Abstract Objectives The aetiopathogenesis of SLE encompasses genetic, environmental and epigenetic factors. We investigated associations between an SLE methylation risk score (MRS), HLA-DRB1*03:01, a non-HLA polygenic risk score (PRS) and clinical and immunological phenotypes. Methods DNA methylation in whole blood from patients fulfilling ≥4 ACR-82 criteria and controls were investigated using the Illumina HM450K array. The discovery cohort included 311 patients and 400 controls, and the replication cohort comprised 175 patients and 187 controls. Seventeen independent, top differentially methylated CpG sites (Δβ of ≥0.1) from case-control comparisons, were used to calculate the MRS. Genotyping was performed using the Immunochip, and the PRS included 57 non-HLA SLE SNVs. Clinical data were collected from patient charts, and serum IFN-α2 was measured using Simoa. Results Higher MRS was strongly associated with serum IFN-α2 levels (p=1.04×10 −14 ). In both cohorts, higher MRS associated with discoid lupus, immunologic involvement, and anti-SSA/SSB/RNP/Sm autoantibodies (all p<0.05), and with higher disease activity in the discovery cohort (p=1.50×10⁻⁴). MRS was also elevated in patients with multiple autoantibodies (p<1.0×10 -15 ) and in HLA-DRB1*03:01 carriers (p<1.0×10 -3 ). In contrast, higher PRS was associated with nephritis, anti-dsDNA positivity, and lower prevalence of anti-SSB antibodies (all p<0.05). No correlation was observed between the MRS and the PRS (p=0.35). Conclusion The MRS defines an interferon-high, HLA-DRB1*03:01-linked SLE subset with multiple autoantibodies, partly distinct from PRS-associated nephritis risk, highlighting potentially divergent pathogenic pathways. These findings underscore the value of integrating genetic and epigenetic data to better understand underlying disease mechanisms in SLE. Key Messages Higher MRS, but not PRS, correlated with increased levels of serum IFN-α. The MRS was associated with discoid rash, hematologic disorder, hypocomplementemia, antibodies including anti-SSA and HLA-DRB1*03:01. Higher PRS was linked to nephritis and anti-dsDNA positivity, and did not associate with the MRS.

OBJECTIVES:The aetiopathogenesis of SLE encompasses genetic, environmental and epigenetic factors. We investigated associations between an SLE methylation risk score (MRS), HLA-DRB1*03:01, a non-HLA polygenic risk score (PRS), and clinical and immunological phenotypes. METHODS:DNA methylation in whole blood from patients fulfilling ≥4 ACR-82 criteria and from controls was investigated using the Illumina HM450K array. The discovery cohort included 311 patients and 400 controls, and the replication cohort comprised 175 patients and 187 controls. Seventeen independent, top differentially methylated CpG sites (Δβ of ≥0.1) from case-control comparisons were used to calculate the MRS. Genotyping was performed using the Immunochip, and the PRS included 57 non-HLA SLE single-nucleotide variants. Clinical data were collected from patient charts, and serum IFN-α2 was measured using Simoa. RESULTS:Higher MRS was strongly associated with serum IFN-α2 levels (P = 1.04 × 10-14). In both cohorts, higher MRS was associated with DLE, immunologic involvement, and anti-SSA/SSB/RNP/Sm autoantibodies (all P < 0.05), and with higher disease activity in the discovery cohort (P = 1.50 × 10-4). MRS was also elevated in patients with multiple autoantibodies (P < 1.0 × 10-15) and in HLA-DRB1*03:01 carriers (P < 1.0 × 10-3). In contrast, higher PRS was associated with nephritis, anti-dsDNA positivity, and lower prevalence of anti-SSB antibodies (all P < 0.05). No correlation was observed between the MRS and the PRS (P = 0.35). CONCLUSION:The MRS defines an IFN-high, HLA-DRB1*03:01-linked SLE subset with multiple autoantibodies, partly distinct from PRS-associated nephritis risk, highlighting potentially divergent pathogenic pathways. These findings underscore the value of integrating genetic and epigenetic data to better understand underlying disease mechanisms in SLE.

OBJECTIVE:To examine B cell-activating factor (BAFF) and type I interferon (IFN) activity at the transcriptional and protein levels in blood and placental tissue in SLE compared with healthy pregnancies to assess their relationship and to determine whether BAFF levels are associated with pregnancy outcomes in SLE. METHODS:In the SLE-Placenta study, we followed women with SLE (n=83) and healthy controls (n=67) throughout pregnancy. Blood samples were collected in all trimesters and at delivery from peripheral blood, placental intervillous blood and cord blood. Postpartum blood samples were obtained from a subset of women with SLE. Bulk messenger RNA (mRNA) sequencing was performed on peripheral blood mononuclear cells (PBMCs) and placental tissue from a subgroup of women with SLE and healthy controls. BAFF concentrations were measured by ELISA and IFNα protein levels by single-molecule array (Simoa). RESULTS:Women with SLE had upregulated BAFF (TNFSF13B) and IFN-stimulated gene expression in PBMCs and placenta compared with controls. BAFF blood levels were consistently and significantly higher in SLE throughout pregnancy and inversely correlated with circulating B cell numbers. SLE pregnancies with IF-ANA or anti-dsDNA positivity displayed higher BAFF levels than antibody-negative pregnancies but BAFF showed no association with disease activity. Both BAFF and IFNα concentrations were higher in placental than peripheral blood in SLE, whereas only BAFF showed additional accumulation in cord blood. Finally, elevated BAFF levels were associated with shorter pregnancy duration in SLE but not in healthy pregnancy. CONCLUSIONS:Pregnant women with SLE exhibited persistently elevated BAFF levels, which were associated with lower B cell numbers, SLE-related autoantibody positivity and shorter pregnancy duration. Together with a disease-specific placental enrichment of IFNα, these findings support the presence of an inflammatory and potentially pathogenic IFN-BAFF signature in SLE pregnancy. Further studies are needed to determine the functional consequences of these immunological alterations on maternal-fetal health in SLE.

OBJECTIVE:Lupus nephritis (LN) is a potentially severe manifestation of SLE, often resulting in permanent kidney damage. We investigated whether polygenic and/or epigenetic risk is associated with time to LN relapse and whether this association differs by initial therapy. METHODS:Caucasian patients with biopsy-verified LN between 1974 and 2022 (n=149) were genotyped using the Illumina Global Screening Array, and DNA methylation was assessed with the Illumina HumanMethylation450k BeadChip. A weighted Polygenic Risk Score (PRS) based on 55 non-Human leukocyte antigen (HLA) SLE risk gene variants and a Methylation Risk Score (MRS) based on the 17 most differentially methylated cytosine-phosphate-guanine sites identified in SLE-control comparison were calculated. Time to LN relapse was analysed using Cox proportional hazards models, adjusted for centre, sex and year of the first LN. RESULTS:Neither PRS nor MRS was associated with time to LN relapse in the overall cohort, irrespective of treatment (PRS: HR=1.00 (0.77-1.30); p=0.99; MRS: HR=1.01 (1.00-1.02); p=0.21). No associations were observed among cyclophosphamide (CYC)-treated patients (PRS: HR=0.81 (0.55-1.20); p=0.29; MRS: HR=1.00 (0.99-1.02); p=0.89). In non-CYC-treated patients, PRS showed a non-significant trend towards shorter time to LN relapse (HR=1.26 (0.88-1.81); p=0.21), with a similar trend for MRS (HR=1.02 (1.00-1.03); p=0.066). Adding MRS to the PRS model improved performance in this subgroup (likelihood ratio test, p=0.028), with significant associations for MRS (PRS: HR=1.56 (0.97-2.50); p=0.066; MRS: HR=1.02 (1.00-1.04); p=0.037). CONCLUSIONS:Genetic and epigenetic susceptibility appears to influence LN relapse risk in a treatment-dependent manner. The absence of an association among patients receiving CYC suggests that initial treatment with CYC may attenuate the impact of genetic and epigenetic predisposition on relapse. These findings support the potential of genetic and epigenetic profiling at LN diagnosis to improve relapse risk stratification and individualise selection of treatment.

OBJECTIVE:The interferon (IFN) system is activated in systemic sclerosis (SSc), and various stimuli may induce IFN in SSc patients. We investigated whether type I and III IFN responses to Toll-like receptor (TLR)-7/8/9 stimulation differ between patients with SSc and healthy individuals, and whether IFN production is associated with clinical features. METHOD:Peripheral blood mononuclear cells (PBMCs), monocyte-depleted PBMCs, and monocytes were prepared from 45 SSc patients and 47 healthy controls. Cells were stimulated with RNA-containing immune complexes (RNA-IC), an RNA oligonucleotide (ORN8L), or inactivated herpes simplex virus (HSV) targeting TLR7, TLR8, and TLR9, respectively. IFN-α, -β, -λ1, and -λ2 levels were measured by immunoassays, and intracellular IFN-α by flow cytometry. RESULTS:SSc PBMCs produced type I and III IFNs in response to all three stimuli, with the strongest response following TLR9 activation. Compared with controls, SSc PBMCs produced less IFN-α (p < 0.02), whereas IFN-β levels were higher in SSc monocytes (342 vs 59.9 pg/mL, p = 0.041) when activated via TLR9. IFN-λ1/2 levels were consistently lower than type I IFNs. IFN-α priming increased TLR7/8-induced IFN production in PBMCs. Strong TLR9-mediated IFN-α production was associated with diffuse cutaneous SSc, anti-RNA-polymerase III autoantibodies, and interstitial lung disease (ILD). CONCLUSION:Leucocytes from SSc patients produce type I and III IFNs in response to a broad range of TLR stimuli. IFN-α priming enhanced TLR7/8-induced IFN production, suggesting that strong IFN inducers, e.g. certain viruses, may amplify IFN synthesis in response to endogenous TLR ligands and contribute to SSc pathogenesis, including the development of ILD.

Lupus erythematosus is a chronic, heterogenous autoimmune disease driven by a complex interplay of genetic, environmental and hormonal factors, which can manifest as systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), or both simultaneously. Plasmacytoid dendritic cells (pDCs) are thought to play a central role in disease pathogenesis following the dysregulated and sustained production of type I interferon (IFN-I), leading to widespread immune system activation and chronic inflammation, resulting in organ and tissue damage characteristic of lupus. While treatment of lupus has shown success with approaches that inhibit the IFN pathway/IFN-I production, targeting pDCs is of great interest due to their accumulation in lesional tissues and key role in IFN-I production. However, SLE and CLE pathogenesis is clouded by unknowns including limited understanding of the pathophysiological loss of IFN pathway negative feedback, the complexity of IFN production by non-pDC cells (including pDC plasticity and role of different subsets/states), the role of other cytokines and chemokines in disease pathogenesis, and differences in pDC activation and downstream effects in SLE and CLE. Conceivably, different IFN-I-producing cells may be important in different organs, patients, and disease stages. As understanding of the role of pDCs in IFN-I production evolves, there is great potential to develop more targeted and effective treatments that can enhance patient outcomes to ensure pathophysiological and chronic inflammation is negated while maintaining physiological immunity to infection.

Renal dysfunction increases cardiovascular (CV) risk. We compared cystatin C-based estimated glomerular filtration rate (eGFRcys), creatinine-based eGFR (eGFRcr), and their ratio (eGFRcys/eGFRcr) in relation to major adverse cardiovascular events (MACE) and all-cause mortality in chronic coronary syndrome, assessing the added prognostic value of the eGFRratio. In this post hoc analysis of 14,513 Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy trial patients, we investigated associations between baseline eGFRcys, eGFRcr, their ratio, and MACE and all-cause death using Cox regression models, unadjusted and adjusted for eGFRcys, eGFRcr, and their combination. Discrimination was assessed using Harrell's C -index; added value by the fraction of new information (FNI). Median age was 65 years; 82% were male. Median eGFRcys was 77 (interquartile range [IQR]: 61–94) and eGFRcr 79 (IQR: 65–91) mL/min/1.73 m 2 . Over 3.7 years, 1449 MACE and 1063 deaths occurred. Lower eGFR values and eGFRratio were associated with increased MACE risk, primarily driven by CV death. For eGFRcys 60 versus 90, the hazard ratio (HR) for MACE adjusted for eGFRcr was 1.77 (95% CI: 1.49–2.09, FNI 54%). In contrast, eGFRcr adjusted for eGFRcys showed no positive association (HR 0.82, 95% CI: 0.68–0.97, FNI 3%). A lower eGFRratio was linked to higher MACE risk (HR 1.99, 95% CI: 1.80–2.21), which remained after eGFRcr adjustment (HR 1.89, 95% CI: 1.70–2.10, FNI 54%) but was attenuated after eGFRcys adjustment (HR 1.29, 95% CI: 1.13–1.46, FNI 5%). In chronic coronary syndrome, lower eGFRcys, eGFRcr, and eGFRratio were associated with higher MACE and mortality risk. eGFRcys had the strongest association; eGFRcr and eGFRratio added limited incremental value.

OBJECTIVES:Around 30% of patients with rheumatoid arthritis (RA) lack rheumatoid factor and anti-citrullinated protein antibodies (ACPA) complicating diagnosis and potentially delaying treatment. We hypothesised that innate immune mechanisms might be more prominent in ACPA- RA. METHODS:We performed single-cell RNA sequencing of mononuclear cells from peripheral blood (PBMC) and synovial fluid (SFMC) of patients with ACPA- and ACPA+ RA (n = 4 per group: discovery cohort; n = 8 per group: validation cohort). Dendritic cells and proinflammatory cytokine production were analysed by flow cytometry on SFMC from patients with ACPA- RA, ACPA+ RA, and psoriatic arthritis. Interferon (IFN) levels in synovial fluid (SF) and serum were measured in these groups. RESULTS:Several macrophage subsets and cDC2 were enriched in ACPA- RA SF whereas the frequency of Tph and B cells was increased in ACPA+ RA SF. Type I IFN-stimulated genes were detected in SFMC, but not PBMC, of patients with ACPA- RA. A type I IFN signature was also observed in synovial tissue from two patients with ACPA- RA in an independent dataset. IFN levels were higher in SF than serum but IFN-α/β production did not differ between ACPA+ and ACPA- RA. CONCLUSIONS:This study identifies a distinct innate cell composition and type I IFN gene response in synovial joints, but not in peripheral blood, of patients with ACPA- RA. Similar IFN levels across groups suggest the IFN signature may have been primed before the cells entered the joints. These findings provide a foundation for future research on type I IFN responses in ACPA- RA.

Background SLE is a systemic autoimmune disease with a large number of common risk gene variants, but several rare gene variants can cause monogenic SLE. The relationship between common and rare variants in SLE is unclear. We therefore investigated the occurrence of rare deleterious variants in patients with childhood-onset SLE (cSLE) and adult-onset SLE (aSLE) and compared the frequency of these variants with their individual SLE polygenic risk score (PRS).Materials and methods Targeted sequencing of 1832 gene regions, including coding regions of 31 genes associated with monogenic SLE, was performed in 958 patients with SLE and 1026 healthy individuals. A total of 116 patients with SLE had disease onset before the age of 18 (cSLE). An SLE common variant PRS was created from 37 SLE genome-wide association study single nucleotide variants (SNVs).Results Rare coding deleterious SNVs (RD SNVs) were observed in 23 of the monogenic SLE-associated genes. Six per cent of patients with cSLE, compared with 3.2% of controls and 4.6% of patients with aSLE, carried rare deleterious alleles. In cSLE, RD SNVs were observed in the C1S, DDX58, IFIH1, IKZF1, RNASEH2A and C8A genes. A PRS analysis showed that patients with cSLE with any of these gene variants had a similar average PRS as control individuals.Conclusion RD SNVs were observed in a small proportion of cSLE and carriers of these RD SNVs had a PRS similar to healthy individuals, suggesting the importance of rare coding heterozygous variants in driving disease risk in a subset of children with SLE.

Objectives Bisphenol A (BPA), a xenoestrogen that can alter DNA methylation status, has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). This study aimed to investigate whether methylation changes at BPA-sensitive 5’-C-phosphate-G-3’ (CpG) sites are associated with SLE and clinical subphenotypes.Methods A discovery cohort (n=747) and a replication cohort (n=388) including Swedish patients with SLE and healthy controls were investigated using the Illumina HM450k bead chip. BPA-sensitive CpG sites were selected if differentially methylated in ≥2 of 7 BPA exposure studies and supported by cell line data. A BPAAll score including 19 CpGs and a BPASLE score based on three CpG sites co-localised in the genome with SLE risk loci were calculated for each individual, analysed for associations with clinical data and then compared with publicly available transcriptomic data from BPA-treated cells.Results Patients with SLE had significantly higher BPASLE score than controls in the discovery (OR 1.34, p=4.6×10-13), replication (OR 1.28, p=1.1×10-5) and meta-analysis (OR 1.32, p=3.3×10-17). Higher BPAAll score was associated with SLE in the discovery cohort (OR 1.05, p=2.3×10-3) but not in the replication cohort (OR 1.04, p=0.12) with a significant difference in the meta-analysis (OR 1.05, p=7.0×10-4). Both scores were associated with prednisolone treatment (p<0.001), and the BPASLE score was associated with serositis and autoantibodies (p<0.05). Transcriptomic analysis of BPA-treated cells revealed enrichment in pathways such as interferon and mitogen-activated protein kinase signalling.Conclusions Our findings reveal a novel association between BPA exposure and DNA methylation changes in SLE, with potential implications for the regulation of immune-related gene expression.

BACKGROUND:Systemic lupus erythematosus (SLE) is an autoimmune disease with a heterogenous clinical picture. This study aimed to link genetic SLE predisposition with relevant clinical manifestations. METHOD:Datasets best corresponding to the 11 American College of Rheumatology 1982 (ACR-82) classification criteria for SLE in a large, public database (FinnGen consortium, >218,000 individuals) were identified. Mendelian randomization analysis was conducted to evaluate the effect of a high genetic SLE predisposition on each manifestation. Next, validation was conducted in a clinical SLE cohort comprising 1487 genotyped Scandinavian patients with detailed clinical data. Based on the public datasets, genetic risk scores (GRSs) for each relevant manifestation were constructed for each patient. Associations between each GRS and the corresponding ACR-82 criterion were evaluated using logistic regression. RESULTS:In the FinnGen biobank, the cumulative effect of the 57 SLE risk SNPs was associated with an increased risk of rosacea, OR 1.09 (1.03-1.16), polyarthropathies, OR 1.10 (1.06-1.14), pleural effusions, OR 1.09 (1.04-1.14), and hemolytic anemia, OR 1.32 (1.10-1.58). In the clinical cohort, 5 of the 11 GRSs generated from the public datasets were associated with their corresponding ACR-82 criterion: arthritis, OR 1.15 (1.02-1.31), renal disorder, OR 1.15 (1.04-1.29), neurologic disorder, OR 1.24 (1.04-1.47), hematologic disorder, OR 1.12 (1.00-1.24), and immunologic disorder, OR 1.37 (1.22-1.56). CONCLUSION:The findings demonstrate that known SLE risk gene variants play a role in the development of at least half of the ACR-82 criteria for SLE, indicating a future possibility of using genetics to predict a variety of disease sub-phenotypes in SLE.

Patients with systemic lupus erythematosus (SLE) display an increased expression of type I interferon (IFN)-regulated genes, a so-called IFN signature. This discovery was preceded by the observation in Uppsala that patients with malignant diseases treated with type I IFN occasionally developed autoimmune diseases, including SLE. The adverse event of IFN treatment was the start of an intensive search for the role of the type I IFN system in patients with spontaneously occurring SLE. A key finding by our group was the detection in patients with SLE of endogenous IFN-inducers that could activate plasmacytoid dendritic cells (pDC) to IFN production. Further studies revealed the mechanisms by which these cells are triggered to a continuous IFN synthesis. We could also identify a large number of risk genes for SLE and several molecules connected to type I IFN production and response. My group early on suggested the possibility that some of these molecules are suitable therapeutic targets in SLE, but also other IFN-driven diseases. Antibodies against the type I IFN receptor (anifrolumab) have recently shown efficacy in clinical trials for SLE, and anifrolumab is now approved as a treatment for this disease. Several other drugs targeting critical molecules in the IFN signaling pathways – including BCDA-2 (Blood Dendritic Cell Antigen 2), TLR7/8 (Toll-like receptor 7/8), and TYK2 (Tyrosine Kinase 2) – are currently in early clinical phases, potentially expanding therapeutic options for SLE. In this review, several important observations regarding the role of the type I IFN system in SLE and therapeutic implications are discussed.

OBJECTIVES:Adverse pregnancy outcomes are more common in women with SLE compared with healthy women, but we lack prognostic biomarkers. Plasma IFN-α protein levels are elevated in a subgroup of pregnant women with SLE, but whether this is associated with pregnancy outcomes is unknown. We investigated the relationship between IFN-α, adverse pregnancy outcomes and the presence of autoantibodies in SLE pregnancy. METHODS:We followed 76 women with SLE prospectively. Protein levels of IFN-α were quantified in plasma collected in the second and third trimester with single-molecule array. Positivity for ANA and aPL antibodies was assessed during late pregnancy with multiplexed bead assay. Clinical outcomes included the adverse pregnancy outcomes small for gestational age (SGA), preterm birth and preeclampsia. RESULTS:During SLE pregnancy, women with SGA infants compared with those without had higher levels of plasma IFN-α protein, and IFN-α positivity was associated with lower birth weight of the infant. Preterm birth was associated with autoantibodies against chromatin. IFN-α protein levels associated positively with autoantibodies against chromatin, Smith/RNP (SmRNP) and RNP, but negatively with aPL antibodies. CONCLUSION:Elevated IFN-α protein in plasma of women with SLE is a potential risk factor for lower birth weight of their infants. The association between IFN-α and lower birth weight warrants further investigation regarding the pathophysiological role of IFN-α during SLE pregnancy.

PV106 / #522 Poster Topic:AS12 - Genetics, Epigenetics, Transcriptomics Using their autoantibody profile, patients with systemic lupus erythematosus (SLE) can be grouped into 4 less heterogeneous subgroups [1] Subgroup 1 was dominated by anti-SSA/SSB, Subgroup 2 by anti-nucleosome/Sm/RNP/dsDNA, Subgroup 3 by aPL, and Subgroup 4 was negative for 13 antibodies tested, but ANA ever positive. Those subgroups differed in cytokine levels, clinical manifestations, andHLA-DRB1gene associations.[1] Therefore, we hypothesize that the pathogenic mechanisms are different among these subgroups of patients. We aimed to evaluate whether there are differences in known SLE genetic risk factors and protein levels among antibody-defined SLE subgroups. We analyzed 448 patients from our previous study[1] to address differences in genetic risk factors. We compared the SLE polygenic risk scores (PRS) previously described by Reid et al 2020.[2] Using a double-sided Mann-Whitney U test, we tested differences in the distribution of 2 PRS: 1 including 4 Single Nucleotide Polymorphisms (SNPs) in theHLAregion or 57 non-HLASNPs across the subgroups. Furthermore, untargeted liquid chromatography-mass spectrometry (LC-MS) was implemented using plasma samples from 100 SLE patients, 25 representing each subgroup. Differential expression analysis was performed using linear modeling with the limma package,[3] and pairwise comparisons were conducted among the subgroups. P-values for both approaches were corrected using a False Discovery Rate (FDR) multiple-testing correction. Adjusted values (P-adj) lower than 0.05 were considered significant. Subgroup 1 displayed significantly higherHLA-PRS compared to subgroup 2 (P-adj = 9.27e-06), subgroup 3 (P-adj =1.65e-10), and subgroup 4 (P-adj =3.64e-04) (Figure 1). Similarly, subgroup 2 had a significantly higher i-PRS than subgroup 3 (P-adj = 7.3e-03). Conversely, for PRS calculated using SNPs outside the HLA region, scores of subgroup 1 were significantly lower than scores from subgroups 2 (P-adj = 3.89e-02) and 3 (P-adj = 3.89e-02), suggesting a higher contribution of SNPs in the HLA region to the overall higher genetic risk of subgroup 1. Differential expression analysis of proteins (n=2625) between the subgroups revealed that an isoform of Complement C4-B (C4B) was significantly overexpressed (P-adj 3.72e0-5) in subgroup 1 compared to subgroup 3, followed by C2, which indicates involvement of the complement system in this subgroup. Likewise, an isoform of Immunoglobulin heavy constant gamma 3 (IGHG3) was significantly overexpressed (P-adj 2.3e-03) in subgroup 2 compared to subgroup 4 (Figure 2). These preliminary results support the concept that unanalyzed or unknown autoantibodies and B cell involvement may be present in patients of subgroup 4. Figure 1. Figure 2. Subgroup 1 exhibited higherHLA-PRS than the other subgroups and elevated C4b levels compared to Subgroup 3. This is consistent with the high linkage disequilibrium betweenC4gene andHLArisk variants. These preliminary findings support the hypothesis of subgroup-specific pathogenic mechanisms among antibody-defined SLE subgroups of patients. We will further analyze this dataset to incorporate PRS relevant to immune cell phenotypes and calculate PRS targeted to SLE subgroups. We will also validate these findings in independent populations.References:[1.] Diaz-Gallo LM. ACR Open Rheumatol 2022;4(1):27-39. [2.] Reid S. Ann Rheum Dis 2020;79(3):363-9. [3.] Ritchie ME. Nucleic Acids Res 2015;43(7):e47.

Anti-cyclic citrullinated peptide2 (CCP2) antibody positivity in rheumatoid arthritis (RA) and in the predisease phase, together with the success of B-cell depletion, support a crucial role for B cells in RA pathogenesis. Yet, knowledge of B cells in the transition from autoimmunity to RA is limited, and therefore we here investigated B-cell changes during the risk-RA phase. B-cell phenotypes in 18 CCP2-positive risk-RA individuals with musculoskeletal complaints were studied, parallel with ten CCP2-positive RA patients and nine healthy controls. Nine of the risk-RA individuals progressed to RA. B-cell phenotypes were investigated using spectral flow cytometry. The results demonstrate that unswitched and switched memory B-cell frequencies in the risk-RA cohort were more similar to controls than RA patients. Yet, risk-RA progressors displayed an early activation profile amongst naïve B cells. Deeper characterization of the memory compartment revealed expansion of CD27-negative IgG+ B cells both in RA compared with controls ( p = 0.0172) and in risk-RA progressors versus non-progressors ( p = 0.0295). Overall, we demonstrate that the phenotypic distribution of B cells is altered in the risk-RA phase. This includes changes in CD27-negative class-switched B cells, which have been attributed to autoreactive and anergic features implicating a possible contribution to RA development.

Fine mapping and bioinformatic analysis of the DDX6-CXCR5 genetic risk association in Sjögren's Disease (SjD) and Systemic Lupus Erythematosus (SLE) identified five common SNPs with functional evidence in immune cell types: rs4938573, rs57494551, rs4938572, rs4936443, rs7117261. Functional interrogation of nuclear protein binding affinity, enhancer/promoter regulatory activity, and chromatin-chromatin interactions in immune, salivary gland epithelial, and kidney epithelial cells revealed cell type-specific allelic effects for all five SNPs that expanded regulation beyond effects on DDX6 and CXCR5 expression. Mapping the local chromatin regulatory network revealed several additional genes of interest, including lnc-PHLDB1-1. Collectively, functional characterization implicated the risk alleles of these SNPs as modulators of promoter and/or enhancer activities that regulate cell type-specific expression of DDX6, CXCR5, and lnc-PHLDB1-1, among others. Further, these findings emphasize the importance of exploring the functional significance of SNPs in the context of complex chromatin architecture in disease-relevant cell types and tissues.