections occurred more commonly in MA (63 vs. 49).Conclusion:In this cohort of subjects with quiescent SLE on long term MMF serious flares occurred infrequently in subjects continuing or withdrawing MMF without differences in time to flare. MMF withdrawal may be considered in subjects with prolonged quiescent disease.Table 1.Baseline and Demographic CharacteristicsMaintenance armWithdrawal armTotalRandomized5052102Female, n (%)39 (78)47 (90)86 (84)White, n (%)25 (50)19 (37)44 (43)Black, n (%)19 (38)22 (42)41 (40)Hispanic/Latino, n (%)10 (20)12 (23)22 (22)Age, Years, mean (SD)42.4 (12.9)41.6 (12.5)42.0 (12.6)Disease Duration, Years, mean (SD)13.6 (8.2)12.2 (7.9)12.9 (8.0)H/O Lupus Nephritis, n (%)40 (80)38 (73)78 (76.5)On Baseline Steroids, n (%)18 (36)23 (44)41 (40)Prednisone Dose, mg, mean (SD)4.8 (2.7)3.3 (1.7)4.0 (2.3)MMF Duration, Years, mean (SD)6.8 (4.3)6.4 (4.3)6.6 (4.3)Baseline MMF Dose, mg, mean1,6121,6681,640SELENA-SLEDAI*, mean (SD)2.4 (1.76)1.9 (1.76)2.2 (1.77)Positive DsDNA, n (%)35 (70)27 (52)62 (61)Low C31, n (%)14 (28)9 (17)23 (23)Low C41, n (%)6 (12)5 (10)11 (11)Figure.Kaplan-Meier Estimates of Flare EndpointsDisclosure of Interests:Eliza Chakravarty: None declared, Tammy Utset: None declared, Diane L Kamen Consultant of: Consulted on SLE survey development for Lilly and consulted on SLE trial protocol development for EMD Serono in 2019, Gabriel Contreras Grant/research support from: Genentech, Merck, Consultant of: Genentech, Merck, William Joseph McCune: None declared, Kenneth C Kalunian: None declared, Cynthia Aranow: None declared, Megan Clowse Grant/research support from: GSK, Pfizer, Consultant of: UCB, Astra-Zeneca, Speakers bureau: UCB, Ellen Goldmuntz: None declared, Jessica Springer: None declared, Lynette Keyes-Elstein: None declared, Bill Barry: None declared, Ashley Pinckney: None declared, Judith James: None declared

Previously published studies revealed that variation in expression of the DNA-binding protein ARID3a in B lymphocytes from patients with systemic lupus erythematosus (SLE) correlated with levels of disease activity ("Disease activity in systemic lupus erythematosus correlates with expression of the transcription factor AT-rich-interactive domain 3A" (J.M. Ward, K. Rose, C. Montgomery, I. Adrianto, J.A. James, J.T. Merrill et al., 2014) [1]). The data presented here compare DNA methylation patterns from SLE peripheral blood mononuclear cells obtained from samples with high numbers of ARID3a expressing B cells (ARID3a(H)) versus SLE samples with normal numbers of ARID3a(+) B cells (ARID3a(N)). The methylation data is available at the gene expression omnibus (GEO) repository, "Gene Expression Omnibus: NCBI gene expression and hybridization array data repository" (R. Edgar, M. Domrachev, A.E. Lash, 2002) [2]. Isolated B cells from SLE ARID3a(H) and ARID3a(N) B samples were also evaluated via qRT-PCR for Type I interferon (IFN) signature and pathway gene expression levels by qRT-PCR. Similarly, healthy control B cells and B cells stimulated to express ARID3a with the TLR agonist, CpG, were also compared via qRT-PCR. Primers designed to detect 6 IFNa subtype mRNAs were tested in 4 IFNa, Epstein-Barr Virus-transformed B cell lines ("Reduced interferon-alpha production by Epstein-Barr virus transformed B-lymphoblastoid cell lines and lectin-stimulated lymphocytes in congenital dyserythropoietic anemia type I" (S.H. Wickramasinghe, R. Hasan, J. Smythe, 1997) [3]). The data in this article support the publication, "Human effector B lymphocytes express ARID3a and secrete interferon alpha" (J.M. Ward, M.L. Ratliff, M.G. Dozmorov, G. Wiley, J.M. Guthridge, P.M. Gaffney, J.A. James, C.F. Webb, 2016) [4].

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a poorly understood preclinical stage of immune dysregulation and symptom accrual. Accumulation of antinuclear autoantibody (ANA) specificities is a hallmark of impending clinical disease. Yet, many ANA-positive individuals remain healthy, suggesting that additional immune dysregulation underlies SLE pathogenesis. Indeed, we have recently demonstrated that interferon (IFN) pathways are dysregulated in preclinical SLE. To determine if other forms of immune dysregulation contribute to preclinical SLE pathogenesis, we measured SLE-associated autoantibodies and soluble mediators in samples from 84 individuals collected prior to SLE classification (average timespan = 5.98 years), compared to unaffected, healthy control samples matched by race, gender, age (± 5 years), and time of sample procurement. We found that multiple soluble mediators, including interleukin (IL)-5, IL-6, and IFN-γ, were significantly elevated in cases compared to controls more than 3.5 years pre-classification, prior to or concurrent with autoantibody positivity. Additional mediators, including innate cytokines, IFN-associated chemokines, and soluble tumor necrosis factor (TNF) superfamily mediators increased longitudinally in cases approaching SLE classification, but not in controls. In particular, levels of B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) were comparable in cases and controls until less than 10 months pre-classification. Over the entire pre-classification period, random forest models incorporating ANA and anti-Ro/SSA positivity with levels of IL-5, IL-6, and the IFN-γ-induced chemokine, MIG, distinguished future SLE patients with 92% (± 1.8%) accuracy, compared to 78% accuracy utilizing ANA positivity alone. These data suggest that immune dysregulation involving multiple pathways contributes to SLE pathogenesis. Importantly, distinct immunological profiles are predictive for individuals who will develop clinical SLE and may be useful for delineating early pathogenesis, discovering therapeutic targets, and designing prevention trials.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell–B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4(+) T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1(hi)CXCR5(–)CD4(+) T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1(hi)CXCR5(–)CD4(+) T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5(–) T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c(+) B cells in SLE patients. PD-1(hi)CD4(+) T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

EgeUn###, Rationale: Several common and rare genetic variants have been associated with idiopathic pulmonary fibrosis, a progressive fibrotic condition that is localized to the lung. Objectives: To develop an integrated understanding of the rare and common variants located in multiple loci that have been reported to contribute to the risk of disease. Methods: We performed deep targeted resequencing (3.69 Mb of DNA) in cases (n = 3,624) and control subjects (n = 4,442) across genes and regions previously associated with disease. We tested for associations between disease and 1) individual common variants via logistic regression and 2) groups of rare variants via sequence kernel association tests. Measurements and Main Results: Statistically significant common variant association signals occurred in all 10 of the regions chosen based on genome-wide association studies. The strongest risk variant is the MUC5B promoter variant rs35705950, with an odds ratio of 5.45 (95% confidence interval, 4.91-6.06) for one copy of the risk allele and 18.68 (95% confidence interval, 13.34-26.17) for two copies of the risk allele (P = 9.60 3 102295). In addition to identifying for the first time that rare variation in FAM13A is associated with disease, we confirmed the role of rare variation in the TERT and RTEL1 gene regions in the risk of IPF, and found that the FAM13A and TERT regions have independent common and rare variant signals. Conclusions: A limited number of common and rare variants contribute to the risk of idiopathic pulmonary fibrosis in each of the resequencing regions, and these genetic variants focus on biological mechanisms of host defense and cell senescence. Copyright © 2019 by the American Thoracic Society, Háskóli Íslands, HI University of Chicago University of Ulsan, UOU Monash University, MU University of Nottingham University of Edinburgh University of Colorado Denver Universitat de Barcelona University of Pittsburgh, Pitt Harvard School of Dental Medicine Massachusetts General Hospital, MGH University of Pennsylvania, Penn University College Cork, UCC City, University of London, City University of California, San Francisco, UCSF School of Medicine, Vanderbilt University Ege Üniversitesi University of Virginia, UV Aarhus Universitetshospital Central Manchester University Hospitals NHS Foundation Trust Columbia University Novartis Sunovion COPD Foundation Pfizer National Heart, Lung, and Blood Institute, NHLBI: R01-HL097163, UH3-HL123442, P01-HL092870 U.S. Department of Defense, DOD: W81XWH-17-1-0597, U01 HL089897, U01 HL089856 AstraZeneca Siemens Foundation North Carolina GlaxoSmithKline Foundation, 1National Jewish Health, Denver, Colorado; 2School of Public Health; 3Department of Medicine, and 54Department of Immunology, University of Colorado Denver, Denver, Colorado; 4Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; 5Brigham and Women’s Hospital, Harvard School of Medicine, Boston, Massachusetts; 6Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma;7Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; 8MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom; 9Respiratory Medicine Unit, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom; 10Biomedical Research Centre, University of Nottingham, Nottingham, United Kingdom; 11Royal Brompton Hospital and Imperial College, London, United Kingdom; 12Simmons Center for Interstitial Lung Disease, University of Pittsburgh, Pittsburgh, Pennsylvania; 13Department of Medicine, University of California, San Francisco, San Francisco, California; 14Gilead Sciences, Foster City, California; 15Department of Medicine, University of Chicago, Chicago, Illinois; 16Department of Medicine, University of Virginia, Charlottesville, Virginia; 17Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania; 18National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka, Japan; 19National Hospital Organization Tokyo National Hospital, Tokyo, Japan; 20Helmholtz Zentrum München, Neuherberg, Germany; 21University Hospital Munich, Munich, Germany; 22Department of Pulmonology, Ege University Hospital, Bornova, Izmir, Turkey; 23Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia; 24Alfred Hospital and Monash University, Melbourne, Australia; 25Pulmonary Medicine, GB Morgagni Hospital, Forlì, Italy; 26Department of Diseases of the Thorax, Ospedale GB Morgagni, Forlì, Italy; 27Université Paris Diderot and Hôpital Bichat, Paris, France; 28Royal Papworth Hospital and Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom; 29Respiratory Department, University Hospital of Bellvitge, University of Barcelona, Barcelona, Spain; 30National University Hospital of Iceland, University of Iceland, Reykjavik, Iceland; 31Department of Medicine, Columbia University Irving Medical Center, New York, New York; 32Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, Massachusetts; 33Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Aarhus, Denmark; 34Instituto Nacional de Enfermedades Respiratorias “Ismael Cosio Villegas,” México City, México; 35Universidad Nacional Autónoma de México, México City, México; 36Cork University Hospital and University College Cork, Cork, Ireland; 37St. Vincent’s University Hospital, Dublin, Ireland; 38School of Medicine, University College Dublin, Dublin, Ireland; 39Department of Respiratory Medicine, First Faculty of Medicine Charles University and Thomayer Hospital, Prague, Czech Republic; 40University of British Columbia, Vancouver, Canada; 41Tokyo Medical and Dental University, Tokyo, Japan; 42Institute for Respiratory Health and; 43Centre for Cell Therapy and Regenerative Medicine, School of Biomedical Sciences, The University of Western Australia, Perth, Australia; 44Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island; 45Advanced Lung Disease and Transplant Program, Inova Fairfax Hospital, Falls Church, Virginia; 46Department of Pulmonary Medicine, Gazi University School of Medicine, Ankara, Turkey; 47Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama; 48Ruhrlandklinik, University Hospital, University of Duisburg-Essen, Essen, Germany; 49Department of Medicine, Tallaght University Hospital, Trinity College Dublin, Dublin, Ireland; 50CardioPulmonary Reserach Center, Alliance Pulmonary Group, Guaynabo, Puerto Rico; 51Department of Internal Medicine, University of Genoa, Genoa, Italy; 52Biomedical Genomics Center, University of Minnesota; Minneapolis, Minnesota; and 53Northwest Genomics Center, University of Washington, Seattle, Washington -- This research was supported by grants from the NHLBI (R01-HL097163, P01-HL092870, and UH3-HL123442) and the Department of Defense (W81XWH-17-1-0597). The COPDGene project was supported by Award Number U01 HL089897 and Award Number U01 HL089856 from the NHLBI. The COPDGene project was also supported by the COPD Foundation through contributions made to an industry advisory board comprised of AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Novartis, Pfizer, Siemens, and Sunovion.

Background Abatacept (ABA) is a fusion protein of the extracellular domain of CTLA4 and the Fc domain of human IgG1, constructed to inhibit B/T cell co-stimulation. Previous studies of ABA in lupus failed to show benefit in overall disease activity, but improvement in arthritis was suggested. Objectives This 6-month randomized, double-blind, placebo-controlled (PBO) study withdrew background medications to facilitate the assessment of ABA in patients with SLE arthritis. Methods Patients were entered with moderate to severe arthritis [BILAG A or B with ≥3 swollen and ≥3 tender joints (28 joint count)]. All DMARDs except prednisone (up to 20mg daily) were withdrawn by baseline and patients were randomized 1:1 to weekly sc ABA or PBO. One or more DepoMedrol injections (≤320mg total) was allowed by end of Month 2. Additional steroids or immune suppressants (IMS) at any time, and transition to open label ABA at Month 3 or later, were allowed, but designated non-response. The primary endpoint was BICLA response at Month 6 compared to screening. Secondary objectives included SRI-4, SLEDAI 4-point improvement, SLEDAI arthritis resolution, BILAG A/B musculoskeletal (MSK) improvement, as well as low disease activity defined by either SLEDAI 2 or Lupus Low Disease Activity State (LLDAS) at Month 6. Flares were evaluated by the modified SELENA-SLEDAI flare index (1) in patients followed after baseline. Results 66 patients were randomized and received at least one dose of the study drug, 31 ABA and 35 PBO. 7 withdrew prior to Month 6. Placebo response rates were lower than in most SLE trials, but no primary or secondary endpoints were met, see table (Fischer’s exact test). Flares were evaluable in 64 patients (31 on ABA, 33 on PBO). 14 (45%) patients on ABA vs. 18 (55%) on PBO had a moderate/severe flare by Month 6. 22 (71%) patients on ABA vs. 23 (70%) on PBO experienced flare or treatment failure (early drop off or off protocol steroids or IMS) by Month 6. Rates of flare and treatment failure by Month 6 did not differ between groups (Log-Rank test, p=0.688 and p=0.631, respectively). The safety profile of ABA was consistent with known effects of the treatment. Conclusion ABA did not demonstrate improvement vs placebo in controlling disease activity or flare in SLE patients in a protocol that mandated IMS withdrawal. Placebo response rates of ≤23% support the validity of these results. Since some evidence suggests potential efficacy of ABA for certain biologic subsets of SLE (2), immunophenotyping may prove helpful. References [1] Thanou A, Chakravarty E, James JA, Merrill JT. Rheumatology (Oxford) 2014;53(12):2175-81. [2] Bandyopadhyay S, Connolly SE, Jabado O, Ye J, Kelly S, Maldonado MA, Westhovens R, Nash P, Merrill JT, Townsend RM. Lupus Sci Med. 2017 4 (1): e000206. Disclosure of Interests Aikaterini Thanou Grant/research support from: Bristol-Myers Squibb, Consultant for: Neovacs, Cristina Arriens Grant/research support from: Bristol-Myers Squibb, GSK/HGS, Consultant for: GSK, AstraZeneca, Teresa Aberle: None declared, Eliza Chakravary: None declared, Samera Vaseer: None declared, Joe Rawdon: None declared, Judith A. James: None declared, Joan Merrill Grant/research support from: Genentech, UCB, GSK, EMD Serono, Pfizer, Celgene, Exagen, Bristol Myers Squibb, Medimmune/Astra Zeneca, Lilly, Amgen, Xencor, Neovacs, Consultant for: Genentech, UCB, GSK, EMD Serono, Pfizer, RemeGen, Celgene, Exagen, Bristol Myers Squibb, Medimmune/Astra Zeneca, Lilly, Immupharma, Amgen, Janssen, Sanofi, Neovacs, Anthera, Speakers bureau: UCB, GSK, EMD Serono, Bristol Myers Squibb, Medimmune/Astra Zeneca, Janssen

Background: XmAb5871 is a humanized anti-CD19 antibody Fc-engineered for increased affinity to FcγRIIb. Co-ligation of CD19 and FcγRIIb inhibits B lineage cells key to SLE pathogenesis. Objectives: This Phase 2 study was designed to minimize background medications and placebo responses to improve interpretation of a small trial in a complex, heterogenous disease. Methods: SLE patients were enrolled with active disease, ameliorated during screening with ≥160 mg of IM Depo-Medrol. Improvement was required before randomization, defined by decrease in SLEDAI ≥4 points or ≥1 grade in a BILAG A or B score. Immunosuppressive drugs were stopped except antimalarials and/or ≤10 mg/day prednisone or equivalent. Subjects were randomized to IV XmAb5871 (5 mg/kg) or placebo and given Depo-Medrol 80 mg IM on Days 1 and 15, after which, steroid impact was expected to withdraw gradually. Study treatments were given Q14 days for up to 16 doses or loss of improvement (LOI), defined as SLEDAI increase ≥4 points OR new BILAG A or B, with investigator-determined significance. At LOI, patients could receive immediate standard treatments. The primary endpoint was the proportion with no LOI by Day 225 in the efficacy evaluable group (those completing Day 225 or withdrawn for LOI or drug-related adverse event). Results: 104 subjects were randomized: 99 female, median age 45 (20-65). The primary endpoint was met by 21 (42%) of XmAb5871-treated patients vs 12 (28.6%) of the placebo group (p=0.18). All but one responder also fulfilled the SRI-4 response definition from screening to completion. Results did not differ in those with or without anti-dsDNA and/or ENA antibodies. Time to flare was significantly longer in the XmAb5871 group (p=0.025) (figure 1). XmAb5871-treated patients with LOI had less recurrent disease after IM steroid cessation than those in the placebo group; 6 (20%) of placebo patients developed BILAG A scores vs 3 (13%) in the active arm. 9 (30%) of worsening placebo patients had SLEDAI increase ≥7 vs 0 in the XmAb5871 group. SLEDAI scores were higher and increased sooner after disease nadir with placebo vs XmAb5871 (figure 2); 16 (30.8%) of XmAb5871 patients vs 7 (13.5%) placebo patients sustained LLDAS (low disease) during months 6-8 (p=0.0453). Transient, infusion-related gastrointestinal side effects occurred in XmAb5871-treated patients during the 1st or 2nd infusion. There were 8 SAEs in 7 XmAb5871-treated subjects, 5 in 4 placebo patients, no opportunistic infections, and no deaths. Infection rate was low compared to other SLE trials. Conclusion: XmAb5871 was well-tolerated. Preliminary data from this small trial indicates suppression of disease recurrence after treatment withdrawal, supporting further evaluation of XmAb5871 in SLE. Disclosure of Interests: Joan Merrill Grant/research support from: Genentech, UCB, GSK, EMD Serono, Pfizer, Celgene, Exagen, Bristol Myers Squibb, Medimmune/Astra Zeneca, Lilly, Amgen, Xencor, Neovacs, Consultant for: Genentech, UCB, GSK, EMD Serono, Pfizer, RemeGen, Celgene, Exagen, Bristol Myers Squibb, Medimmune/Astra Zeneca, Lilly, Immupharma, Amgen, Janssen, Sanofi, Neovacs, Anthera, Speakers bureau: UCB, GSK, EMD Serono, Bristol Myers Squibb, Medimmune/Astra Zeneca, Janssen, Joshua June: None declared, Fotios Koumpouras: None declared, Wambui Machua: None declared, Mohammad Faisal Khan: None declared, Anca Askanase: None declared, Arezou Khosroshahi: None declared, Saira Sheikh: None declared, Judith A. James: None declared, Joel Guthridge: None declared, Gaurav Rathi Shareholder of: Xencor Inc, Employee of: Xencor Inc, Bart Burington Shareholder of: Xencor Inc, Employee of: Xencor Inc, Paul Foster Shareholder of: Xencor Inc, Employee of: Xencor Inc, Debra Zack Shareholder of: Xencor Inc, Employee of: Xencor Inc

Background Autoantibodies (AAbs) are a hallmark of systemic lupus erythematosus (SLE). Common SLE AAbs show differences in specificities, levels and associations with clinical characteristics of disease which may be influenced by ethnicity.(1-2) NA SLE patients do not always exhibit classical AAb profiles.(2) Understanding how AAb profiles associate with clinical disease, including in NA patients, is important to better inform disease management. Objectives To identify patient subsets enriched for renal disease by clustering NA, AA and EA SLE patients into more homogeneous disease subsets based on expanded AAb profiles. Methods Serum samples from 49 NA, 49 AA, and 49 EA age, sex and ANA titer-matched SLE patients who met ACR classification and 10 sex and age matched controls from each ethnicity were tested for AAb reactivity by autoantigen microarray. Normalized fluorescence intensity (NFI) for AAb binding was determined for each individual for all autoantigens. Ratios of NFIs to total IgG were calculated and converted to Z-scores. Kruskal-Wallis analysis of Z-score transformed AAb ratios were used to identify AAbs that differed significantly between patients and controls for each ethnicity; these AAbs were used for hierarchal clustering of SLE subjects within each ethnicity. The enrichment of SLE clinical criteria within each cluster was determined by Fisher’s exact test. Results AAbs to nucleic acids and Ro52 were present in SLE patients of all three ethnicities. On average, NA SLE patients, but not EA or AA SLE patients, had lower levels of AAbs against extracellular matrix (ECM) antigens compared to controls. AA and EA, but not NA, SLE patients had significantly higher levels of AAbs to Sm, RNP and histones compared to matched controls. In each ethnicity four clusters of SLE subjects were identified by ethnicity-specific AAbs. All three groups had a cluster of subjects enriched in nucleic acid-specific AAbs and a cluster of subjects enriched in Ro52 AAbs. The NA group also had two clusters enriched in ECM AAbs. The AA group had an Sm/RNP AAb enriched cluster and a nucleolin/histone AAb enriched cluster. Two additional EA SLE clusters had similar levels of nucleic acid-specific AAbs and Ro52 AAbs, but were distinguished by significant differences in histone 2A (H2A) AAb levels. Renal manifestations were significantly enriched in the NA Ro52 cluster and the AA nucleolin/histone cluster compared to other SLE patient clusters of the same ethnicity. Conclusion Expanded AAb profiles can be used to identify SLE subsets that are more likely to have renal manifestations of disease. References [1] Sanchez E, et al. Impact of genetic ancestry and sociodemographic status on the clinical expression of systemic lupus erythematosus in American Indian-European populations. Arthritis Rheum. 2012;64(11):3687-94. [2] Kheir JM, et al. Unique clinical characteristics, autoantibodies and medication use in Native American patients with systemic lupus erythematosus. Lupus Sci. & Med. 2018;5:e000247. Acknowledgement This research supported by the National Institute of Allergy and Infectious Disease (U19AI082714), National Institute of Arthritis, Musculoskeletal and Skin Diseases (P30AR053483, P30AR073750), and National Institute of General Medical Sciences (U54GM104938) of the US NIH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the United States government. Disclosure of Interests None declared

Background Lupus Nephritis (LN) usually presents within five years of SLE diagnosis. Male gender, Hispanic and African American race, and childhood-onset SLE portend greater risk of early nephritis. However, patients with late-occurring nephritis are poorly characterized. Since proliferative nephritis is a more aggressive class of nephritis than membranous and mesangial, we hypothesized that female gender, Caucasian race, adult-onset SLE, and membranous pathology would be associated with a later onset of nephritis. Methods A single large urban medical center was the source for retrospective record review of patients who underwent kidney biopsy between 1999 and 2015. Patients with biopsy-proven LN were evaluated to determine whether gender, race, age at SLE diagnosis, age at first biopsy, or histopathologic classification were associated with time between SLE diagnosis and first renal biopsy. Results 630 patients were screened. After elimination of duplicates, those without initial biopsy, biopsy findings other than LN, and unknown date of SLE diagnosis, 293 subjects had adequate data available for analysis. Those with late onset SLE, diagnosis after age 50, were excluded from further analysis due to known differences in this subgroup. The remaining 278 patients had characteristics shown in table 1. Twenty-nine percent of patients in this cohort developed nephritis more than 5 years after initial SLE diagnosis. Multivariable linear regression identified greater age at first biopsy and race as the strongest predictors of time between SLE diagnosis and first renal biopsy. Hispanic patients were more likely to present with LN earlier in their course and Asian patients were more likely to present later, with African American and Caucasian groups between them. The model accounts for 27% of the variability in the time elapsed between diagnosis and biopsy. Histopathologic class did not make a significant contribution, and proliferative nephritis was noted in similar numbers of patients who developed LN after 5 years and those in the early presenting group. Conclusions Surprisingly, more than a quarter of known LN patients at this large US center present after 5 years of SLE, higher than expected based on low numbers of late onset nephritis in other cohorts. This appears to be more common in Caucasian and Asian subgroups. Older patients are more likely to develop nephritis later, but aggressive histopathology is not less likely in later presentations. The data underscore the importance of remaining vigilant about nephritis risk in patients of diverse demographics five years and more after diagnosis with SLE. Funding Source(s): Mallinckrodt Research Fellowship, NIH:T32DK007257,UL1TR001105,U54GM104938,P30AR053483,U01AI101934,U19AI082714

Background Classification and treatment decisions in lupus nephritis (LN) are largely based on renal histology. Single-cell RNA sequencing (scRNAseq) analysis may accurately differentiate types of renal involvement at the transcriptomic level, and better inform treatment decisions and prognosis. Methods scRNAseq was performed on kidney and non-lesional skin tissue collected from 20 SLE patients undergoing a clinically indicated renal biopsy. Cell types were determined using principal component analysis and t-distributed stochastic neighbor embedding (tSNE) plotting, resulting in the definitive identification of keratinocytes, tubular cells, mesangial cells, fibroblasts, endothelial cells, and leukocytes. Results LN patients expressed upregulated IFN response genes in both their tubular cells and keratinocytes. This IFN response signature in tubular cells predicted poor response to therapy 6 months post-biopsy. Tubular cells of non-responder patients also expressed upregulated extracellular matrix proteins and fibrotic markers (figure 1A and 1B). Using logistic regression analysis, a 4-gene tubular fibrosis score was created and able to predict response to treatment with an area under curve of 0.9 (figure 1C). Keratinocytes of non-responders also upregulated certain extracellular matrix genes and this response was not observed in peripheral blood mononuclear cells. Differential expression analysis between histology classes indicated an upregulation of IFN and TNF signaling in the tubular cells of patients with proliferative LN compared with membranous. Conclusions scRNAseq from 2–10 mm of renal biopsy tissue in SLE can differentiate between the different classes of LN, and provide important insights into potential pathogenic mechanisms. Further, changes in the skin of LN patients can provide a useful source of biomarkers and may reflect important information concerning concurrent kidney pathological events. Funding Source(s): This work was supported by the Accelerating Medicines Partnership (AMP) in Rheumatoid Arthritis and Lupus Network. AMP is a public-private partnership (AbbVie, Arthritis Foundation, Bristol-Myers Squibb, Foundation for the National Institutes of Health, Lupus Foundation of America, Lupus Research Alliance, Merck Sharp and Dohme, National Institute of Allergy and Infectious Diseases, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Pfizer, Rheumatology Research Foundation, Sanofi, and Takeda Pharmaceuticals) created to develop new ways of identifying and validating promising biological targets for diagnostics and drug development. Funding was provided through grants from the National Institutes of Health (UH2-AR067676, UH2-AR067677, UH2-AR067679, UH2-AR067681, UH2-AR067685, UH2-AR067688, UH2-AR067689, UH2-AR067690, UH2-AR067691, UH2-AR067694, and UM2-AR067678).

Background Systemic lupus erythematosus (SLE) is an autoimmune disorder with a variable clinical presentation and periods of waxing and waning disease. Heterogeneity in SLE is influenced by genetic and non-genetic susceptibility found in different ethnicities that drive disease expression and severity. The immune pathways that contribute to heightened disease activity in lupus and immune variation by race are critical to understanding SLE disease mechanisms and outcomes. Methods Peripheral whole blood samples of European or African American healthy controls (n=18) and SLE patients with either high (SLEDAI4) (n=20) or low (SLEDAI Results European American SLE patients with high disease activity were differentiated from patients with low disease activity by reduced frequencies of peripheral B cells, specifically naive B cells (CD27-IgD+CD24 lo) (p=0.0101) and double negative B cells (CD27-IgD-) (p=0.0220), while African American patients with high disease activity had elevated frequencies of memory B cells (CD27 +IgD CD38+) (p Conclusions Our results support a model where race influences heightened SLE disease activity mechanisms with alterations in B cell signaling, and greater dysregulation in phospho-signaling and pro-inflammatory soluble mediators observed in African American patients. Funding Source(s): NIH (U19AI082714, U19AI082719, U54GM104938, P30GM103510, U01AI101934)

Background A universal hallmark of systemic lupus erythematosus (SLE) is the presence of antinuclear antibodies (ANAs). While their cognate antigens and ANA levels can associate with SLE subphenotypes, many individuals develop ANAs years prior to autoimmune disease manifestations or never develop any clinical symptoms. At present, the mechanisms governing the transition from autoantibody production to disease onset remain unknown. Methods To ascertain differences in immune cell populations and their transcriptional state, we performed scRNA-seq on peripheral blood mononuclear cells from 30 patients, including African and European American individuals with lupus-associated autoantibodies (n=10), SLE patients (n=10), and ANA- healthy controls (n=10). Transcriptomes were analyzed using canonical correlation analysis, the results of which were used by a smart local moving algorithm to identify cell population clusters and by Uniform Manifold Approximation and Projection for visualization. Differential expression of genes was assessed using Model-based Analysis of Single Cell Transcriptomics. Results Distinct cell populations could be identified for each disease classification. EA ANA +individuals showed a decrease in the number of naive B cells relative to SLE and healthy individuals; in contrast, AA ANA +individuals had higher numbers of centrocytes. Additionally, AA SLE patients demonstrated an enrichment of plasma cells relative to both ANA +and healthy individuals while FCRL5, a marker of dysfunctional B cells, was increased only in SLE memory B cells. Overall, AA ANA +cell populations showed an upregulation in a number of heat shock genes in ANA+ (HSPA6, HSPA1A, DNAJB1). Multipotent progenitor (MPP) cells from SLE patients of both ethnicities were marked by upregulation of interferon stimulated genes; ANA +MPPs, however, differed in that cells from AA individuals had an upregulation of housekeeping genes (GAPDH, PGK1) while anti-inflammatory NFKB1A was increased in EA MPPs. Comparison of a manually curated list of regulatory factors demonstrated dysregulation of several genes, including upregulation of TGFB across all AA ANA +cell clusters and an increase in CD46 in EA ANA +clusters. Similarly, gene set enrichment analysis of differentially regulated genes indicated activation of stress response pathways in AA and EA ANA +individuals. Conclusions These data indicate that substantial transcriptional and cell population differences exist among immune cells from ANA+, SLE, and healthy individuals, including an activated stress response and enhanced immune regulation in the cells from ANA +individuals. Our results suggest that cells from ANA +individuals may be regulating reactions to stimulation, and exhaustion of that response may allow for transition to SLE. Funding Source(s): NIH U54GM104938, U01AI101934, U19AI082714, and P30AR053483.

Background Anti-nuclear antibody (ANA) positivity is a principal feature of individuals with an autoimmune disease, yet up to one in five healthy individuals are ANA-positive (ANA+) and will never develop overt disease. Understanding differences in immune cell physiology between ANA+healthy individuals and individuals with clinical SLE remains a critical goal in the understanding of SLE pathogenesis across ethnicities. Methods Blood specimens and information on disease activity were collected from European (EA) and African American (AA) individuals classified and matched in groups as ANA- healthy controls (n=24), ANA +healthy (n=24) or SLE patients (n=24). Single-cell analysis of cell surface markers was completed by mass cytometry on PBMCs and cellular heterogeneity was visualized using tSNE (figure 1A–B) and manual gating. Further, phospho-specific flow cytometry was used to measure basal levels of pERK, pPLCg2 and p38 and expression following CD3/CD28 (TCR) and anti-IgG and IgM (BCR) stimulation. Whole genome RNA-sequencing was performed on flow cytometry sorted T cells, B cells and monocytes from 35 matched ANA-, ANA +and SLE patients followed by weighted correlation network analysis (WGCNA) and pathway enrichment analyses. Results Both European and African American SLE patients were distinguished from healthy individuals by T cell proliferation (p=0.002) (figure 1C), plasmacytoid dendritic cell activation (p=0.021) and elevated stem cell factor (p=0.0003). EA ANA+healthy individuals exhibited greater immune regulation with reduced T cell numbers (p=0.002) (figure 1C), decreased activation of dendritic cells (p=0.039) and transitional B cells (0.033), and elevated expression of the inhibitory receptor CD85j (p=0.042) on specific immune cell subsets compared to ANA- healthy subjects. Further, a module associated with hematopoiesis, T cell activation and intrinsic apoptosis signaling pathways is expressed at a higher level in T cells of EA ANA+individuals. In contrast, AA ANA+healthy individuals had elevated plasma levels of IL-6 (p=0.018) and reduced inhibitory receptor expression (p=0.0089) compared to ANA- healthy controls. Gene expression modules associated with viral responses and type I IFN pathway activation were identified in AA SLE patient B cells, while similar expression modules were only found in the monocytes of European American SLE patients. Conclusions These results highlight the importance of stem cell factor and T cell expansion in SLE pathogenesis, and suggest that mechanisms of SLE pathogenesis differ by ethnicity. ANA +European Americans may have more effective regulatory mechanisms in place to prevent transition to classified autoimmune disease. Funding Source(s): This work was supported by the NIH under award numbers U54GM104938, U01AI101934, U19AI082714, and P30AR053483.

Background Although systemic lupus erythematosus (SLE) disproportionately affects minority racial groups, they are significantly under-represented in clinical trials. This produces underpowered conclusions in race-based sub-group analyses. The decision to participate in clinical research is complex. Primary care providers (PCPs) have the ability to introduce the idea of clinical trials and to refer to specialists who participate in clinical trials. We evaluated SLE knowledge and implicit bias in clinical research participation in both PCPs and lupus patients. Methods Lupus patients and PCPs completed a pre-test consisting of knowledge and belief questions followed by an educational program about lupus, clinical research, and human subjects protections. The same questions were repeated as part of the post-test. Responses to knowledge questions were analyzed by Fishers exact test for between group (patients vs. PCPs) comparisons or McNemars test for within group (pre-test vs. post-test) comparisons. Belief questions were scored on a Likert scale and analyzed by Mann-Whitney or Wilcoxon matched pairs for between group and within group comparisons. Results 55 providers and 32 lupus patients completed the questionnaires and educational program. Knowledge topics included 1) triggers of SLE, 2) lupus racial differences, and 3) informed consent. There was a statistically significant difference between PCPs and patients on the pre-test informed consent topic (PCP 46% correct and patient 15%, p=0.0005). Post-test results were similar between groups. The education program resulted in improvement in knowledge scores for PCPs (Q1 42% to 85%, p Conclusions Beliefs about race, education, and poverty may impact referral to clinical trial centers and clinical trial participation. More than a third of PCPs believe that indigent people are poor candidates for clinical trials. Despite a shift away from this attitude following an educational program, providers remained much more likely than lupus patients to hold this opinion. Funding Source(s): HHS-OMH-CPI-MP-17-002-1

Detecting autoimmune diseases at an early stage is crucial for effective treatment and disease management to slow disease progression and prevent irreversible organ damage. In many autoimmune diseases, disease-specific autoantibodies are produced by B cells in response to soluble autoantigens due to defects in B cell tolerance mechanisms. Autoantibodies accrue early in disease development, and several are so disease-specific they serve as classification criteria. In this study, we established a high-throughput, sensitive, intact serum autoantibody analysis platform based on the optimization of a one dimensional ultra-high-pressure liquid chromatography top-down mass spectrometry platform (1D UPLC-TDMS). This approach has been successfully applied to a 12 standard monoclonal antibody antigen-binding fragment (Fab) mixture, demonstrating the feasibility to separate and sequence intact antibodies with high sequence coverage and high sensitivity. We then applied the optimized platform to characterize total serum antibody Fabs in a systemic lupus erythematosus (SLE) patient sample and compared it to healthy control samples. From this analysis, we show that the SLE sample has many dominant antibody Fab-related mass features unlike the healthy controls. To our knowledge, this is the first top-down demonstration of serum autoantibody pool analysis. Our proposed approach holds great promise for discovering novel serum autoantibody biomarkers that are of interest for diagnosis, prognosis, and tolerance induction, as well as improving our understanding of pathogenic autoimmune processes.

Systemic lupus erythematosus (SLE) is a highly variable autoimmune disease that can involve severe organ-threatening symptoms, such as lupus nephritis. Certain drugs, such as mycophenolate mofetil (MMF), are effective at reducing morbidity associated with nephritis; however, the immune pathways associated with disease suppression are poorly defined. Here, we provide evidence that MMF inhibits phosphorylation of STAT3 and other associated immune pathways. Using mass cytometry and bead-based or ELISA assays, the systemic phenotype of SLE patients not taking (MMF–) or taking (MMF+) MMF were studied. MMF+ SLE patients had significant reductions in total numbers of transitional B cells, plasmablasts, and T cells, specifically CD4(+) Th17-type and CD4(+) Treg-type cells, compared with MMF– patients. Plasma soluble mediators were decreased in MMF+ patients including chemokines (MIG/CXCL9 and SDF-1α/CXCL12) and growth factors (VEGF-A and PDGF-BB). Soluble mediators and cell subsets grouped by functional properties revealed significant modifications associated with STAT3 and B cell pathways. Further, healthy PBMCs treated with IL-6 revealed a reduction in p-STAT3 following the addition of mycophenolic acid (the active metabolite of MMF). In conclusion, the inhibition of STAT3 phosphorylation by MMF may explain the effectiveness of this treatment in SLE patients, since increased levels of p-STAT3 are associated with disease pathology.

Memory B cells that are generated during an infection or following vaccination act as sentinels to guard against future infections. Upon repeat antigen exposure memory B cells differentiate into new antibody-secreting plasma cells to provide rapid and sustained protection. Some pathogens evade or suppress the humoral immune system, or induce memory B cells with a diminished ability to differentiate into new plasma cells. This leaves the host vulnerable to chronic or recurrent infections. Single cell approaches coupled with next generation antibody gene sequencing facilitate a detailed analysis of the pathogen-specific memory B cell repertoire. Monoclonal antibodies that are generated from antibody gene sequences allow a functional analysis of the repertoire. This review discusses what has been learned thus far from analysis of diverse pathogen-specific memory B cell compartments and describes major differences in their repertoires. Such information may illuminate ways to advance the goal of improving vaccine and therapeutic antibody design.

Infections remain a significant source of morbidity and mortality in systemic lupus erythematosus (SLE). Both the underlying disease process and therapeutic immunosuppression can increase susceptibility to a wide variety of pathogens while influencing the clinical course of infections. Because of the atypical presentation of many infections and the varied array of baseline symptoms, distinguishing an acute infection from a lupus flare is a common clinical challenge in treating SLE patients. This challenge is compounded by the need to suppress the immune system enough to prevent autoimmune complications without making patients overly vulnerable to infection. This chapter reviews the impact of infections on lupus morbidity and mortality, risk factors for infection, the clinical spectrum of infections in lupus, proposed mechanisms of infection susceptibility, and biomarkers or other factors that may assist the clinician when differentiating active infection from an SLE disease flare.

SLE is a clinically heterogeneous disease characterized by an unpredictable relapsing-remitting disease course. Although the etiology and mechanisms of SLE flares remain elusive, Epstein-Barr virus (EBV) reactivation is implicated in SLE pathogenesis. This study examined the relationships between serological measures of EBV reactivation, disease activity, and interferon (IFN)-associated immune pathways in SLE patients. Sera from adult SLE patients (n = 175) and matched unaffected controls (n = 47) were collected and tested for antibodies against EBV-viral capsid antigen (EBV-VCA; IgG and IgA), EBV-early antigen (EBV-EA; IgG), cytomegalovirus (CMV; IgG), and herpes simplex virus (HSV-1; IgG). Serological evidence of EBV reactivation was more common in SLE patients compared to controls as demonstrated by seropositivity to EBV-EA IgG (39% vs 13%; p = 0.0011) and EBV-VCA IgA (37% vs 17%; p = 0.018). EBV-VCA, CMV1, and HSV-1 IgG seropositivity rates did not differ between SLE patients and controls. Furthermore, concentrations of EBV-VCA (IgG and IgA) and EBV-EA (IgG) were higher in SLE patients. SLE patients with high disease activity had increased concentrations of EBV-VCA IgA (mean ISR 1.34 vs. 0.97; p = 0.041) and EBV-EA IgG levels (mean ISR 1.38 vs. 0.90; p = 0.007) compared with those with lower disease activity. EBV reactivation was associated with enhanced levels of the IFN-associated molecule IP-10 (p, Highlights • Serologic markers of EBV reactivation are more common in SLE patients. • Elevated EBV reactivation is associated with higher SLE disease activity. • EBV serologic reactivation correlates with elevated IP-10, IL-10, and BLyS levels. • EBV reactivation occurs in SLE clusters with robust inflammatory and IFN responses.

Objective Antinuclear antibodies (ANAs) are detected in ∼18% of females, yet autoimmune disease develops in only 5–8%. Immunologic differences between ANA-positive healthy individuals and patients with systemic lupus erythematosus (SLE) may elucidate the regulatory mechanisms by which ANA-positive individuals avoid transition to clinical autoimmune disease. Methods Healthy individuals (n = 790) were screened for autoantibodies specific for 11 antigens associated with lupus, systemic sclerosis, and Sjogren's syndrome. From this screening, 31 European American ANA-positive healthy individuals were selected and demographically matched to ANA-negative controls and SLE patients. Serum cytokine profiles, leukocyte subset frequency, and reactivity were analyzed by multiplex assays, immunophenotyping, and phosphospecific flow cytometry. Results Of 790 individuals screened, 57 (7%) were ANA-positive. The majority of proinflammatory cytokines, including interferon-γ (IFNγ), tumor necrosis factor, interleukin-17 (IL-17), and granulocyte colony-stimulating factor, exhibited a stepwise increase in serum levels from ANA-negative controls to ANA-positive healthy individuals to SLE patients (P < 0.0001). IFNα, IFNβ, IL-12p40, and stem cell factor/c-Kit ligand were increased in SLE patients only (P < 0.05). B lymphocyte stimulator (BlyS) was elevated in SLE patients but decreased in ANA-positive individuals (P < 0.001). Further, IL-1 receptor antagonist (IL-1Ra) was down-regulated in SLE patients only (P < 0.0001). ANA-positive individuals had increased frequencies of monocytes, memory B cells, and plasmablasts and increased levels of pSTAT-1 and pSTAT-3 following IFNα stimulation compared with ANA-negative controls (P < 0.05). Conclusion ANA-positive healthy individuals exhibit dysregulation in multiple immune pathways yet differ from SLE patients by the absence of elevated IFNs, BLyS, IL-12p40, and stem cell factor/c-Kit ligand. Further, severely decreased levels of IL-1Ra in SLE patients compared with ANA-positive individuals may contribute to disease development. These results highlight the importance of IFN-related pathways and regulatory elements in SLE pathogenesis.

Although the U.S. National Academy of Sciences concluded that anthrax vaccine adsorbed (AVA) has an adverse event (AE) profile similar to those of other adult vaccines, 30 to 70% of queried AVA vaccinees report AEs. AEs appear to be correlated with certain demographic factors, but the underlying immunologic pathways are poorly understood. We evaluated a cohort of 2,421 AVA vaccinees and found 153 (6.3%) reported an AE. Females were more likely to experience AEs (odds ratio [OR] = 6.0 [95% confidence interval {CI} = 4.2 to 8.7]; P < 0.0001). Individuals 18 to 29 years of age were less likely to report an AE than individuals aged 30 years or older (OR = 0.31 [95% CI = 0.22 to 0.43]; P < 0.0001). No significant effects were observed for African, European, Hispanic, American Indian, or Asian ancestry after correcting for age and sex. Additionally, 103 AEs were large local reactions (LLRs), whereas 53 AEs were systemic reactions (SRs). In a subset of our cohort vaccinated 2 to 12 months prior to plasma sample collection ( n = 75), individuals with LLRs ( n = 33) had higher protective-antigen (PA)-specific IgE levels than matched, unaffected vaccinated individuals ( n = 50; P < 0.01). Anti-PA IgE was not associated with total plasma IgE, hepatitis B-specific IgE, or anti-PA IgG in individuals who reported an AE or in matched, unaffected AVA-vaccinated individuals. IP-10 was also elevated in sera of individuals who developed LLRs ( P < 0.05). Individuals reporting SRs had higher levels of systemic inflammation as measured from C-reactive protein ( P < 0.01). Thus, LLRs and SRs are mediated by distinct pathways. LLRs are associated with a vaccine-specific IgE response and IP-10, whereas SRs demonstrate increased systemic inflammation without a skewed cytokine profile.

Objective To perform a genome-wide DNA methylation study to identify differential DNA methylation patterns in subchondral bone underlying eroded and intact cartilage from patients with hip osteoarthritis (OA) and to compare these with DNA methylation patterns in overlying cartilage. Methods Genome-wide DNA methylation profiling using Illumina HumanMethylation 450 arrays was performed on eroded and intact cartilage and subchondral bone from within the same joint of 12 patients undergoing hip arthroplasty. Genes with differentially methylated CpG sites were analyzed to identify shared pathways, upstream regulators, and overrepresented gene ontologies, and these patterns were compared with those of the overlying cartilage. Histopathology was graded by modified Mankin score and assessed for correlation with DNA methylation. Results We identified 7,316 differentially methylated CpG sites in subchondral bone underlying eroded cartilage, most of which (∼75%) were hypomethylated, and 1,397 sites in overlying eroded cartilage, 126 of which were shared. Samples clustered into 3 groups with distinct histopathologic scores. We observed differential DNA methylation of genes including the RNA interference-processing gene AGO2, the growth factor TGFB3, the OA suppressor NFATC1, and the epigenetic effector HDAC4. Among known susceptibility genes in OA, 32 were differentially methylated in subchondral bone, 8 were differentially methylated in cartilage, and 5 were shared. Upstream regulator analysis using differentially methylated genes in OA subchondral bone showed a strong transforming growth factor β1 signature (P = 1 × 10(-40) ) and a tumor necrosis factor family signature (P = 3.2 × 10(-28) ), among others. Conclusion Our data suggest the presence of an epigenetic phenotype associated with eroded OA subchondral bone that is similar to that of overlying eroded OA cartilage.

Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145 μg/mL kappa, 2.82 μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies.

Objective Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a strong genetic component. We undertook the present work to perform the first genome-wide association study on individuals from the Americas who are enriched for Native American heritage. Methods We analyzed 3,710 individuals from the US and 4 countries of Latin America who were diagnosed as having SLE, and healthy controls. Samples were genotyped with HumanOmni1 BeadChip. Data on out-of-study controls genotyped with HumanOmni2.5 were also included. Statistical analyses were performed using SNPtest and SNPGWA. Data were adjusted for genomic control and false discovery rate. Imputation was performed using Impute2 and, for classic HLA alleles, HiBag. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Results The IRF5–TNPO3 region showed the strongest association and largest OR for SLE (rs10488631: genomic control–adjusted P [Pgcadj] = 2.61 × 10−29, OR 2.12 [95% CI 1.88–2.39]), followed by HLA class II on the DQA2–DQB1 loci (rs9275572: Pgcadj = 1.11 × 10−16, OR 1.62 [95% CI 1.46–1.80] and rs9271366: Pgcadj = 6.46 × 10−12, OR 2.06 [95% CI 1.71–2.50]). Other known SLE loci found to be associated in this population were ITGAM, STAT4, TNIP1, NCF2, and IRAK1. We identified a novel locus on 10q24.33 (rs4917385: Pgcadj = 1.39 × 10−8) with an expression quantitative trait locus (eQTL) effect (Peqtl = 8.0 × 10−37 at USMG5/miR1307), and several new suggestive loci. SLE risk loci previously identified in Europeans and Asians were corroborated. Local ancestry estimation showed that the HLA allele risk contribution is of European ancestral origin. Imputation of HLA alleles suggested that autochthonous Native American haplotypes provide protection against development of SLE. Conclusion Our results demonstrate that studying admixed populations provides new insights in the delineation of the genetic architecture that underlies autoimmune and complex diseases.

Objective Autoantibodies reactive with Ro52 (tripartite motif−containing protein 21 [TRIM21]) are detected in 70% of patients with primary Sjogren's syndrome (SS). TRIM21 belongs to a 34-member C-IV family of TRIM proteins. Although autoantibodies against other TRIM proteins within the C-IV family have been detected in the sera of patients with primary SS, their clinical relevance remains unclear. This study was undertaken to investigate the frequency of anti-TRIM38 in patients with primary SS and evaluate its association with various clinical measures of the disease. Methods Serum samples from patients with primary SS (n = 235) and controls (n = 50) were analyzed for reactivity with in vitro−transcribed and −translated 35S-methionine−labeled TRIM38 protein. The associations of anti-TRIM38 with various laboratory and clinical measures of primary SS were evaluated. Reactivity of anti-TRIM38 with different structural domains of TRIM38 was analyzed. Affinity-purified anti-TRIM38 antibodies were used to immunoprecipitate TRIM21. Results TRIM38-reactive autoantibodies were detected in the sera of 24 of the 235 patients with primary SS and 2 of the 50 controls. Anti-TRIM38 positivity was significantly associated with the presence of anti-Ro60, anti-Ro52, anti-La, rheumatoid factor, and hypergammaglobulinemia. Clinically, anti-TRIM38 was associated with significantly higher ocular surface staining scores, lower Schirmer's test scores, and minor labial salivary gland biopsy focus scores of ≥3.0. Anti-TRIM38 antibodies mainly recognized the cortactin-binding protein 2 (CortBP-2; amino acids 128–238) and the B30.2/SPRY (amino acids 268–465) domains on TRIM38. Affinity-purified antibodies to TRIM38–CortBP-2 and TRIM38–B30.2/SPRY domains reacted with TRIM21. Conclusion Our data demonstrate that anti-TRIM38 specificity arising in a subset of patients with primary SS is associated with increased severity of the disease.

Secreted toxin B (TcdB) substantially contributes to the pathology observed during Clostridium difficile infection. To be successfully incorporated into a vaccine, TcdB-based immunogens must stimulate the production of neutralizing antibody (Ab)-encoding memory B cells (Bmem cells). Despite numerous investigations, a clear analysis of Bmem cellular responses following vaccination against TcdB is lacking. B6 mice were therefore used to test the ability of a nontoxigenic C-terminal domain (CTD) fragment of TcdB to induce Bmem cells that encode TcdB-neutralizing antibody. CTD was produced from the historical VPI 10463 strain (CTD1) and from the hypervirulent strain NAP1/BI/027 (CTD2). It was then demonstrated that CTD1 induced strong recall IgG antibody titers, and this led to the development of functional Bmem cells that could be adoptively transferred to naive recipients. Bmem cell-driven neutralizing Ab responses conferred protection against lethal challenge with TcdB1. Further experiments revealed that an experimental adjuvant (Imject) and a clinical adjuvant (Alhydrogel) were compatible with Bmem cell induction. Reactivity of human Bmem cells to CTD1 was also evident in human peripheral blood mononuclear cells (PBMCs), suggesting that CTD1 could be a good vaccine immunogen. However, CTD2 induced strong Bmem cell-driven antibody titers, and the CTD2 antibody was neutralizing in vitro , but its protection against lethal challenge with TcdB2 was limited to delaying time to death. Therefore, CTD from different C. difficile strains may be a good immunogen for stimulating B cell memory that encodes in vitro neutralizing Ab but may be limited by variable protection against intoxication in vivo .

Leptin is abnormally elevated in the plasma of patients with systemic lupus erythematosus (SLE), where it is thought to promote and/or sustain pro-inflammatory responses. Whether this association could reflect an increased genetic susceptibility to develop SLE is not known, and studies of genetic associations with leptin-related polymorphisms in SLE patients have been so far inconclusive. Here we genotyped DNA samples from 15,706 SLE patients and healthy matched controls from four different ancestral groups, to correlate polymorphisms of genes of the leptin pathway to risk for SLE. It was found that although several SNPs showed weak associations, those associations did not remain significant after correction for multiple testing. These data do not support associations between defined leptin-related polymorphisms and increased susceptibility to develop SLE. © 2015 Elsevier Inc.

Earlier studies showed that the DNA-binding protein, Bright/ARID3a bound to a subset of human and mouse immunoglobulin heavy chain promoters where it enhanced expression. Indeed, mice with transgenic expression of ARID3a in all B lymphocytes have expanded MZ B cells and produce anti-nuclear antibodies (ANAs). Consistent with our findings in mice, we observed that human systemic lupus erythematosus (SLE) patients had expanded numbers of peripheral blood ARID3a+ B cells that were associated with increased disease activity (p = 0.0038). We hypothesized that ARID3a+ naïve B cells would eventually produce autoantibodies, explaining associations between ARID3a expression and disease activity in lupus. Unlike healthy controls, ARID3a was expressed in the naïve B cell population in SLE patients, and we hypothesized that these might represent expansions of autoreactive cells. Therefore, monoclonal antibodies were generated from single-sorted naïve B cells derived from patients with normal (ARID3aN) and high (ARID3aH) numbers of ARID3a+ B cells. We found that ARID3a expression did not correlate with autoantibody expression. Furthermore, measures of antigen specificities of autoreactive antibodies did not reveal skewing toward particular proteins. These data suggest that the association of increased disease activity in SLE with numbers of ARID3a+ B lymphocytes may be mediated by an antibody-independent mechanism.

Background/Purpose High serum interferon alpha (IFN-α) is an important heritable phenotype in systemic lupus erythematosus (SLE) which is involved in primary disease pathogenesis. High vs. low levels of IFN-α are associated with disease severity and account for some of the biological heterogeneity between SLE patients. The aim of the study was to replicate and fine-map previously detected genetic associations with serum IFN-α in SLE. Methods We previously undertook a case-case genome-wide association study of SLE patients stratified by ancestry and extremes of phenotype in serum IFN-α. Single nucleotide polymorphisms (SNPs) in seven loci identified in this screen were selected for follow up in a large independent cohort of 1370 SLE patients (703 European-ancestry, 432 African ancestry, and 235 Amerindian ancestry). Each ancestral background was analyzed separately, and ancestry-informative markers were used to control for ancestry and admixture. Results We find a rare haplotype spanning the promoter region of EFNA5 that is strongly associated with serum IFN-α in both African-American and European-American SLE patients (OR = 3.0, p = 3.7 × 10−6). We also find SNPs in the PPM1H, PTPRM, and NRGN regions associated with IFN-α levels in European-American, Amerindian, and African-American SLE patients respectively. Many of these associations are within regulatory regions of the gene, suggesting an impact on transcription. Conclusion This study demonstrates the power of molecular sub-phenotypes to reveal genetic factors involved in complex autoimmune disease. The distinct associations observed in different ancestral backgrounds emphasize the heterogeneity of molecular pathogenesis in SLE.

Background:Sjögren’s syndrome (SS) is a complex autoimmune disease with exocrine gland dysfunction leading to substantial morbidity. There are 10 published genetic susceptibility loci.Objectives:Our genome-wide association study (GWAS) aimed to identify additional risk loci of genome-wide significance (GWS; pMethods:A total of 3232 cases and 17481 controls genotyped on GWAS arrays and 619 cases and 6171 controls genotyped on ImmunoChip (IC) arrays were imputed after quality control. Logistic regression was calculated adjusting for ancestry using the first 4 principal components to identify SS-associated SNPs. GWAS and IC results were meta-analyzed using weighted Z-scores. Bayesian statistics were used to assign posterior probabilities and define credible SNP sets for each locus. Bioinformatic analyses were used to predict functionality.Results:Seven novel loci exceeded GWS in the GWAS analysis:NAB1,MIR146A-PTTG1,XKR6,MAPT-CRHR1,RPTOR-CHMP6-BAIAP2,TYK2andSYNGR1. Meta-analysis with IC data identified three more novel loci exceeding GWS:CD247,PRDM1-ATG5andTNFAIP3. Several additional loci with suggestive association (pADAMTSL2,CGNL1andPHRF1.Several identified loci have reported functional implications in immune regulation and autoimmune disease. In lupus, rs2431697 correlated with rs2431098, which was shown to alterMIR146Aexpression, resulting in type I interferon pathway imbalance. Similarly,TYK2risk association reportedly drives interferon, IL10 and RET signaling pathways.PRDM1encodes Blimp-1, a master regulator of immune cell differentiation.CD247encodes the zeta subunit of the T cell receptor complex.XKR6is implicated in apoptotic cell ingestion.ATG5is also involved in apoptosis, as well as autophagy and antigen presentation.Additional bioinformatics analyses (Haploreg, Regulome DB, ENCODE, etc.) revealed immune-relevant functional implications for each risk locus. The SS-associated credible set included variants downstream ofTNFAIP3in a region reported to abolish looping between an enhancer and theTNFAIP3promoter in lupus and a coding variant that has been shown to alter NF-kB activity and neutrophil extra-cellular traps. The rs2293765 in the 5’ UTR ofNAB1showed evidence of enhancer/promoter activities. The rs2069235 in theSYNGR1locus showed enhancer and transcription start site activities in B and T cells. The rs7210219 in theMAPT-CRHR1locus showed enhancer/promotor activities in various tissues.Conclusion:We have identified ten novel genetic susceptibility loci associated with SS pathology. Our finding increases the current number of GWS regions in SS patients of European origin, from 10 to 20. Future work is needed to identify and characterize the functional variants in each region.Disclosure of Interests:Bhuwan Khatri: None declared, Tove Ragna Reksten: None declared, Kandice L Tessneer: None declared, Astrid Rasmussen Speakers bureau: Novartis, ThermoFischer, R Hal Scofield Grant/research support from: Pfizer, Simon J. Bowman Consultant of: Astrazeneca, Biogen, BMS, Celgene, Medimmune, MTPharma, Novartis, Ono, UCB, xtlbio, Glapagos, Speakers bureau: Novartis, Joel Guthridge Grant/research support from: Xencor, Bristol Myers Squibb, DXterity, Judith A. James Grant/research support from: Progentec Diagnostics, Inc, Consultant of: Abbvie, Novartis, Jannsen, Lars Ronnblom Grant/research support from: AZ, Speakers bureau: AZ, Blake M Warner: None declared, Xavier Mariette: None declared, Roald Omdal: None declared, Javier Martin Ibanez: None declared, Maria Teruel: None declared, Janicke Liaaen Jensen: None declared, Lara A Aqrawi: None declared, Øyvind Palm: None declared, Marie Wahren-Herlenius: None declared, Torsten Witte: None declared, Roland Jonsson: None declared, Maureen Rischmueller: None declared, A Darise Farris Speakers bureau: Biogen, Marta Alarcon-Riquelme: None declared, Wan-fai Ng: None declared, Kathy L Sivils: None declared, Gunnel Nordmark: None declared, Christopher Lessard: None declared

Background:Sjögren’s syndrome (SS) is a chronic, heterogenous autoimmune disease characterized by inflammatory destruction of the exocrine glands. Long non-coding RNAs (lncRNAs) have emerged as a functionally diverse class of non-protein coding RNA (ncRNA) with increasing implications in interferon signaling, immune cell regulation, and autoimmune disease pathology. The potential role of lncRNAs in SS pathogenesis is unknown.Objectives:To identify and characterize candidate lncRNAs with potential relevance to SS pathology.Methods:RNA-seq was used on whole blood from SS patients (n=30 antibody negative (Ro-); n=27 antibody positive (Ro+)) and healthy controls (HC, n=27) to identify differentially expressed (DE) lncRNAs (log2 fold change (FC) ≥ 2 or ≤ 0.5; padjIn vitrotime course experiments in HSB2 T cell lymphoblasts stimulated with PMA/Ionomycin (PMA/I) or type I interferon (IFN) were used to assess biological relevance.LINC01871function was further investigated by RNA-seq on a single cell clone of HSB2 with confirmed CRISPR-targetedLINC01871deletion (LINC01871-/-).Results:We identified a total of 1054 unique DE ncRNAs between Ro+, Ro-and/or a combined analysis relative to HC; of these, 45 (1 long intergenic ncRNA (lincRNA), 1 antisense, 43 pseudogenes) were overexpressed in all 3 SS subsets. To begin investigating the function of the previously undescribed lincRNA,LINC01871(SSRo-: FC=2.85; padj=1.1x10-4), we performed a correlation analysis of the SSRo-transcriptome, which found several co-expressed protein coding RNAs involved in immune regulation (THEMIS,TBX21,IL10RA,IL2RB,among many others). Similarly, Ingenuity Pathway Analysis of the SS transcriptome compared to HC, as well as several gene ontology enrichment analyses of publicly available RNA expression correlation databases, identified shared immune-related pathways including cytotoxic T cell, natural killer cell, and T cell regulation. To further study the role ofLINC01871in cytotoxic T cells, we used qRT-PCR to resolve the effects of PMA/I or type I IFN stimulation onLINC01871expression in the T lymphoblastoid HSB2 cells.LINC01871expression was downregulated after PMA/I stimulation, but unchanged with type I IFN stimulation. To explore the regulatory function ofLINC01871in T cells, we targetedLINC01871in HSB2 cells using CRISPR. To this end, we generated a single cellLINC01871-/-clone with no RNA expression by qPCR and confirmed homozygous deletion using DNA sequencing. RNA-seq analysis ofLINC01871-/-compared to unmodified HSB2 cells identified 1166 DE transcripts. Pathway analyses clustered the DE transcripts into similar immune regulatory, cytotoxic and T cell pathways identified in SSRo-whole blood RNA-seq and publicly available RNA-seq databases. Further, several prominent T cell regulatory transcripts that exhibited correlated upregulation withLINC01871in SSRo-whole blood RNA-seq also demonstrated downregulation afterLINC01871deletion:CD109(FC=-9.7; padj=5.3x10-16),IL22(FC=-8.1; padj=7.6x10-11),PDCD1(FC=-6.2; padj=1.1x10-6),THEMIS(FC=-3.8; padj=2.7x10-165) andTBX21(FC=-2.1; padj=3.3x10-25).Conclusion:LncRNAs are emerging as important regulators of immune function with increasing evidence of autoimmune disease relevance. Here, we leveraged RNA-seq, extensive bioinformatic data, and CRISPR technology to identify and functionally characterizeLINC01871as a potential mediator of the dysregulated T cell inflammatory response pathways implicated in SS pathogenesis.Disclosure of Interests:Michelle L Joachims: None declared, Bhuwan Khatri: None declared, Kandice L Tessneer: None declared, Anna M Stolarczyk: None declared, Graham B Wiley: None declared, Astrid Rasmussen Speakers bureau: Novartis, ThermoFischer, Joel Guthridge Grant/research support from: Xencor, Bristol Myers Squibb, DXterity, Judith A. James Grant/research support from: Progentec Diagnostics, Inc, Consultant of: Abbvie, Novartis, Jannsen, R Hal Scofield Grant/research support from: Pfizer, Kathy L Sivils: None declared, Indra Adrianto: None declared, Christopher Lessard: None declared

Background:Sjögren’s Syndrome (SS) and Systemic Lupus Erythematosus (SLE) are distinct chronic, complex autoimmune diseases with shared characteristics such as autoantibodies, heightened interferons, and polyarthritis. SS and SLE genome-wide association studies (GWAS) report strong associations with theDDX6-CXCR5risk interval. DDX6 suppresses interferon stimulated gene expression and CXCR5 regulates T cell functions implicated in autoimmunity.Objectives:To identify functional variants that impact regulation in theDDX6-CXCR5interval.Methods:Fine-mapping was done using ImmunoChip data from 3785 SLE, 1916 SS cases and 6893 population controls of European ancestry that were imputed and tested for SNP-trait association. Bayesian statistics assigned posterior probabilities to SNPs and defined a credible set of risk variants. Bioinformatic analyses further prioritized variants with predicted functionality. Electrophoretic mobility shift assays (EMSAs) and luciferase expression were used to validate predicted SNPs in EBV transformed B (EBV B) cells.Results:While some differences were observed, the overall SS and SLE association signals were similar. SNP-SS rs9736016 nearCXCR5and SNP-SLE rs76409436 nearDDX6were the most significant but did not show evidence of functionality. Bayesian statistics defined credible sets of variants in strong D’ in common between both SS and SLE. Bioinformatics analyses (Haploreg, RegulomeDB, ENCODE data, etc) further refined the credible set and identified 5 common SNPs with strong evidence of functionality in immune cell types: rs4938572, rs4936443, rs57494551, rs7117261 and rs4938573. EMSAs showed a significant increase in protein binding to the risk allele of rs57494551 (p=0.0001), rs7117261 (p=0.0001) and rs4938573 (p=0.0003), but not the others, using nuclear lysates from EBV B cells. Luciferase vectors with a minimal promoter or no promoter were used to test for enhancer or promoter activity, respectively. To this end, the rs57494551 risk allele exhibited a significant increase in enhancer activity (p=0.0001). In contrast, the rs7117261 risk allele decreased enhancer activity (p=0.018). The rs4938573 risk allele decreased enhancer (p=0.043) and promoter (p=0.024) activity. While rs7117261 or rs4938573 were not reported in eQTL databases, GTex data reported rs57494551 as an eQTL that altersDDX6expression in whole blood (p=1.8E-7). Additionally, these functional SNPs have been associated with looping events to several proximal promoters in nearby genes in immune cells.Conclusion:SS and SLE have similar genomic architecture across theDDX6-CXCR5risk interval. Multiple variants in the credible set exhibited allele specific changes in protein binding, as well as modified enhancer activity, promoter activity or both. Ongoing studies will use Cas9 in EBV B cells to determine which other loci are within the local regulatory network.Disclosure of Interests:Mandi M Wiley: None declared, Bhuwan Khatri: None declared, Kandice L Tessneer: None declared, Michelle L Joachims: None declared, Anna M Stolarczyk: None declared, Astrid Rasmussen Speakers bureau: Novartis, ThermoFischer, Simon J. Bowman Consultant of: Astrazeneca, Biogen, BMS, Celgene, Medimmune, MTPharma, Novartis, Ono, UCB, xtlbio, Glapagos, Speakers bureau: Novartis, Lida Radfar: None declared, Roald Omdal: None declared, Marie Wahren-Herlenius: None declared, Blake M Warner: None declared, Torsten Witte: None declared, Roland Jonsson: None declared, Maureen Rischmueller: None declared, Patrick M Gaffney: None declared, Judith A. James Grant/research support from: Progentec Diagnostics, Inc, Consultant of: Abbvie, Novartis, Jannsen, Lars Ronnblom Grant/research support from: AZ, Speakers bureau: AZ, R Hal Scofield Grant/research support from: Pfizer, Xavier Mariette: None declared, Wan-fai Ng: None declared, Kathy L Sivils: None declared, Gunnel Nordmark: None declared, Betty Tsao: None declared, Christopher Lessard: None declared

INTRODUCTION Sjögren’s syndrome (SS) is a rheumatic autoimmune disease characterized by focal lymphocytic infiltrates in the lacrimal and salivary glands, severe dry mouth and eyes, pain and debilitation. Diagnosis requires autoantibodies to ubiquitous Ro antigens or a lip biopsy positive for focal lymphocytic infiltrates. Here we used human proteome arrays to identify novel antibodies in plasma from Ro positive and Ro/La antibody negative SS patients compared with healthy controls. METHODS Anti-Ro positive (n=15) and anti-Ro negative (n=15) cases meeting 2016 ACR/EULAR classification criteria for SS were age, race, and sex matched with each other and healthy controls (n=15). Plasma IgG binding to human proteome arrays containing >19,500 recombinant human proteins representing >80% of the human proteome (HuProt v3.2 arrays, CDI Laboratories) was assessed. Data were normalized by the Robust Linear Model using the PAA Bioconductor Package in R and log intensity values for each protein generated. Thresholds of mean + 4SD were established using the controls. Antigens bound by IgG more frequently in cases compared to controls (p RESULTS IgG from Ro positive SS cases significantly bound 18 proteins, including the canonical SS antigens Ro60 and Ro52. IgG from Ro negative SS cases significantly bound 4 proteins compared to controls, 3 of which were shared with the Ro positive group. Binding to any one of 4 novel proteins identified 73% of the Ro negative SS cases. CONCLUSION A total of 17 novel antigen specificities were identified in SS, with 4 antigens being bound by plasma IgG from Ro/La negative SS cases. These antigens may be useful for diagnosing SS without a lip biopsy.

ObjectivesTo evaluate the association between lupus severity and cell-bound complement activation products (CB-CAPs) or low complement proteins C3 and C4.MethodsAll subjects (n=495) fulfilled the American College of Rheumatology (ACR) classification criteria for SLE. Abnormal CB-CAPs (erythrocyte-bound C4d or B-lymphocyte-bound C4d levels >99th percentile of healthy) and complement proteins C3 and C4 were determined using flow cytometry and turbidimetry, respectively. Lupus severity was estimated using the Lupus Severity Index (LSI). Statistical analysis consisted of multivariable linear regression and groups comparisons.ResultsAbnormal CB-CAPs were more prevalent than low complement values irrespective of LSI levels (62% vs 38%, respectively, p2=0.145).ConclusionAbnormalities in complement activation as measured by CB-CAPs are associated with increased LSI.

Multiple sclerosis (MS) is an autoimmune demyelinating disease with progressive neurodegeneration and complex etiology likely involving genetic and environmental factors. MS has been associated with Epstein Barr virus (EBV) infection, with patients often showing enhanced responses to EBV antigens. To determine whether abnormal EBV nuclear antigen-1 (EBNA-1) humoral immunity can serve as an initiator of autoimmune responses in MS, we investigated the fine specificities of the humoral immune response against EBNA-1 in MS patients using solid phase epitope mapping. Antibodies from MS patients recognized an EBNA-1 epitope spanning amino acids 411–426, previously unknown to be recognized specifically by untreated MS patients. Antibodies against this epitope cross-reacted to myelin basic protein (MBP). Furthermore, animals immunized with this EBNA-1 polypeptide mounted a response against MBP and developed signs of experimental autoimmune encephalitis (EAE). These data support a link between MS and EBV through antibodies that cross-react between EBV proteins and the MBP autoantigen.

Epstein Barr virus (EBV) is a gamma herpes virus associated with certain malignancies and autoimmune diseases. EBV maintains latency in B cells with occasional reactivation, in part by overcoming the host immune response with viral homologs of several human proteins. EBV interleukin 10 (vIL-10), a lytic phase protein, is a homolog of human IL-10 (hIL-10). The effect of vIL-10 on human monocytes, which are one of the first immune cells to respond to infection, is not known. To understand the role of vIL-10, monocytes from peripheral blood mononuclear cells were stimulated with hIL-10 or vIL-10. Human IL-10 stimulated STAT3 phosphorylation, which is required for suppression of inflammatory responses. However, vIL-10 induced significantly lower phosphorylation of STAT3 compared to hIL-10, and was less efficient in downregulating inflammatory genes. vIL-10 significantly reduced the expression of scavenger receptor CD163 on monocytes, suggesting inhibition of M2 polarization. Furthermore, uptake of apoptotic cells was reduced in vIL-10-stimulated monocytes compared to hIL-10-stimulated monocytes. A neutralizing antibody to IL-10R1 inhibited STAT3 phosphorylation induced by either hIL-10 or vIL-10, suggesting that vIL-10 signals through IL-10R1. Interestingly, vIL-10 suppressed hIL-10-induced STAT3 phosphorylation and inhibited upregulation of suppressors of inflammatory response by hIL-10. We further show that vIL-10 levels were significantly higher in plasma samples from systemic lupus erythematosus (SLE) patients compared to matched unaffected controls. vIL-10 levels did not correlate with hIL-10 levels, but were associated with levels of IgA antibodies to EBV viral capsid antigen, which is an indirect measure of viral reactivation. We propose that the suppression of hIL-10- induced anti-inflammatory genes by vIL-10, together with an increase in inflammatory gene expression, may overcome the anti-inflammatory effects of hIL-10 and exacerbate autoimmune responses in systemic autoimmune diseases.

ObjectiveExisting methods for grading lupus flares or improvement require definition-based thresholds as increments of change. Visual analogue scales (VAS) allow rapid, continuous scaling of disease severity. We analysed the performance of the SELENA SLEDAI Physician’s Global Assessment (SSPGA) and the Lupus Foundation of America-Rapid Evaluation of Activity in Lupus (LFA-REAL) as measures of improvement or worsening in SLE.MethodsWe evaluated the agreement between prospectively collected measures of lupus disease activity [SLE Disease Activity Index (SLEDAI), British Isles Lupus Assessment Group Index 2004 (BILAG 2004), Cutaneous Lupus Area and Severity Index (CLASI), SSPGA and LFA-REAL] and response [(SLE Responder Index (SRI)-4 and BILAG-Based Combined Lupus Assessment (BICLA)] in a clinical trial.ResultsFifty patients (47 females, mean age 45 (±11.6) years) were assessed at 528 consecutive visits (average 10.6 (±4.1) visits/patient). Changes in disease activity compared with baseline were examined in 478 visit pairs. SSPGA and LFA-REAL correlated with each other (r=0.936), and with SLEDAI and BILAG (SSPGA: r=0.742 (SLEDAI), r=0.776 (BILAG); LFA-REAL: r=0.778 (SLEDAI), r=0.813 (BILAG); all pConclusionSSPGA and LFA-REAL are reliable surrogates of common SLE trial end points and could be used as continuous or dichotomous response measures. Additionally, LFA-REAL can provide individualised scoring at the symptom or organ level.Trial registration numberNCT02270957.