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Additional file 2: Supplementary Table 1. Numbers of collected blood samples. Supplementary Table 2. Antibodies used for flow cytometry.

Additional file 1: Supplementary figure 1. (A) Expression of CD15 and CD14 on low-density granulocytes (LDG) from two pregnant women with SLE (two left panels, samples from trimester three) and two healthy pregnant controls (HC, two right panels, one sample from trimester three and one sample from trimester one). (B) Analysis of proportions of LDG and shedding of CD62L by NDG in blood from pregnant women with SLE and healthy pregnant controls between 17 and 24 h post venipuncture i.e., the time span when all samples in the study were analyzed. (C) Analysis of total number of granulocytes, proportions of LDG and shedding of CD62L by normal-density granulocytes (NDG) from one pregnant woman with SLE in trimester three and one healthy pregnant control in trimester one at 5 and 24 h post venipuncture. Supplementary figure 2. Comparison of (A) proportions of low-density granulocytes (LDG), (B) proportions of LDG that have shed CD62L, (C) proportions of normal-density granulocytes (NDG) that have shed CD62L and (D) total granulocyte counts during compared to after pregnancy among women with SLE from whom late postpartum samples were collected. *p < 0.05, **p < 0.01 and ***p < 0.001, Kruskal-Wallis followed by Dunn’s multiple comparison test. Supplementary figure 3. Comparison of LDG proportions in pregnant women with SLE with or without a moderate/high disease activity (SLEDAI-2K ≥ 4) in (A) trimester one, (B) trimester two and (C) trimester three. Mann-Whitney U test. Supplementary figure 4. Comparison of (A) LDG proportions, (B) LDG activation by CD62L shedding, (C) NDG activation by CD62L shedding and (D) IFNα protein levels in SLE and healthy pregnancy for each trimester. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Mann-Whitney U test. Supplementary figure 5. Comparison of the proportions of low-density granulocytes (LDG) and normal-density granulocytes (NDG) that have shed CD62L in pregnant women with SLE and in healthy pregnant controls (HC). **** p < 0.0001, Mann-Whitney U test. Supplementary figure 6. Concentrations of G-CSF (A) and GM-CSF (B) in plasma during pregnancy (first, second and third trimester) compared to the late postpartum period among women with SLE from whom late postpartum samples were collected. Concentrations of G-CSF (C) and GM-CSF (D) in plasma in the first, second and third trimester in SLE compared to healthy pregnancies. Supplementary figure 7. (A) OPLS loading column plot depicting granulocyte-related immune variables positively or negatively associated with prednisone use in SLE pregnancy, and low-density proportions in pregnant women with SLE who were treated with prednisone compared to those who were not treated. (B) OPLS plot depicting granulocyte-related immune variables positively or negatively associated with azathioprine use in SLE pregnancy and CD62L shedding of normal-density granulocytes in pregnant women with SLE who were treated with azathioprine compared to those who were not treated. (C) OPLS plot depicting granulocyte-related immune variables positively or negatively associated with low molecular weight heparin use in SLE pregnancy. * p < 0.05, Mann-Whitney U test. Supplementary figure 8. IFNα plasma protein concentrations in trimester one, two and three from pregnant women with SLE who were treated or not with prednisone (A), azathioprine (B) or low molecular weight heparin (C). Supplementary figure 9. IFNα plasma protein concentrations in trimester one, two and three from pregnant women with SLE who were anti-SSA positive compared to those who were negative. ** p < 0.01, Mann-Whitney U test. Supplementary figure 10. (A) Gestational age at birth among women with SLE with induced compared to spontaneous delivery. (B) Gestational age at birth in women with SLE with induced or spontaneous delivery who were positive or negative for anti-CL, anti-β2GPI or LAC. (C) Correlations between gestational age at birth and proportions of LDG in trimester three, two and one among women with SLE with induced or spontaneous delivery. * p < 0.05, (B) Mann-Whitney U test and (C) Spearman rank correlation test.

Objective Complete genetic deficiency of the complement component C2 is a strong risk factor for monogenic systemic lupus erythematosus (SLE), but whether heterozygous C2 deficiency adds to the risk of SLE or primary Sjogrens syndrome (SS) has not been studied systematically. This study was undertaken to investigate potential associations of heterozygous C2 deficiency and C4 copy number variation with clinical manifestations in patients with SLE and patients with primary SS. Methods The presence of the common 28-bp C2 deletion rs9332736 and C4 copy number variation was examined in Scandinavian patients who had received a diagnosis of SLE (n = 958) or primary SS (n = 911) and in 2,262 healthy controls through the use of DNA sequencing. The concentration of complement proteins in plasma and classical complement function were analyzed in a subgroup of SLE patients. Results Heterozygous C2 deficiency-when present in combination with a low C4A copy number-substantially increased the risk of SLE (odds ratio [OR] 10.2 [95% confidence interval (95% CI) 3.5-37.0]) and the risk of primary SS (OR 13.0 [95% CI 4.5-48.4]) when compared to individuals with 2 C4A copies and normal C2. For patients heterozygous for rs9332736 with 1 C4A copy, the median age at diagnosis was 7 years earlier in patients with SLE and 12 years earlier in patients with primary SS when compared to patients with normal C2. Reduced C2 levels in plasma (P = 2 x 10(-9)) and impaired function of the classical complement pathway (P = 0.03) were detected in SLE patients with heterozygous C2 deficiency. Finally, in a primary SS patient homozygous for C2 deficiency, we observed low levels of anti-Scl-70, which suggests a risk of developing systemic sclerosis or potential overlap between primary SS and other systemic autoimmune diseases. Conclusion We demonstrate that a genetic pattern involving partial deficiencies of C2 and C4A in the classical complement pathway is a strong risk factor for SLE and for primary SS. Our results emphasize the central role of the complement system in the pathogenesis of both SLE and primary SS. Funding Agencies|Swedish Research Council for Medicine and Health; Swedish Rheumatism Association; Swedish Heart-Lung Foundation; Swedish Society for Medical Research; Swedish Society of Medicine; Gustafsson Family Foundation; Wallenberg Scholarship; King Gustaf Vs 80-Year Foundation; Stockholm County and Region Ostergotland

ObjectiveLupus nephritis (LN) is a common and severe manifestation of SLE. The genetic risk for nephritis and progression to end-stage renal disease (ESRD) in patients with LN remains unclear. Herein, we aimed to identify novel genetic associations with LN, focusing on subphenotypes and ESRD.MethodsWe analysed genomic data on 958 patients with SLE (discovery cohort: LN=338) with targeted sequencing data from 1832 immunological pathway genes. We used an independent multiethnic cohort comprising 1226 patients with SLE (LN=603) as a replication dataset. Detailed functional annotation and functional epigenomic enrichment analyses were applied to predict functional effects of the candidate variants.ResultsA genetic variant (rs56097910) within theMERTKgene was associated with ESRD in both cohorts, meta-analysis OR=5.4 (2.8 to 10.6); p=1.0×10-6. We observed decreased methylation levels in peripheral blood cells from SLE patients with ESRD, compared with patients without renal SLE (p=2.7×10-4), at one CpG site (cg16333401) in close vicinity to the transcription start site ofMERTKand located in a DNAse hypersensitivity region in T and B cells. Rs56097910 is linked to alteredMERTKexpression in kidney tissue in public eQTL databases. Two loci were replicated for association with proliferative LN:PRDM1(rs6924535, pmeta=1.6×10-5, OR=0.58) andAPOA1BP(NAXE) (rs942960, pmeta=1.2×10-5, OR=2.64).ConclusionWe identified a novel genetic risk locus,MERTK, associated with SLE-ESRD using the data from two large SLE cohorts. Through DNA methylation analysis and functional annotation, we showed that the risk could be mediated through regulation of gene expression. Our results suggest that variants in theMERTKgene are important for the risk of developing SLE-ESRD and suggest a role forPRDM1andAPOA1BPin proliferative LN.

The genetic background of lupus nephritis (LN) has not been completely elucidated. We performed a case-only study of 2886 SLE patients, including 947 (33%) with LN. Renal biopsies were available from 396 patients. The discovery cohort (Sweden, n = 1091) and replication cohort 1 (US, n = 962) were genotyped on the Immunochip and replication cohort 2 (Denmark/Norway, n = 833) on a custom array. Patients with LN, proliferative nephritis, or LN with end-stage renal disease were compared with SLE without nephritis. Six loci were associated with LN (p < 1 x 10(-4), NFKBIA, CACNA1S, ITGA1, BANK1, OR2Y, and ACER3) in the discovery cohort. Variants in BANK1 showed the strongest association with LN in replication cohort 1 (p = 9.5 x 10(-4)) and proliferative nephritis in a meta-analysis of discovery and replication cohort 1. There was a weak association between BANK1 and LN in replication cohort 2 (p = 0.052), and in the meta-analysis of all three cohorts the association was strengthened (p = 2.2 x 10(-7)). DNA methylation data in 180 LN patients demonstrated methylation quantitative trait loci (meQTL) effects between a CpG site and BANK1 variants. To conclude, we describe genetic variations in BANK1 associated with LN and evidence for genetic regulation of DNA methylation within the BANK1 locus. This indicates a role for BANK1 in LN pathogenesis. Funding Agencies|Swedish Research Council for Medicine and Health [521-2014-2263, 521-2014-3954, 2016-01982, 2018-02399]; Swedish Rheumatism Association; Swedish Society for Medical Research; Swedish Society of Medicine; Ingegerd Johansson donation; King Gustav Vs 80-year Foundation; Science for Life Laboratory; Swedish Research Council (VR-RFI)Swedish Research Council; Uppsala University; Knut and Alice Wallenberg Foundation

Objectives Systemic lupus erythematosus (SLE) is an autoimmune disease with extensive heterogeneity in disease presentation between patients, which is likely due to an underlying molecular diversity. Here, we aimed at elucidating the genetic aetiology of SLE from the immunity pathway level to the single variant level, and stratify patients with SLE into distinguishable molecular subgroups, which could inform treatment choices in SLE. Methods We undertook a pathway-centred approach, using sequencing of immunological pathway genes. Altogether 1832 candidate genes were analysed in 958 Swedish patients with SLE and 1026 healthy individuals. Aggregate and single variant association testing was performed, and we generated pathway polygenic risk scores (PRS). Results We identified two main independent pathways involved in SLE susceptibility: T lymphocyte differentiation and innate immunity, characterised by HLA and interferon, respectively. Pathway PRS defined pathways in individual patients, who on average were positive for seven pathways. We found that SLE organ damage was more pronounced in patients positive for the T or B cell receptor signalling pathways. Further, pathway PRS-based clustering allowed stratification of patients into four groups with different risk score profiles. Studying sets of genes with priors for involvement in SLE, we observed an aggregate common variant contribution to SLE at genes previously reported for monogenic SLE as well as at interferonopathy genes. Conclusions Our results show that pathway risk scores have the potential to stratify patients with SLE beyond clinical manifestations into molecular subsets, which may have implications for clinical follow-up and therapy selection. Funding Agencies|AstraZeneca-Science for Life Laboratory Research Collaboration grant (DISSECT); Swedish Research Council for Medicine and Health [2018-02399, 2018-02535]; Swedish Rheumatism Association; King Gustav Vs 80-year Foundation; Swedish-Heart-Lung foundationSwedish Heart-Lung Foundation; Wallenberg Scholar Award; Swedish Society of Medicine; Science for Life Laboratory; Swedish Research Council (VR-RFI)Swedish Research Council; Uppsala University; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation

By performing whole-genome sequencing in a Swedish cohort of 71 parent-offspring trios, in which the child in each family is affected by systemic lupus erythematosus (SLE, OMIM 152700), we investigated the contribution of de novo variants to risk of SLE. We found de novo single nucleotide variants (SNVs) to be significantly enriched in gene promoters in SLE patients compared with healthy controls at a level corresponding to 26 de novo promoter SNVs more in each patient than expected. We identified 12 de novo SNVs in promoter regions of genes that have been previously implicated in SLE, or that have functions that could be of relevance to SLE. Furthermore, we detected three missense de novo SNVs, five de novo insertion-deletions, and three de novo structural variants with potential to affect the expression of genes that are relevant for SLE. Based on enrichment analysis, disease-affecting de novo SNVs are expected to occur in one-third of SLE patients. This study shows that de novo variants in promoters commonly contribute to the genetic risk of SLE. The fact that de novo SNVs in SLE were enriched to promoter regions highlights the importance of using whole-genome sequencing for identification of de novo variants. Funding Agencies|Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; Swedish Research Council for Medicine and Health [2018-02399, 2017-02000]; Swedish Rheumatism Association; King Gustaf Vs 80-year Foundation; Swedish Society of Medicine; Ingegerd Johansson donation

Objectives To investigate associations between a high genetic disease risk and disease severity in patients with systemic lupus erythematosus (SLE). Methods Patients with SLE (n=1001, discovery cohort and n=5524, replication cohort) and healthy controls (n=2802 and n=9859) were genotyped using a 200K Immunochip single nucleotide polymorphism array. A genetic risk score (GRS) was assigned to each individual based on 57 SLE risk loci. Results SLE was more prevalent in the high, compared with the low, GRS-quartile (OR 12.32 (9.53 to 15.71), p=7.9x10(-86) and OR 7.48 (6.73 to 8.32), p=2.2x10(-304) for the discovery and the replication cohorts, respectively). In the discovery cohort, patients in the high GRS-quartile had a 6-year earlier mean disease onset (HR 1.47 (1.22 to 1.75), p=4.3x10(-5)), displayed higher prevalence of damage accrual (OR 1.47 (1.06 to 2.04), p=2.0x10(-2)), renal disorder (OR 2.22 (1.50 to 3.27), p=5.9x10(-5)), anti-dsDNA (OR 1.83 (1.19 to 2.81), p=6.1x10(-3)), end-stage renal disease (ESRD) (OR 5.58 (1.50 to 20.79), p=1.0x10(-2)), proliferative nephritis (OR 2.42 (1.30 to 4.49), p=5.1x10(-3)), anti-cardiolipin-IgG (OR 1.89 (1.13 to 3.18), p=1.6x10(-2)), anti-beta 2-glycoprotein-I-IgG (OR 2.29 (1.29 to 4.06), p=4.8x10(-3)) and positive lupus anticoagulant test (OR 2.12 (1.16 to 3.89), p=1.5x10(-2)) compared with patients in the low GRS-quartile. Survival analysis showed earlier onset of the first organ damage (HR 1.51 (1.04 to 2.25), p=3.7x10(-2)), first cardiovascular event (HR 1.65 (1.03 to 2.64), p=2.6x10(-2)), nephritis (HR 2.53 (1.72 to 3.71), p=9.6x10(-7)), ESRD (HR 6.78 (1.78 to 26.86), p=6.5x10(-3)) and decreased overall survival (HR 1.83 (1.02 to 3.30), p=4.3x10(-2)) in high to low quartile comparison. Conclusions A high GRS is associated with increased risk of organ damage, renal dysfunction and all-cause mortality. Our results indicate that genetic profiling may be useful for predicting outcomes in patients with SLE.

Natural killer (NK) cell cytotoxicity toward self-cells is restrained by the inhibitory HLA class I-binding receptors CD94/NKG2A and the killer cell immunoglobulin-like receptors (KIRs). CD94/NKG2A and KIRs are also essential for NK cell education, which is a dynamic functional maturation process where a constitutive binding of inhibitory receptors to cognate HLA class I molecules is required for NK cells to maintain their full cytotoxic capacity. Previously, we described autoantibodies to CD94/NKG2A in patients with systemic lupus erythematosus (SLE). In this study we analyzed sera from 191 patients with SLE, 119 patients with primary Sjogren's syndrome (pSS), 48 patients with systemic sclerosis (SSc), and 100 healthy donors (HD) for autoantibodies to eight different KIRs. Anti-KIR autoantibodies were identified in sera from 23.0% of patients with SLE, 10.9% of patients with pSS, 12.5% of patients with SSc, and 3.0% of HD. IgG from anti-KIR-positive SLE patients reduced the degranulation and cytotoxicity of NK cells toward K562 tumor cells. The presence of anti-KIR-autoantibodies reacting with >3 KIRs was associated with an increased disease activity (p < 0.0001), elevated serum levels of IFN-alpha (p < 0.0001), nephritis (p = 0.001), and the presence of anti-Sm (p = 0.007), and anti-RNP (p = 0.003) autoantibodies in serum. Together these findings suggest that anti-KIR autoantibodies may contribute to the reduced function of NK cells in SLE patients, and that a defective NK cell function may be a risk factor for the development of lupus nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with heterogeneous clinical presentation and complex etiology involving the interplay between genetic, epigenetic, environmental and hormonal factors. Many common SNPs identified by genome wide-association studies (GWAS) explain only a small part of the disease heritability suggesting the contribution from rare genetic variants, undetectable in GWAS, and complex epistatic interactions. Using targeted re-sequencing of coding and conserved regulatory regions within and around 215 candidate genes selected on the basis of their known role in autoimmunity and genes associated with canine immune-mediated diseases, we identified a rare regulatory variant rs200395694:G > T located in intron 4 of the MEF2D gene encoding the myocyte-specific enhancer factor 2D transcription factor and associated with SLE in Swedish cohorts (504 SLE patients and 839 healthy controls, p = 0.014, CI = 1.1–10). Fisher’s exact test revealed an association between the genetic variant and a triad of disease manifestations including Raynaud, anti-U1-ribonucleoprotein (anti-RNP), and anti-Smith (anti-Sm) antibodies (p = 0.00037) among the patients. The DNA-binding activity of the allele was further studied by EMSA, reporter assays, and minigenes. The region has properties of an active cell-specific enhancer, differentially affected by the alleles of rs200395694:G > T. In addition, the risk allele exerts an inhibitory effect on the splicing of the alternative tissue-specific isoform, and thus may modify the target gene set regulated by this isoform. These findings emphasize the potential of dissecting traits of complex diseases and correlating them with rare risk alleles with strong biological effects. Funding agencies: Knut and Alice Wallenberg Foundation; Swedish Research Council; Swedish Research Council FORMAS; Swedish Rheumatism Foundation; King Gustaf Vs 80-Year Foundation; Swedish Institute scholarship; King Gustav V 80th years Foundation scholarship; Kazan Federa

Objectives: To performa cross-comparative analysis of DNA methylation in patients with systemic lupus erythematosus (SLE), patients with primary Sjogrens syndrome (pSS), and healthy controls addressing the question of epigenetic sharing and aiming to detect disease-specific alterations. Methods: DNA extracted from peripheral blood from 347 cases with SLE, 100 cases with pSS, and 400 healthy controls were analyzed on the Human Methylation 450k array, targeting 485,000 CpG sites across the genome. A linear regression model including age, sex, and blood cell type distribution as covariates was fitted, and association p-values were Bonferroni corrected. A random forest machine learning classifier was designed for prediction of disease status based on DNA methylation data. Results: We established a combined set of 4,945 shared differentially methylated CpG sites (DMCs) in SLE and pSS compared to controls. In pSS, hypomethylation at type I interferon induced genes was mainly driven by patients who were positive for Ro/SSA and/or La/SSB autoantibodies. Analysis of differential methylation between SLE and pSS identified 2,244 DMCs with a majority of sites showing decreased methylation in SLE compared to pSS. The random forest classifier demonstrated good performance in discerning between disease status with an area under the curve (AUC) between 0.83 and 0.96. Conclusions: The majority of differential DNA methylation is shared between SLE and pSS, however, important quantitative differences exist. Our data highlight neutrophil dysregulation as a shared mechanism, emphasizing the role of neutrophils in the pathogenesis of systemic autoimmune diseases. The current study provides evidence for genes and molecular pathways driving common and disease-specific pathogenic mechanisms. Funding Agencies|Knut and Alice Wallenberg Foundation [KAW 2011.0073]; Swedish Research Council for Medicine and Health [VR-MH Dnr 521-2014-2263, Dnr 2018-02399, Dnr 2016-01982]; Gustaf V and Queen Victorias Freemasons Foundation; County Council of Ostergotland; Swedish Rheumatism Association; King Gustaf Vs 80-year Foundation; Swedish Society of Medicine; Science for Life Laboratory, Uppsala University; Swedish Research Council (VR-RFI); Ingegerd Johansson donation; Knut and Alice Wallenberg Foundation

Objectives: Systemic lupus erythematosus (SLE) is a chronic autoimmune condition with heterogeneous presentation and complex aetiology where DNA methylation changes are emerging as a contributing factor. In order to discover novel epigenetic associations and investigate their relationship to genetic risk for SLE, we analysed DNA methylation profiles in a large collection of patients with SLE and healthy individuals. Methods: DNA extracted from blood from 548 patients with SLE and 587 healthy controls were analysed on the Illumina HumanMethylation 450 k BeadChip, which targets 485 000 CpG sites across the genome. Single nucleotide polymorphism (SNP) genotype data for 196 524 SNPs on the Illumina ImmunoChip from the same individuals were utilised for methylation quantitative trait loci (cis-meQTLs) analyses. Results: We identified and replicated differentially methylated CpGs (DMCs) in SLE at 7245 CpG sites in the genome. The largest methylation differences were observed at type I interferon-regulated genes which exhibited decreased methylation in SLE. We mapped cis-meQTLs and identified genetic regulation of methylation levels at 466 of the DMCs in SLE. The meQTLs for DMCs in SLE were enriched for genetic association to SLE, and included seven SLE genome-wide association study (GWAS) loci: PTPRC (CD45), MHC-class III, UHRF1BP1, IRF5, IRF7, IKZF3 and UBE2L3. In addition, we observed association between genotype and variance of methylation at 20 DMCs in SLE, including at the HLA-DQB2 locus. Conclusions: Our results suggest that several of the genetic risk variants for SLE may exert their influence on the phenotype through alteration of DNA methylation levels at regulatory regions of target genes.

Dogs can spontaneously develop complex systemic autoimmune disorders, with similarities to human autoimmune disease. Autoantibodies directed at self-antigens are a key feature of these autoimmune diseases. Here we report the identification of interleukin enhancer-binding factors 2 and 3 (ILF2 and ILF3) as autoantigens in canine immune-mediated rheumatic disease. The ILF2 autoantibodies were discovered in a small, selected canine cohort through the use of human protein arrays; a method not previously described in dogs. Subsequently, ILF3 autoantibodies were also identified in the same cohort. The results were validated with an independent method in a larger cohort of dogs. ILF2 and ILF3 autoantibodies were found exclusively, and at a high frequency, in dogs that showed a speckled pattern of antinuclear antibodies on immunofluorescence. ILF2 and ILF3 autoantibodies were also found at low frequency in human patients with SLE and Sjögren’s syndrome. These autoantibodies have the potential to be used as diagnostic biomarkers for canine, and possibly also human, autoimmune disease.

Objectives Patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) have increased risk of cardiovascular disease (CVD). We investigated whether single nucleotide polymorphisms (SNPs) at autoimmunity risk loci were associated with CVD in SLE and RA. Methods Patients with SLE (n=1045) were genotyped using the 200K Immunochip SNP array (Illumina). The allele frequency was compared between patients with and without different manifestations of CVD. Results were replicated in a second SLE cohort (n=1043) and in an RA cohort (n=824). We analysed publicly available genetic data from general population, performed electrophoretic mobility shift assays and measured cytokine levels and occurrence of antiphospholipid antibodies (aPLs). Results We identified two new putative risk loci associated with increased risk for CVD in two SLE populations, which remained after adjustment for traditional CVD risk factors. An IL19 risk allele, rs17581834(T) was associated with stroke/myocardial infarction (MI) in SLE (OR 2.3 (1.5 to 3.4), P=8.5x10(-5)) and RA (OR 2.8 (1.4 to 5.6), P=3.8x10(-3)), meta-analysis (OR 2.5 (2.0 to 2.9), P=3.5x10(-7)), but not in population controls. The IL19 risk allele affected protein binding, and SLE patients with the risk allele had increased levels of plasma-IL10 (P=0.004) and aPL (P=0.01). An SRP54-AS1 risk allele, rs799454(G) was associated with stroke/transient ischaemic attack in SLE (OR 1.7 (1.3 to 2.2), P=2.5x10(-5)) but not in RA. The SRP54-AS1 risk allele is an expression quantitative trait locus for four genes. Conclusions The IL19 risk allele was associated with stroke/MI in SLE and RA, but not in the general population, indicating that shared immune pathways may be involved in the CVD pathogenesis in inflammatory rheumatic diseases.

Objectives Genetic variants in the transcription factor STAT4 are associated with increased susceptibility to systemic lupus erythematosus (SLE) and a more severe disease phenotype. This study aimed to clarify how the SLE-associated intronic STAT4 risk allele rs7574865[T] affects the function of immune cells in SLE. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 52 genotyped patients with SLE. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) in response to interferon (IFN)-α, IFN-γ or interleukin (IL)-12, total levels of STAT4, STAT1 and T-bet, and frequency of IFN-γ+ cells on IL-12 stimulation were determined by flow cytometry in subsets of immune cells before and after preactivation of cells with phytohaemagglutinin (PHA) and IL-2. Cellular responses and phenotypes were correlated to STAT4 risk allele carriership. Janus kinase inhibitors (JAKi) selective for TYK2 (TYK2i) or JAK2 (JAK2i) were evaluated for inhibition of IL-12 or IFN-γ-induced activation of SLE PBMCs. Results In resting PBMCs, the STAT4 risk allele was neither associated with total levels of STAT4 or STAT1, nor cytokine-induced pSTAT4 or pSTAT1. Following PHA/IL-2 activation, CD8+ T cells from STAT4 risk allele carriers displayed increased levels of STAT4 resulting in increased pSTAT4 in response to IL-12 and IFN-α, and an augmented IL-12-induced IFN-γ production in CD8+ and CD4+ T cells. The TYK2i and the JAK2i efficiently blocked IL-12 and IFN-γ-induced activation of PBMCs from STAT4 risk patients, respectively. Conclusions T cells from patients with SLE carrying the STAT4 risk allele rs7574865[T] display an augmented response to IL-12 and IFN-α. This subset of patients may benefit from JAKi treatment.

Systemic lupus erythematosus (SLE) is an autoimmune, inflammatory disease characterized by autoantibody production and an activated type I interferon system. Cardiovascular disease (CVD) is as a major cause of morbidity and mortality. The aim of this thesis was to identify genetic risk factors for CVD in SLE. The role of T cells in regulation of the interferon-α (IFNα) production by plasmacytoid dendritic cells (pDCs) was also investigated. In paper I, a thicker intima, thinner media and increased intima/media ratio was found in young premenopausal women with SLE compared to healthy controls indicating increased cardiovascular risk. As traditional ultrasound assessment of the common carotid intima-media thickness (CCA-IMT) in SLE has given conflicting results separate measurement of the intima and media can be a useful tool to identify SLE patients at increased risk of CVD. In paper II, an association was demonstrated in SLE between a STAT4 risk allele and ischemic cerebrovascular disease and presence of anti-phospholipid antibodies (aPL). The association remained after adjustment for traditional CVD risk factors. A possible mechanism for this association is that the risk allele leads to increased production of aPL, which promotes thromboembolism. In paper III, a genetic locus in IRF8 was identified to be associated to coronary heart disease (CHD) in SLE. The association remained after adjustment of other CHD risk factors. Patients with the IRF8 risk variant had increased CCA-IMT, more carotid plaques and reduced frequency of circulating B cells. Weaker binding of nuclear protein to the risk allele was demonstrated, suggesting a regulatory function of the IRF8 risk variant. In paper IV, activated T cells were found to strongly enhance the IFNα production by pDC stimulated with RNA-containing immune complexes via GM-CSF and IL-3. Activated SLE T cells enhanced the IFNα production to the same extent as T cells from healthy controls. This finding together with previous observations in SLE of increased levels of GM-CSF and IL-3 suggests that T cells contribute to the activated type I interferon system in SLE. In conclusion, this thesis demonstrates that genetic predisposition is important for CVD in SLE and describes a new role for T cells in the pathogenesis of SLE.