Sera Of SLE Patients Research Articles

P224 Anti-PARP1 as a novel autoantibody in myositis

Abstract Background/Aims Idiopathic inflammatory myopathies (IIM) are multisystem diseases characterised by muscle inflammation. Over 60% of patients with IIM have a myositis-related autoantibody. Our laboratory specialises in autoantibody testing. We offer an extended spectrum autoantibody testing diagnostic service and have also screened over 3000 myositis patients enrolled in research studies for the presence of autoantibodies. We noticed a recurring pattern following K562 cell radio-immunoprecipitation that was present in several myositis patient sera and in a handful of samples that came to us via the diagnostic service. We set out to determine the antigenic target of this novel autoantibody. Methods We have previously screened 1319 serum/plasma samples from IIM patients enrolled in the UKMyoNet and 380 from patients with juvenile onset IIM enrolled in the JDCBS for autoantibodies by immunoprecipitation. Additional cohorts similarly investigated include >150 healthy control sera and >400 SLE patient sera. Patients with the novel autoantibody of interest were identified by a distinctive 120kDa band associated with a ‘smear’ on autoradiography following K562 cell immunoprecipitation and separation of autoantigens by SDS PAGE. ESI-QTOF mass spectrometry was used to identify the antigenic target in an example serum. Using a commercial anti-PARP1 antibody as a control, western blotting of K562 and PARP1 overexpressed cell lysate was used to confirm the antigenic target in remaining samples of interest. Indirect immunofluorescence was performed on HEp-2 cells according to manufacturers’ instructions. Results 11 patient samples were identified as having the 120 complex ‘smear’ pattern of interest: six had been received via the diagnostic service and five were enrolled in UKMyoNet. Prevalence in the UKMyoNet cohort was 0.4%. Mass spectrometry identified PARP1 as the antigenic target. This was confirmed in all remaining samples by western blot. Table 1 shows available clinical data. Conclusion PARP1 is an autoantigen target in myositis patients in addition to those with other rheumatic diseases. Where data was available, patients with anti-PARP1 had features suggesting an anti-synthetase syndrome phenotype, but this may primarily relate to the other autoantibodies present. Further work is needed to determine the prevalence of anti-PARP1 in other cohorts and its clinical associations. Disclosure V. Riddell: Grants/research support; Bath Institute of Rheumatic Disease. Z. Betteridge: None. E. Bowler: None. H. Chinoy: None. P. Gordon: None. L. Wedderburn: None. S. Bagby: None. N. Mchugh: None. S. Tansley: None.

Immune complexes containing serum B-cell activating factor and immunoglobulin G correlate with disease activity in systemic lupus erythematosus

Levels of serum BAFF, IgG anti-BAFF and BAFF-IgG complexes were quantified by enzyme-linked immunosorbent assay. IgG anti-BAFF and BAFF-IgG complexes were further characterized using serum fractions obtained by fast protein liquid chromatography. To study the association of serum BAFF, IgG anti-BAFF and BAFF-IgG complex levels with SLE manifestations, 373 visits from 178 patients prospectively included in the Swiss SLE Cohort Study were analysed. While IgG anti-BAFF levels were not associated with clinical manifestations of SLE, serum BAFF levels correlated with disease activity and were higher in patients with renal involvement. Interestingly, we could also demonstrate the occurrence of BAFF-IgG complexes of different sizes in the sera of SLE patients, which were not due to treatment with belimumab and differed from complexes constructed in vitro. Most strikingly, the levels of these BAFF-IgG complexes were found to strongly correlate with overall disease activity, low complement levels and a history of lupus nephritis. BAFF-IgG complexes strongly correlate with disease activity in SLE patients, suggesting a pathogenic role in SLE.

Protoarray analysis reveals novel autoantigens targeted by autoantibodies associated with DNA-repair pathway in systemic erythematosus lupus (HUM2P.330)

Abstract Systemic erythematosus lupus (SLE) is an autoimmune disease characterized by presence of autoantibodies (autoAbs) against a broad spectrum of self-antigens. In order to identify novel autoAbs associated with SLE, we utilized a Protoarray bearing 9,500 antigens to screen IgG and IgM autoAbs in sera of SLE patients. 446 IgG and 1218 IgM autoAbs were identified to be significantly elevated in SLE patients compared with healthy controls (p<0.05). Protoarray revealed not only the previously described autoAbs such as antibodies against dsDNA, SSA/SSB, Histone and Sm/RNP, but also uncovered autoAbs against a broad range of novel antigens including 151 nuclear-associated antigens and 150 cytoplasma or membrane-associated antigens. The Protoarray results were further validated by ELISA in a larger cohort of SLE patients and controls. Besides, 65 of the IgG autoAb-targeted proteins were also disregulated in SLE on mRNA level by microarray analysis. Pathway analysis recognized significant enrichment of antigens involved in cell proliferation and DNA repair pathways which were targeted by IgG autoAbs including APEX1, AURKA, CSNK1G1, EIF2C1, HMGB1, IFIT5, MAPKAPK3, PADI4, PRKRA, RALGPS1, UBE2S and VRK1. The elevated IgG autoAbs to APEX1 and other proteins in the pathway may reflect the compromised DNA-repair activity during DNA replication in SLE. In conclusion, identification of novel autoAbs by protoarray may shed light on some of the pathogenic pathways leading to disease in SLE.

Identification of novel autoantibodies in systemic autoimmunity using 10,000-antigen proteome arrays (P4006)

Abstract Systemic erythematosus lupus (SLE) is an autoimmune disease characterized by presence of autoantibodies against a broad spectrum of self-antigens. In order to identify novel serum autoantibodies associated with SLE, we utilized a protoarray bearing 10,000 antigens to screen IgG and IgM autoAbs in sera of SLE patients. 446 IgG and 1218 IgM autoAbs were significantly increased in SLE compared with health controls (p<0.05), in which 367 autAbs overlapped in IgG and IgM. Except the autoAbs previously identified in SLE patients, such as anti-DNA, Ro, La, etc., the protoarray also uncovered a group of novel autoAbs against a broad range of antigens. The increased expression of 16 novel autoantibodies in SLE, including IgG autoantibodies against APEX1, AURKA, BAG3, CSNK1G1, EIF2C1, HMGB1, IFIT5, MAPKAPK3, PADI4, PKRKA, RALGPS1, RGS3, SRP19, STIP1, UBE2S and VRK1 were further confirmed by ELISA. Gene expression analysis revealed that some of the self-antigens also significantly upregulated on transcription level. In addition to uncovering ~500 novel autoantibody specificities, the novel arrays also shed light on some of the pathogenic pathways leading to disease in systemic lupus. In conclusion, protoarray provide a powerful tool for identification of novel autoantibodies in system autoimmune diseases.

Presence of IgG-CD4 Complexes in the Circulation

Many proteins of various origins are able to form complexes with Immunoglobulin G using reaction sites formed by residues of constant domains. Studies of IgG complexes of non-immune nature are important as biologically active proteins could escape from the circulation forming complexes with IgG as well as with other serum proteins whose concentration in serum is high. A quantitative characterization of circulating complexes in various diseases could give valuable information on the development of pathological processes. Immunoglobulins G are widely used for the treatment of a number of diseases and it is essential to understand what proteins are present in the preparations beside IgG. In the present study CD4 T-cell membrane glycoprotein was found in complexes with human IgG molecules isolated from donor sera as well as from sera of SLE patients via a sensitive quantitative dot-blot assay. According to immunoblotting experiments the CD4 part of the complexes had a molecular mass of about 50 kDa and is composed from all four extracellular domains. The CD4 content of the complexes varied among the studied human sera. There was no difference in IgG-CD4 complex concentration between the SLE patients and healthy controls. The data support the assumption that IgG molecules are able to act as scavengers and eliminate various proteins from the circulation including soluble CD4 protein.

Proteomic analysis of autoantibodies in neuropsychiatric systemic lupus erythematosus patient with white matter hyperintensities on brain MRI

The pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) may be related to autoantibody-mediated neural dysfunction, vasculopathy and coagulopathy. We encountered an NPSLE patient whose brain showed characteristic diffuse symmetrical hyperintensity lesions in the cerebral white matter, cerebellum and middle cerebellar peduncles on T2-weighted magnetic resonance (MR) images. In this study, we investigated all the antigens that reacted strongly with autoantibodies in this patient's serum by two-dimensional electrophoresis (2DE), followed by western blotting (WB) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using rat brain proteins as the antigen source. As a result, we identified four antigens as beta-actin, alpha-internexin, 60 kDa heat-shock protein (Hsp60) and glial fibrillary acidic protein (GFAP). There are several reports on the detection of anti-endothelial cell antibodies (AECAs) in an SLE patients. Recently, one of the antigens reacting with AECAs in SLE patient's sera has been identified as human Hsp60. We speculated that the abnormal findings on brain MR images of our patient may be due to impairment of microcirculation associated with vascular endothelial cell injury mediated by the antibody against Hsp60. This proteomic analysis is a useful tool for identifying autoantigens in autoimmune diseases involving autoantibodies.

Epstein–Barr Virus and Cytomegalovirus in Autoimmune Diseases

To date, it is believed that the origin of autoimmune diseases is one of a multifactorial background. A genetic predisposition, an immune system malfunction or even backfire, hormonal regulation, and environmental factors all play important roles in the pathogenesis of autoimmune diseases. Among these environmental factors, the role of infection is known to be a major one. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are considered to be notorious as they are consistently associated with multiple autoimmune diseases. A cohort of 1595 serum samples, of 23 different autoimmune disease groups, was screened for evidence of prior infection with EBV and CMV. All samples were screened for antibodies against EBV nuclear antigen-1 (IgG), EBV viral capsid antigen (IgG and IgM), EBV early antigen (IgG), EBV heterophile antibody, and CMV (IgG and IgM) antibodies using Bio-Rad's BioPlex 2200. A new association is proposed between EBV and polymyositis, as results show a significant increase in titers of various EBV target analytes when compared with healthy controls. Our results also support prior information suggesting the association between EBV and multiple autoimmune diseases, including SLE, antiphospholipid syndrome, rheumatoid arthritis, multiple sclerosis, pemphigus vulgaris, giant cell arthritis, Wegener's granulomatosis, and polyarteritis nodosa (PAN). Elevated CMV IgG titers were observed in sera of SLE patients. Our data support the theory that EBV is notoriously associated with many autoimmune diseases. CMV appears to be associated to autoimmune diseases as well, yet establishing this theory requires further investigation.

Antiphospholipid Antibodies in Patients with Lupus Nephritis

The aim of this study was to compare the prevalence of anticardiolipin antibodies with other types of antiphospholipid antibodies (aPL) (antiphosphatidylserine—aPS, antiphosphatidylinositol—aPI, antiphosphatidylethanolamine—aPE) in patients with lupus nephritis and to find if the examination of a panel of various aPL is valuable for further diagnosis of patients. Additionally we determined the levels of autoantibodies against β2-glycoprotein I (β2GPI) and oxidized low-density lipoprotein (anti-oxLDL) and also investigated the relationship between antibodies against β2GPI and oxLDL, which were assessed by ELISA methods. Twenty-two patients with lupus nephritis were studied. The control group consisted of 62 healthy blood donors. A statistically significant higher occurrence of all aPLs in the patients with lupus nephritis in comparison to the control group was found. The prevalence of polyspecific antibodies, which reacted with at least two various phospholipids, was 82% in the group of SLE patients. Significantly higher levels of IgG anti-β2GPI in the sera of SLE patients (p = 0.0003) was detected. The levels of anti-oxLDL in the sera of the patients group did not differ significantly from the control one. Some positive samples for anti-β2GPI and negative for aCL or anti-oxLDL and vice versa were found. It can be concluded that the production of aPL including anti-β2GPI and anti-oxLDL in the lupus nephritis patients is higher in comparison with healthy blood donors. We assume that the estimation of various types of aPL may be important in the selection of the group patients with renal diseases. The synthesis of aPL can reflect the spreading of the autoimmune response for several antigens modified on the vessel wall.

Identification of Human ST2 Protein in the Sera of Patients with Autoimmune Diseases

Soluble human ST2 protein (IL1RL1-a) in the sera of patients with various autoimmune diseases was identified by a newly developed procedure using specific monoclonal antibodies. After immunoprecipitation and subsequent immunoblotting, a glycosylated protein of about 60 kDa was detected in the sera of SLE patients, but not in the sera of healthy controls. The experiments using gel filtration and SDS–PAGE under a nonreducing condition indicated the existence of the ST2 multimer in serum. The mobility of the natural protein was slower than that of the recombinant human ST2 protein produced by COS7 cells in SDS–PAGE, suggesting a difference of glycosylation between humans and monkeys. The identification of the natural human ST2 protein should be important both to fundamental researches and the further clarification of the clinical implications of the ST2 protein.

Increased Level of Soluble HLA Class I Antigens in Systemic Lupus Erythematosus: Correlation with Anti-DNA Antibodies and Leukopenia

The concentration of soluble HLA class I (sHLA-I) was measured by ELISA in serum samples from 30 well-characterised SLE patients at high and low disease activity states and from 100 healthy controls. HLA-A allotypes in the patients were analysed by a PCR-based typing technique. A higher level of sHLA-I was found in SLE patient sera both at high and low disease activity than in controls (P< 0.001). The sHLA-I level was further increased during active disease (P< 0.01). Concentrations of sHLA-I correlated with anti-dsDNA antibodies at high disease activity, but not with disease activity as analysed by a modified SLEDAI. Numbers of leukocytes and lymphocytes, as well as levels of C1q and C3 correlated inversely with sHLA-I concentration. In five serial samples from ten patients the sHLA-I level co-varied with disease activity. Presence of HLA allotype A9 was associated with higher sHLA-I levels in both patients (P< 0.001) and controls (P< 0.001). We conclude that the increased sHLA-I concentration in SLE patients was related to several laboratory parameters reflecting disease activity suggesting that sHLA-I molecules are connected with the disease process. Increased sHLA-I level due to HLA-A allotype was not a disease susceptibility factor for SLE.

Immunochemical Determination of DNA in Immune Complexes Present in the Circulation of Patients with Systemic Lupus Erythematosus

The presence of DNA antigen in circulating immune complexes formed with anti-DNA antibodies of IgG class was determined in sera of 138 SLE patients and 23 healthy donors, employing monoclonal anti-dsDNA antibodies in a quantitative dot blot assay. About half of the SLE patients had elevated amounts of DNA antigen in the immune complexes (presumably as nucleosomes). The number of patients with the SLE manifestations was not higher in the group with the high amount of DNA in immune complexes. Elevated levels of DNA in immune complexes was found only in sera of SLE patients with the active, as well as quiescent form, of the disease and not in sera of healthy donors. The presence of increased amounts of DNA antigen in circulating immune complexes could indicate the presence of SLE pathology even if no manifestations of SLE are found. The level of the circulating anti-DNA antibodies was not correlated with the amount of DNA in immune complexes.

Cellular mechanism of the conduction abnormalities induced by serum from anti-Ro/SSA-positive patients in rabbit hearts.

In this study, IgG fractions from sera of SLE patients with anti-Ro/SSA or anti-Ro/SSA and anti-La/SSB activity were tested in Langendorff preparations of adult rabbit hearts, aiming to reproduce the cardiac manifestations observed in neonatal lupus in an experimental model. The hearts were perfused with normal Tyrode's solution for 30 min, followed by perfusion with Tyrode's containing 0.3 mg/ml of anti-Ro/SSA- (or anti-Ro/La-) positive IgG (nine sera), anti-ribonucleoprotein (RNP)-positive IgG (five sera), or IgG fractions from normal donors (five sera). In one third of the experiments done with anti-Ro/La-positive IgG, heart block was observed. With the remaining fractions, a decrease in heart rate of 17.1% was observed, but normal sinus rhythm was maintained. The IgG fractions with anti-RNP activity (five experiments) and from normal sera (six experiments) reduced heart rates by 12.9 and 3.3%, respectively, but heart block was not observed. To further characterize the cellular mechanisms involved in the conduction disturbances observed in the whole rabbit hearts, we conducted experiments with ventricular myocytes isolated from young rabbit hearts, studied by whole cell patch-clamp technique. In these experiments, the slow inward currents were analyzed during the superfusion of the cell with normal Tyrode's solution and 5 min after superfusion with Tyrode's solution containing 0.3 mg/ml of anti-Ro/SSA- (or anti-Ro/La-) positive IgG (five sera), anti-RNP-positive IgG (three sera), or IgG from normal donors (four sera). Resting and action potential amplitudes were not affected by any of the sera used. The anti-Ro/SSA IgG fraction induced a mean reduction in the peak slow inward current of 31.6%. IgG fractions with anti-RNP activity reduced slow inward current by 4.4%, whereas IgG fractions from normal donors increased this current by 3.3%. IgG-free fractions from sera of patients with anti-Ro/SSA activity did not alter the peak slow inward current. These results show, for the first time, that the presence of anti-Ro/SSA or anti-Ro/SSA and anti-La/SSB antibody activity in IgG fractions from lupus patients' sera can induce cardiac conduction disorders similar to those observed in neonatal lupus.

Anti-nuclear antibodies associated with schistosomiasis and anti-schistosomal antibodies associated with SLE.

Various autoantibodies to nuclear antigens were detected by ELISA in pooled sera of 15 ICR mice 9 weeks after infection with schistosoma. The binding of this serum with acute infection to cercarial extract was inhibited by prior incubation of the sera with DNA, polynucleotides and cercarial extract. Sera from 9 BALB/c mice with experimentally induced lupus, reacted with cercarial extract. No binding was noted with normal mouse sera (NMS). Similarly, sera of SLE patients (N = 113) reacted with the parasite extracts in a higher incidence (14.2%) compared to NHS (3.7%) (P < 0.05). Immunoblot analysis showed common features to SLE sera reacting with two antigenic "groups". The first are antigens that also were recognized by sera of mice infected with schistosomiasis (60, 85, and 94 kDa cercarial proteins). The second group entailed proteins that bound with SLE sera only (10-18 kDa, 29 kDa and 52 kDa). It can be concluded that antinuclear autoantibodies can be found in sera of acute infected mice with schistosomiasis, while SLE sera may react with 3 schistosomal extracts in a higher incidence than NHS (P < 0.05). These results suggest an autoimmune response in schistosomiasis. It is possible that a parasite infection (as bilharzia) may be one of the "trigger" mechanisms for SLE in a subject with the suitable immunogenetic and hormonal background.

Polyspecific reactivity of a murine monoclonal antibody that binds to nuclear matrix-associated, chromatin-bound autoantigens

To investigate polyspecific autoantibody interactions, we have characterized the binding of a cloned murine monoclonal IgM antibody termed (RTE-23) of strain BALB c origin. By indirect immunofluorescence this antibody displayed a nuclear speckled and peripheral pattern in interphase cells from human and rodent cell lines. In contrast, in mitotic cells, antibody RTE-23 bound to the periphery of individual chromosomes. Immunoblot analysis of soluble and insoluble nuclear proteins from purified rat fibroblast nuclei showed that antibody TRE-23 bound to molecules of 28, 29, and 33 kDa. Further-more, antibody RTE-23 demonstrated marked polyspecificity and reacted with cytoskeletal proteins (vimentin, keratin, actin), single-stranded DNA, specific synthetic polynucleotides, and cardiolipin. Antibody RTE-23 also showed a lupus anticoagulant-like activity. Screening of sera of autoimmune disease patients with antinuclear antibodies revealed two patients, both with SLE, whose sera blocked antibody RTE-23 binding to nuclei and recognized nuclear proteins identical to those recognized by antibody RTE-23. These results suggested that antibody RTE-23 displays a pattern of self-antigen binding that is represented as well in SLE patient sera.

Complement activation by antibodies to DNA in systemic lupus erythematosus measured by enzyme immunoassay

An enzyme immunoassay to detect complement-fixing antibodies to DNA (CF-antiDNA) was developed. Of SLE sera, 64% had these antibodies as did 6% of 50 rheumatoid arthritis and 3.2% of 93 normal human sera. The mean CF-antiDNA level was higher in the sera of SLE patients with renal disease than those SLE patients who had no renal disease ( P < 0.0001), and higher in those SLE patients with active rather than inactive renal disease ( P = 0.006). CF-antiDNA was more closely associated with renal activity than total IgG-antiDNA or CH50. These observations suggest that both the quality and quantity of anti-DNA antibodies play a role in the pathogenesis of renal disease, and that modern enzyme immunoassays help distinguish the relative importance of complement-fixing antibodies to anti-DNA from that of total anti-DNA.

Influence of assay conditions on ELISA determinations of anti-DNA antibodies

The influence of assay conditions on anti-DNA determinations by an enzyme-linked immunosorbent assay (ELISA) was investigated to evaluate the detection of various DNA antigenic specificities. Among 4 monoclonal anti-DNA antibodies of MRL- lpr/lpr strain origin, 2 showed higher titers in 100 mM NaCl-50 mM Tris, pH 7.5 (Tris-NaCl) than in phosphate-buffered saline (PBS). The determination of ‘polyspecificity’ for these monoclonal products also depended on the set of conditions used for assay with inhibitory activity of polynucleotides differing in the 2 buffers. The buffer effects were not confined to the monoclonal antibodies as increases in anti-DNA titers were demonstrated for certain SLE patient sera when assayed in Tris-NaCl rather than PBS; sera of MRL- lpr/lpr mice showed an opposite effect, however, with enhancement of anti-DNA activity by PBS. These results suggest that the representation of antigenic sites on DNA may be variably affected by the conditions of assay, altering quantitative and qualitative assessment of this important serological marker.

Detection of masked anti-DNA antibodies in lupus sera by a monoclonal anti-idiotype.

We report here the use of a monoclonal anti-idiotype 3I to human anti-DNA antibodies to detect in serum idiotype-positive antigen-binding antibodies lacking DNA-binding activity as measured by conventional antigen binding assays. We studied paired serum samples from 13 patients with systemic lupus obtained at two times in the course of their disease: in each patient, one serum sample has anti-DNA activity and the second serum sample has no anti-dsDNA activity detectable by Millipore filter, ELISA, or Crithidia assay. Reactivity with 3I as detected with a radioimmunoassay (RIA) was present in all 13 sera with anti-dsDNA activity. Six patients showed a decrease in 3I reactivity to normal levels in the second serum sample, in which anti-dsDNA antibodies were not detectable by conventional antigen-binding assays. The other seven patients' second serum sample continued to show elevated 3I reactivity by RIA even though no anti-dsDNA activity was apparent. When the 3I-reactive antibodies from these latter patients' sera were eluted from a 3I affinity column, they revealed DNA-binding activity. Furthermore, dsDNA binding by these sera was apparent when they were displayed on Western blots of isoelectric focusing gels run in 8 M urea and incubated with radiolabeled dsDNA. These results indicate that the 3I anti-idiotype can detect anti-DNA antibodies in some sera of SLE patients that lack anti-DNA activity by ordinary assays. These antibodies may be inhibited in binding dsDNA by excess antigen or autologous anti-idiotype, and their DNA binding activity can be unmasked by procedures promoting immune complex dissociation.

Immunoglobulin deposits in skin in systemic lupus erythematosus

Immunoglobulin deposits in the dermal-epidermal junction of clinically normal skin from patients with SLE were eluted by acid buffer. The eluates contained antinuclear and antibasement membrane antibody activities. The anti-BM antibody reacted with skin and esophageal but not glomerular basement membrane. Enzymatic studies indicated that the antibody reacted with carbohydrate moeities in the basement membrane. The anti-BM antibody was not present in corresponding sera of SLE patients.

Cigarette dependence: II. Doctor's role in management.

<h3>Background</h3> Identification of non-invasive differentially regulated markers in SLE are valuable to improve diagnosis, prediction of response to treatment(s) or classification of disease subpopulations. Our goal was to assess a broad range of serum protein markers in a large cohort of SLE patients and further characterize the role of autoantibodies in serum of these patients for their functional capacity as specific immune complexes to activate the IFN axis. <h3>Methods</h3> In this multicenter study, we have characterised a large cohort of mostly pediatric SLE patients (n=150) with longitudinal sera samples (total n=269). Serum cytokines/chemokines levels and their extractable nuclear antigen antibody (ENA) and antinuclear antibody (ANA) profiles measured from SLE patients and healthy subjects using a 30-panel Multiplex and individual enzyme-linked immunosorbent assays. Patient sera containing autoantibodies and specific antigens activating the IFN pathway quantified on human primary cells using AlphaLisa technology. Statistics for significance Mann-Whitney (*p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001). <h3>Results</h3> Differentially regulated cytokines/chemokines were elevated in sera of SLE patients compared to healthy individuals. Notably, IFN-related proteins (IFNalpha*, IFNgamma* and IP10****), B-cell and monocytes chemokines including CXCL13**** and MCP-1***, as well as Th1/17 activating cytokines (IL-12****, IL-17**) and those involved in antibody production by B lymphocytes (IL-10**, IL-13**, IL-6*) being more significant. Collectively, these peripheral markers may serve to help inform diagnosis and support the choice treatment in the clinic. We established a miniaturized functional assay and show that the vast majority of SLE patient sera containing common ENAs (against RNP/Sm, SS-A) and ANAs (dsDNA) could specifically activate the type I IFN pathway as an immune-complex on healthy peripheral blood mononuclear cells (PBMCs) (figure 1). We demonstrate that SLE containing immune-complexes (with specific antigens) can trigger IFN release potentially via nucleic-acid RNA/DNA sensors. Clustering patient autoantibody ENAs profiles reveal that most of the SLE patients in this cohort display a stronger IFN functional activation profile. <h3>Conclusions</h3> Given the high incidence of elevated IFN signatures in SLE patients, it is not surprising that increased IFN-related proteins and ENA/ANAs that trigger IFN responses are more evident in SLE patient serum. The prevalence of ENA/ANAs in patient sera that have the functional capacity to activate IFN downstream of nucleic acid sensors may help to define subpopulations of SLE. <h3>Funding Source(s):</h3> National Council for Scientific and Technological Development (CNPq 157534/2015–4) PBMCs were prepared from fresh healthy human blood, stimulated with individual patient SLE serum in combination with defined antigens corresponding to their ENA or ANA titer profile. After 20 hour, IFNalpha protein levels were quantified and expressed relative to agonist ssRNA40. Data shown are from 2 more independent donors with total SLE patients numbers indicated in parenthesis. ****p&lt;0.0001 (unpaired t-test).