BACKGROUND:Systemic autoimmune rheumatic diseases (SARDs) are a heterogeneous group of autoimmune conditions characterized by immune system dysregulation leading to chronic inflammation and tissue damage. The overlapping clinical manifestations make differential diagnosis challenging, highlighting the need for novel biomarkers to facilitate early diagnosis, stratification, and personalized treatment. METHODS:Five SARDs including idiopathic inflammatory myopathies (n = 210), rheumatoid arthritis (n = 84), systemic sclerosis (n = 100), Sjögren disease (n = 99), and systemic lupus erythematosus (n = 99), as well as healthy controls (n = 400) and controls with acute infectious diseases (n = 218) were selected for plasma protein profiling using Olink Explore 1536. Differential abundance analysis and machine learning were used to identify proteins with both known and novel association to SARDs. RESULTS:The five SARDs share hundreds of proteins with consistently altered abundance compared to both healthy and infectious controls, reflecting common underlying molecular dysregulation. Despite the overlap, we identify multiple proteins with higher abundance specific to individual SARDs. Machine learning further enables accurate classification of the five SARDs, identifying a panel of 48 proteins with high discriminatory performance, several of which are also supported by differential abundance analysis. CONCLUSIONS:Altogether, this explorative cross-sectional study demonstrates the importance of a pan-disease approach, including also infectious and healthy controls, to identify robust and disease-informative protein panels for improved classification of SARDs. Protein levels from this study are available open access through the Human Protein Atlas, facilitating further plasma proteome research on autoimmune disease.

OBJECTIVE:Tissue-specific biomarkers that are reliable and associated with clinical manifestations of idiopathic inflammatory myopathy (IIM) are lacking. The blood circulation in individuals serves as a central conduit, allowing communication between tissues and facilitating clearance and recycling of tissue-derived proteins. Thus, we applied a dual antibody-based proximity extension assay to profile >1000 proteins in subtypes of patients with IIM. METHODS:Plasma samples from 201 patients diagnosed with IIM at Karolinska University Hospital were analyzed using Olink Proximity Extension Assay technology. Clinical manifestations, disease activity measurements, and laboratory results were collected. First, we evaluated differential protein abundance across IIM subtypes. Then we calculated tissue scores for each disease subtype using tissue gene expression databases. Finally, interferon (IFN) pathway scores were calculated. RESULTS:Plasma from patients with dermatomyositis was enriched for IFN signalling and skin associated proteins, anti-synthetase syndrome (ASyS) for lung associated proteins, immune-mediated necrotizing myopathy (IMNM) for muscle associated proteins, and inclusion body myositis for T cell response proteins. Patients with IMNM showed the highest tissue score for skeletal muscles, and ASyS for lung tissue score. Skeletal muscle scores correlated strongly with muscle disease activity visual analog scale scores, creatine kinase levels, and muscle weakness score. Patients with interstitial lung disease along with anti-MDA5 and those with anti-Jo1 autoantibodies showed differential enrichment of surfactant proteins and IFN pathway activation. CONCLUSIONS:Circulatory proteome is a promising tool to capture distinct tissue-specific responses that correlate with disease activity and tissue injury in patients with IIM.

Background: Autoantibodies are found in up to 80 % of patients with idiopathic inflammatory myopathies (IIM) and are associated with distinct clinical phenotypes. Autoantibodies targeting cytosolic 5 '-nucleotidase 1A (antiNT5C1A) are currently the only known serum biomarker for the subgroup inclusion body myositis (IBM), although detected even in other autoimmune diseases. The aim of the study was to identify new autoimmune targets in IIM. Methods: In a first cross-sectional exploratory study, samples from 219 IIM (108 Polymyositis (PM), 80 Dermatomyositis (DM) and 31 IBM) patients, 349 Systemic Lupus Erythematosus (SLE) patients and 306 population controls were screened for IgG reactivity against a panel of 357 proteins using an antigen bead array. All samples were identified in the local biobank of the Rheumatology clinic, Karolinska University Hospital. Positive hits for the IBM subgroup were then validated in an independent larger cohort of 287 patients with IBM followed at nine European rheumatological or neurological centers. IBM serum samples were explored by antigen bead array and results validated by Western blot. As controls, sera from 29 patients with PM and 30 with DM, HLA-matched with the Swedish IBM cohort, were included. Demographics, laboratory, clinical, and muscle biopsy data of the IBM cohort was retrieved. Results: In the exploratory study, IgG reactivity towards NADH dehydrogenase 1 alpha subcomplex 11 (NDUFA11), a subunit of the membrane-bound mitochondrial respiratory chain complex I, was discovered with higher frequency in the IBM (9.7 %) than PM (2.8 %) and DM samples (1.3 %), although the difference was not statistically significant. Anti-NDUFA11 IgG was also found in 1.4 % of SLE and 2.0 % of population control samples. In the validation study, anti-NDUFA11 autoantibodies were detected in 10/287 IBM patients (3.5 %), 0/29 p.m. and 0/30 DM patients. Reactivity against NDUFA11 could be confirmed by Western blot. No statistically significant differences were found between patients with and without anti-NDUFA11 antibodies when comparing clinical, laboratory and histological data. However, we observed a trend of higher frequency of distal lower extremity muscle weakness, ragged red fibers and higher CK levels at time of diagnosis in the anti-NDUFA11 positive group. Co-existence of anti-NDUFA11 and anti-NT5C1A antibodies was not observed in any IBM patient. Conclusion: Our results reveal a new autoimmune target in the mitochondrial respiratory chain complex I that might be specifically associated with IBM. This is of particular interest as mitochondrial abnormalities are known histological findings in muscle biopsies of IBM patients.

Background Anti-synthetase syndrome (ASSD), a sub-group of idiopathic inflammatory myopathies (IIM) is characterized by the presence of autoantibodies targeting aminoacyl tRNA synthetases (aaRS) and specific clinical manifestations such as myositis and interstitial lung disease (ILD) [1]. Some of the most common anti-aaRS autoantibodies in ASSD are anti-Jo1, -PL7, -PL12 and-EJ. In addition, many anti-aaRS positive patients are also positive for anti-Ro52. Having the combination of anti-Jo1 and anti-Ro52 increases the risk of developing ILD [2]. The presence of autoantibodies is an important part of the classification of ASSD, however only autoantibodies of IgG isotype are usually analyzed in the clinical setting. In rheumatoid arthritis there is evidence that anti-citrullinated protein/peptide antibodies (ACPA) can be found as IgG, IgA and IgM, and importantly, specific isotypes might correlate with disease activity [3, 4]. Objectives To verify if other autoantibody isotypes, besides IgG, might be present in sera of patients with IIM/ASSD and to compare with the corresponding frequencies in population controls (PC). Methods Stored sera collected from consecutive 366 IIM patients and 156 age/gender matched PC at Karolinska University Hospital were retrospectively selected. The serum samples were screened for the presence of autoantibodies of isotypes IgG, IgA and IgM, against a panel of 20 antigens representing Jo1 (HisRS), PL7 (ThrRS), PL12 (AlaRS), EJ (GlyRS), and Ro52 (TRIM21) using a multiplex bead array assay. Results We identified IIM patients with autoantibodies of different isotypes, and a low frequency in PC (Figure 1). For anti-Jo1 autoantibodies we could detect IIM patients with only IgG (n=13), only IgM (n=8) and only IgA (n=4), but the majority had a combination of two (n=32) or three isotypes (n=16). For the other anti-aaRS autoantibodies the distribution was more equal to each of the three isotypes with anti-PL12 and anti-PL7 being represented by a slightly higher frequency of IgG and only a few patients had antibodies of more than one isotype targeting PL12, PL7 or EJ. The majority of anti-Ro52 positive IIM patients (n=52) only harbored IgG isotype. The combination of anti-Ro52 and anti-aaRS autoantibodies was identified in 28 patients (anti-Jo1 (n=19), -PL12 (n=2), -PL7 (n=3), and -EJ (n=4)). Most patients with such combination had anti-Ro52 IgG together with anti-aaRS IgG or IgG in combination with IgA and/or IgM. The exception was observed for three anti-Jo1 positive patients who had the combination anti-Ro52 IgG with only anti-Jo1 IgM and one anti-PL7 positive patient who had anti-Ro52 IgA together with anti-PL7 IgA and IgG. Figure 1. Venn diagrams showing reactivity in idiopathic inflammatory myopathies (IIM) (top) and population controls (PC) (bottom) for the three autoantibody isotypes IgG, IgA and IgM against five myositis antigens: Jo1 (HisRS), PL12 (AlaRS), ThrRS (PL7), EJ (GlyRS) and Ro52 (TRIM21). Conclusion The frequency of the different autoantibody isotypes seems to be autoantigen dependent. Our results suggest that for anti-aaRS autoantibodies it could be important to investigate additional autoantibody isotypes, as some patients only harbor autoantibodies of IgM or IgA isotypes but not IgG. The clinical relevance of the different antibody isotypes still needs to be determined. References [1]Mahler, M., et al., Rev, 2014. 13 (4-5): p. 367-71. [2]Huang, H.L., et al., J Clin Neurosci, 2020. [3]Arlestig, L., et al., Ann Rheum Dis, 2012. 71 (6): p. 825-9. [4]Roos Ljungberg, K., et al., Arthritis Res Ther, 2020. 22 (1): p. 274. Table 1. Total number of individuals and percentage (n (%)) in each group for each of the isotypes and antigens. anti-Jo1 anti-PL12 anti-PL7 anti-EJ anti-Ro52 IIM PC IIM PC IIM PC IIM PC IIM PC IgG 61 (16.7) 1 (0.6) 7 (1.9) 0 (0.0) 7 (1.9) 0 (0.0) 3 (0.8) 0 (0.0) 54 (14.8) 5 (3.2) IgA 20 (5.5) 0 (0.0) 2 (1.2) 1 (0.6) 3 (0.8) 2 (1.3) 1 (0.3) 1 (0.6) 3 (0.8) 1 (0.6) IgM 56 (15.3) 1 (0.6) 1 (0.3) 2 (1.3) 7 (1.9) 0 (0.0) 1 (0.3) 0 (0.0) 3 (0.8) 2 (1.3) Acknowledgements SciLifeLab facilities Autoimmunity and Serology Profiling and Human Antibody Therapeutics (Drug Discovery and Development). IMI project EUbOPEN, This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 875510. The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA and Ontario Institute for Cancer Research, Royal Institution for the Advancement of Learning McGill University, Kungliga Tekniska Hoegskolan, Diamond Light Source Limited. Disclosure of Interests Charlotta Preger Grant/research support from: IMI project EUbOPEN, Grant no 875510, Antonella Notarnicola: None declared, Cecilia Hellström: None declared, Edvard Wigren Grant/research support from: IMI project EUbOPEN, Grant no 875510, Ingrid E. Lundberg Shareholder of: Roche and Novartis, Consultant of: Corbus Pharmaceuticals Inc, Astra Zeneca, Bristol Myer´s Squibb, Corbus Pharmaceutical, EMD Serono Research & Development Institute, Argenx, Octapharma, Kezaar, Orphazyme, and Janssen, Grant/research support from: Astra Zeneca, Per-Johan Jakobsson Shareholder of: Gesynta Pharma, Consultant of: UCB, Grant/research support from: Gesynta Pharma, Helena Persson Employee of: Affibody AB, Susanne Gräslund Grant/research support from: IMI project EUbOPEN, Grant no 875510

Background To address the reactivity and affinity against histidyl-transfer RNA synthetase (HisRS) autoantigen of anti-Jo1 autoantibodies from serum and bronchoalveolar lavage fluid (BALF) in patients with idiopathic inflammatory myopathies/anti-synthetase syndrome (IIM/ASSD). To investigate the associations between the reactivity profile and clinical data over time. Methods Samples and clinical data were obtained from (i) 25 anti-Jo1(+) patients (19 sera with 16 longitudinal samples and 6 BALF/matching sera at diagnosis), (ii) 29 anti-Jo1(-) patients (25 sera and 4 BALF/matching sera at diagnosis), and (iii) 27 age/gender-matched healthy controls (24 sera and 3 BALF/matching sera). Reactivity towards HisRS full-length (HisRS-FL), three HisRS domains (WHEP, antigen binding domain (ABD), and catalytic domain (CD)), and the HisRS splice variant (SV) was tested. Anti-Jo1 IgG reactivity was evaluated by ELISA and western blot using IgG purified from serum by affinity chromatography. In paired serum-BALF, anti-Jo1 IgG and IgA reactivity was analyzed by ELISA. Autoantibody affinity was measured by surface plasmon resonance using IgG purified from sera. Correlations between autoantibody reactivity and clinical data were evaluated at diagnosis and longitudinally. Results Anti-Jo1 IgG from serum and BALF bound HisRS-FL, WHEP, and SV with high reactivity at the time of diagnosis and recognized both conformation-dependent and conformation-independent HisRS epitopes. Anti-HisRS-FL IgG displayed high affinity early in the disease. At the time of IIM/ASSD diagnosis, the highest autoantibody levels against HisRS-FL were found in patients ever developing interstitial lung disease (ILD) and arthritis, but with less skin involvement. Moreover, the reactivity of anti-WHEP IgG in BALF correlated with poor pulmonary function. Levels of autoantibodies against HisRS-FL, HisRS domains, and HisRS splice variant generally decreased over time. With some exceptions, longitudinal anti-HisRS-FL antibody levels changed in line with ILD activity. Conclusion High levels and high-affinity anti-Jo1 autoantibodies towards HisRS-FL were found early in disease in sera and BALF. In combination with the correlation of anti-HisRS-FL antibody levels with ILD and ILD activity in longitudinal samples as well as of anti-WHEP IgG in BALF with poor pulmonary function, this supports the previously raised hypothesis that the lung might have a role in the immune reaction in anti-Jo1-positive patients.

Mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer's disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signaling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterized. Here, we present phage display generated antibody single-chain variable fragments (scFvs) to human TREM2 immunoglobulin-like domain. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species, which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterization of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.

ABSTRACT Objectives Autoantibodies are thought to play a key role in the pathogenesis of idiopathic inflammatory myopathies (IIM). However, up to 40% of IIM patients, even those with clinical manifestations of anti-synthetase syndrome (ASSD), test seronegative to all known myositis-specific autoantibodies (MSAs). We hypothesized the existence of new potential autoantigens among human cytoplasmic aminoacyl tRNA synthetases (aaRS) in patients with IIM. Methods Plasma samples and clinical data from 217 patients with, 50 patients with ASSD, 165 without, and two with unknown ASSD status were included retrospectively, as well as serum from 156 age/sex-matched population controls. Samples were screened using a multiplex bead array assay for presence of autoantibodies against a panel of 118 recombinant protein variants, representing 33 myositis-related proteins, including all 19 cytoplasmic aaRS. Results We identified reactivity towards 16 aaRS in 72 of the 217 patients. Twelve patients displayed reactivity against nine novel aaRS. The novel autoantibody specificities were detected in four patients previously seronegative for MSAs and in eight with previously detected MSAs. We also confirmed reactivity to four of the most common aaRS (Jo1, PL12, PL7, and EJ (n=45)) and identified patients positive for anti-Zo, -KS, and -HA (n=10) that were not previously tested. A low frequency of anti-aaRS autoantibodies was detected in controls. Conclusion Our results suggest that most, if not all, cytoplasmic aaRS may become autoantigenic. Autoantibodies against new aaRS may be found in plasma of patients previously classified as seronegative with potential high clinical relevance.

Background: Idiopathic inflammatory myopathies (IIM) are rare chronic inflammatory diseases associated with high mortality and morbidity [1]. One sub-group of IIM, anti-synthetase syndrome (ASS), is characterized by the presence of autoantibodies that target aminoacyl transfer(t) RNA synthetases (aaRS), together with specific clinical manifestations such as myositis, interstitial lung disease (ILD), arthritis, mechanic’s hand, Raynaud’s syndrome and fever [2]. The most common anti-aaRS autoantibody, anti-Jo1 targeting histidyl tRNA synthetase (HisRS), is present in up to 20-30% of patients with IIM, and up to 90% of patients with myositis and ILD [3, 4]. Besides Jo1, there are today seven other identified autoantigens within the aaRS family. Objectives: A large part of patients with IIM, including individuals with clinical manifestations indicating ASS, test seronegative to all known myositis specific autoantibodies. However, these patients could potentially harbor autoantibodies against targets not tested for in clinic. In this study, we aimed at extending the detection of autoantibodies by including all cytoplasmic aaRS in the analysis of patients with IIM. We hypothesized the existence of new potential autoantigens within this protein family. Methods: The presence of anti-aaRS autoantibodies was determined using a multiplex suspension bead array assay on 242 IIM patients from the Karolinska University Hospital myositis cohort. A panel of 186 recombinant constructs, representing 57 proteins that included full-length or partial sequence overlaps between constructs of all cytoplasmic aaRS as well as other myositis related proteins, were coupled to magnetic color-coded beads and each plasma sample was tested against the complete antigen panel. Results: By the use of this multiplex method we identified patients with autoantibodies against many of the tested aaRS. Autoantibodies binding to HisRS have previously been shown to bind with higher reactivity to the WHEP domain of HisRS and this was also confirmed in this study. We confirmed reactivity against three of the other aaRS tested for in the clinic (PL-12, PL-7, and EJ). In addition, we identified patients positive for anti-Zo, -KS and -HA, autoantibodies usually not screened for in routine. Finally, our data indicates that there are autoantibodies binding to other aaRS than the previously known eight autoantigens, which will be presented. Conclusion: In this study, we could detect autoantibodies in plasma from patients with IIM, both against the most common aaRS autoantigens, but also against other aaRS that are usually not tested for in clinic. We conclude that it is important to continue the studies of anti-aaRS autoantibodies, and their correlation to clinical manifestations, and in the long run also include more aaRS autoantigens in clinical practice. References: [1]Dobloug, G.C., et al., Mortality in idiopathic inflammatory myopathy: results from a Swedish nationwide population-based cohort study. Ann Rheum Dis, 2018. 77(1): p. 40-47. [2]Barsotti, S. and I.E. Lundberg, Myositis an evolving spectrum of disease. Immunol Med, 2018. 41(2): p. 46-54. [3]Vencovsky, J., H. Alexanderson, and I.E. Lundberg, Idiopathic Inflammatory Myopathies. Rheum Dis Clin North Am, 2019. 45(4): p. 569-581. [4]Richards, T.J., et al., Characterization and peripheral blood biomarker assessment of anti-Jo-1 antibody-positive interstitial lung disease. Arthritis Rheum, 2009. 60(7): p. 2183-92. Disclosure of Interests: Charlotta Preger: None declared, Antonella Notarnicola: None declared, Cecilia Hellström: None declared, Edvard Wigren: None declared, Catia Cerqueira: None declared, Peter Nilsson: None declared, Ingrid E. Lundberg Grant/research support from: Bristol Meyer Squibb, Corbus Pharmaceuticals, Inc and Astra Zeneca, Helena Persson: None declared, Susanne Gräslund: None declared, Per-Johan Jakobsson Shareholder of: Gesynta Pharma, Grant/research support from: Gesynta Pharma, AstraZeneca,

Background: Autoantibodies that target aminoacyl transfer(t) RNA synthetases (aaRS) represent the serological marker of the anti-synthetase syndrome (ASS), a major subgroup of the idiopathic inflammatory myopathies (IIM) (1). Among the anti-aaRS, anti-histidyl tRNA synthetase (HisRS) autoantibodies (anti-Jo1) are the most common. Up to 90% of IIM/ASS patients diagnosed with interstitial lung disease (ILD) harbor anti-Jo1 autoantibodies (2). Objectives: Reactivity and affinity of anti-Jo1 autoantibodies from serum and broncheoalveolar lavage fluid (BALF) were investigated against HisRS autoantigen. Associations with clinical data from patients IIM/ASS were addressed. Methods: Total IgGs were purified by affinity chromatography. Samples and clinical data were obtained from: i) 26 anti-Jo1 + patients (19 at diagnosis, 16/19 at follow-up, 7 BALF/matching serum at baseline; ii) 29 anti-Jo1 - (25 serum at diagnosis, 4 BALF/matching serum at baseline); iii) 24 age/gender matched healthy controls. Anti-Jo1 IgG and IgA response against HisRS was evaluated by ELISA and western blot. Affinity was measured by surface plasmon resonance. HisRS full-length (HisRS-FL), two HisRS domains (ABD and CD), and two HisRS splice variants (WHEP and WHEP + ABD splice variant (SV)) were tested. Correlations between autoantibody reactivity and clinical data, at baseline and over disease course, were evaluated. Results: Anti-Jo1 autoantibodies from serum and lung bound HisRS-FL, WHEP and SV with high reactivity and affinity already at diagnosis and recognized both conformational and linear HisRS epitopes (Fig. 1). Levels of autoantibodies (against HisRS-FL, -domains and -splice variants) varied among patients and overtime. Patients with ILD, arthritis and less skin involvement presented higher anti-Jo1 titers compared to those with lower anti-Jo1 titers and to the anti-Jo1 negative group (Fig. 2). Anti-WHEP reactivity in BALF strongly correlated with poor pulmonary function. Conclusion: High reactivity and affinity at time of diagnosis indicates that autoimmunity against HisRS is most likely initiated before IIM/ASS diagnosis. Reactivity to specific splice variants of HisRS may be employed as diagnostic and prognostic markers. References: [1]Marguerie C, Bunn CC, Beynon HL, Bernstein RM, Hughes JM, So AK, Walport MJ: Polymyositis, pulmonary fibrosis and autoantibodies to aminoacyl-tRNA synthetase enzymes. Q J Med 1990, 77(282):1019-1038 [2]Richards TJ, Eggebeen A, Gibson K, Yousem S, Fuhrman C, Gochuico BR, Fertig N, Oddis CV, Kaminski N, Rosas IO et al: Characterization and peripheral blood biomarker assessment of anti-Jo-1 antibody-positive interstitial lung disease. Arthritis Rheum 2009, 60(7):2183-2192. Fig. 1. Anti-Jo1 reactivity in total IgG purified from the first available serum sample Fig. 2. Reactivity of total anti-Jo1+ IgG purified from the first available serum close to IIM/ASS diagnosis in relation to clinical data Disclosure of Interests: Antonella Notarnicola: None declared, Charlotta Preger: None declared, Susanna Lundström: None declared, Nuria Renard: None declared, Edvard Wigren: None declared, Eveline Van Gompel: None declared, Angeles Shunashy Galindo-Feria: None declared, Helena Persson: None declared, Maryam Fathi: None declared, Johan Grunewald: None declared, Per-Johan Jakobsson Shareholder of: Gesynta Pharma, Grant/research support from: Gesynta Pharma, AstraZeneca,, Susanne Gräslund: None declared, Ingrid E. Lundberg Grant/research support from: Bristol Meyer Squibb, Corbus Pharmaceuticals, Inc and Astra Zeneca, Catia Cerqueira: None declared

Aminoacyl-tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have noncanonical functions, and several of the aaRSs have been linked to autoimmune diseases, cancer, and neurological disorders. Deciphering these roles has been challenging because of a lack of tools to enable their study. To help solve this problem, we have generated recombinant high-affinity antibodies for a collection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top-performing binders were tested in immunoprecipitation followed by MS for their ability to capture the endogenous protein from mammalian cell lysates. For antibodies targeting individual members of the multi-tRNA synthetase complex, we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a subset of binders for each target was also confirmed using immunofluorescence. The sequences of these proteins have been deposited in publicly available databases and repositories. We anticipate that this open source resource, in the form of high-quality recombinant proteins and antibodies, will accelerate and empower future research of the role of aaRSs in health and disease.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10−9, 3 × 10−6, and 5 × 10−6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.

Antibody fragments have great potential as crystallization chaperones for structural biology due to their ability to either stabilise targets, trap certain conformations and/or promote crystal packing. Here we present an example of using a single-chain variable fragment (scFv) to determine the previously unsolved structure of the multidomain protein SP140. This nuclear leukocyte-specific protein contains domains related to chromatin-mediated gene expression and has been implicated in various disease states. The structure of two of the domains (PHD-bromodomain) was solved by crystallizing them as a complex with a scFv generated by phage display technology. SP140 maintains a similar overall fold to previous PHD-bromodomains and the scFv CDR loops predominately interact with the PHD, while the framework regions of the scFv makes numerous interactions with the bromodomain. Analysis of our and other complex structures suggest various protein engineering strategies that might be employed to improve the usefulness of scFvs as crystallization chaperones.