Identification of new candidate drugs for primary Sjögren's syndrome using a drug repurposing transcriptomic approach.

OBJECTIVES: To date, no immunomodulatory drug has demonstrated its efficacy in primary SS (pSS). We sought to analyse potential commonalities between pSS transcriptomic signatures and signatures of various drugs or specific knock-in or knock-down genes. METHODS: Gene expression from peripheral blood samples of patients with pSS was compared with that of healthy controls in two cohorts and three public databases. In each of the five datasets, we analysed the 150 most up- and downregulated genes between pSS patients and controls with regard to the differentially expressed genes resulting from the biological action on nine cell lines of 2837 drugs, 2160 knock-in and 3799 knock-down genes in the Connectivity Map database. RESULTS: We analysed 1008 peripheral blood transcriptomes from five independent studies (868 patients with pSS and 140 healthy controls). Eleven drugs could represent potential candidate drugs, with histone deacetylases and PI3K inhibitors among the most significantly associated. Twelve knock-in genes were associated with a pSS-like profile and 23 knock-down genes were associated with a pSS-revert profile. Most of those genes (28/35, 80%) were interferon-regulated. CONCLUSION: This first drug repositioning transcriptomic approach in SS confirms the interest of targeting interferons and identifies histone deacetylases and PI3K inhibitors as potential therapeutic targets.

Stratified genetic analysis reveals sex differences in MPO-ANCA-associated vasculitis.

OBJECTIVE: To identify and genetically characterize subgroups of patients with ANCA-associated vasculitides (AAV) based on sex and ANCA subtype. METHODS: A previously established SNP dataset derived from DNA sequencing of 1853 genes and genotyping of 1088 Scandinavian cases with AAV and 1589 controls was stratified for sex and ANCA subtype and analysed for association with five top AAV SNPs. rs9274619, a lead variant at the HLA-DQB1/HLA-DQA2 locus previously associated with AAV positive for myeloperoxidase (MPO)-ANCA, was analysed for association with the cumulative disease involvement of ten different organ systems. RESULTS: rs9274619 showed a significantly stronger association to MPO-ANCA-positive females than males [P = 2.0 × 10-4, OR = 2.3 (95% CI 1.5, 3.5)], whereas proteinase 3 (PR3)-ANCA-associated variants rs1042335, rs9277341 (HLA-DPB1/A1) and rs28929474 (SERPINA1) were equally associated with females and males with PR3-ANCA. In MPO-ANCA-positive cases, carriers of the rs9274619 risk allele were more prone to disease engagement of eyes [P = 0.021, OR = 11 (95% CI 2.2, 205)] but less prone to pulmonary involvement [P = 0.026, OR = 0.52 (95% CI 0.30, 0.92)]. Moreover, AAV with both MPO-ANCA and PR3-ANCA was associated with the PR3-ANCA lead SNP rs1042335 [P = 0.0015, OR = 0.091 (95% CI 0.0022, 0.55)] but not with rs9274619. CONCLUSIONS: Females and males with MPO-ANCA-positive AAV differ in genetic predisposition to disease, suggesting at least partially distinct disease mechanisms between the sexes. Double ANCA-positive AAV cases are genetically similar to PR3-ANCA-positive cases, providing clues to the clinical follow-up and treatment of these patients.

Biological treatment in severe psoriasis: Influence on peripheral blood dendritic cells.

In-depth immunophenotyping by flow cytometry of peripheral blood dendritic cell (DC) populations of psoriasis vulgaris without (PsO; N = 23) or with psoriatic arthritis (PsA; N = 15), before (T1) and after 12 months (T2) therapy with the anti-TNF drugs infliximab, etanercept, the anti-IL-17A secukinumab and the anti-IL12/IL-23 ustekinumab. Compared to healthy donors (N = 38), patients with PsA displayed lower frequencies of dendritic cell subsets pDC, cDC1 and cDC2, which were normalized following treatment except pDC. In contrast, patients with PsO only displayed lower frequencies of pDC which were normalized following treatment. Figure created with BioRender.com.

Mass cytometry analysis of blood immune cells from psoriasis patients on biological therapy.

Psoriasis is a chronic immune-mediated skin disease accompanied by systemic inflammation and comorbidities. We analyzed peripheral blood mononuclear cells (PBMCs) in the search for immune signatures and biomarkers related to psoriasis severity and treatment effect. Thirty-two patients with psoriasis and 10 matched healthy controls were included. PBMCs were collected before and after initiation of anti-TNF, anti-IL-17 or anti-IL-12/23 treatment and analyzed utilizing 26-parameter mass cytometry. The number of circulating Th17, Th22, Th9, and cytotoxic T cells were increased in severe psoriasis. Intracellular pp38 and pERK in T helper cells were associated with disease severity. Differences between responders and nonresponders regarding cell composition and intracellular signaling were identifiable already at inclusion. Biological treatment induced memory cells, restored inhibitory PD-1 function of T cells, and reduced a potential pro-atherogenic profile in monocytes. In conclusion, these results indicate amelioration of systemic inflammation in psoriasis after biological treatment. Such broad immune profiling may enable prospective stratification of patients regarding future treatment response. Successful early intervention may lead to a healthier trajectory with favorable implications on later comorbidities.

Identification and functional characterization of a novel susceptibility locus for small vessel vasculitis with MPO-ANCA.

OBJECTIVE: To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV). METHODS: Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay. RESULTS: PR3-ANCA+ AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 × 10-61, odds ratio (OR) 0.10; rs9277341, P = 1.5 × 10-44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 × 10-10, OR 2.9). MPO-ANCA+ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 × 10-25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 × 10-7, OR 3.0), the latter a novel susceptibility locus for MPO-ANCA+ granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele. CONCLUSION: We identified a novel susceptibility locus for MPO-ANCA+ AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types.

Disease mechanisms in Sjögren's syndrome: What do we know?

Why should we explore and study disease mechanisms? This is particularly important when we are dealing with complex pathogenesis without a direct causal agent, for example, syndromes with multiple organ involvements. Sjögren's syndrome is definitely such an entity. Also, there are a number of reasons for such studies such as disclosing the aetiology, to identify biomarkers for diagnosis and assessment of the disease process and monitor response to treatment, to determine targets for treatment, to define critical items in classification criteria, amongst others. Samples available for the study of disease mechanisms in Sjögren's syndrome have included serum (autoantibodies, cytokines), DNA (gene profiling, GWAS), cells (phenotypes/flow cytometry, proportion of cells/CyTOF), tissue (focal inflammation, germinal centres, mass cytometry), and saliva (proteomics, biochemistry, mucosal immunity). An original explanatory concept for the pathogenesis of Sjögren's syndrome proposed a specific and self-perpetuating immune-mediated loss of exocrine tissue as the principal cause of glandular hypofunction. This hypothesis however falls short of accommodating several Sjögren's syndrome-related phenomena and experimental findings. Today, the emergence of advanced bio-analytical platforms has further enabled the identification of central pathogenic processes and potential biomarkers. The purpose of this minor review is to highlight a selection of previous but also recent and novel aspects on the disease mechanisms in Sjögren's syndrome.

Molecular pathways in patients with systemic lupus erythematosus revealed by gene-centred DNA sequencing.

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.

Genetic and clinical basis for two distinct subtypes of primary Sjögren's syndrome.

OBJECTIVES: Clinical presentation of primary Sjögren's syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints. METHODS: We performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls. RESULTS: We found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2×10-62) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS. CONCLUSION: Two subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups.

Viral antigens elicit augmented immune responses in primary Sjögren's syndrome.

OBJECTIVES: Infections have been suggested in the pathogenesis of primary SS (pSS). Systematic studies of immune responses to microbial antigens in vivo may be performed during vaccination. In the present study, we therefore longitudinally followed patients with pSS and controls during split-virion influenza vaccination to identify pSS-specific cellular, transcriptomic and serological responses. METHODS: Patients without treatment (pSSUntr, n = 17), on hydroxychloroquine-treatment (pSSHCQ, n = 8), and healthy controls (n = 16) were included. Antibody titres were determined by ELISA. Plasma proteins were measured by proximity extension assay. Monocyte gene expression was assessed by Nanostring. Routine laboratory tests were performed and clinical disease symptoms were registered by questionnaires. RESULTS: pSSUntr developed higher vaccine-specific IgG titres compared with controls. Notably, anti-Ro52 autoantibody titres increased in pSSUntr but remained unchanged in pSSHCQ. No changes in disease symptoms including EULAR Sjögren's Syndrome Patient Reported Index score were registered. Twenty-four hours after vaccination, the leucocyte count in pSSUntr decreased, with a concomitant increase of CCL7 in plasma. Transcriptomic analysis in monocytes revealed differential vaccination-related expression of the NEMO/IKBKG gene, and its higher induced expression in pSSUntr associated with higher serological vaccine responses. Moreover, titres of vaccine-specific antibodies were associated with higher vaccination-induced NF-κB signalling and higher steady-state IFN signatures in monocytes, and with the levels of several plasma proteins with soluble PD-1 displaying the strongest association. CONCLUSION: We observed augmented innate and adaptive immune responses in pSS following viral antigen exposure suggesting an underlying hyper-responsiveness to immune challenges, supporting a role for infections driving the immunopathology and acting as environmental risk factor for pSS.

A brief history of Scandinavian society for immunology.

The Scandinavian Society for Immunology (SSI) was established with the purpose to advance the study of immunology in Scandinavia and to facilitate contacts between individuals and laboratories working within the field. To fulfill this the Society should organize scientific meetings and laboratory courses and take any other measure to support the development of immunology. A second objective was to establish contact and scientific exchange with other societies in Europe and overseas. By joining five national societies from the Nordic countries (Denmark, Finland, Iceland, Norway and Sweden) into one umbrella society this has given SSI a more powerful voice in international organizations such as European Federation of Immunological Societies (EFIS) and International Union of Immunological Societies (IUIS). The Scandinavian Foundation for Immunology and the Scandinavian Journal of Immunology has greatly facilitated the annual meetings to be of international high quality and by attracting more participants. Thus, SSI provides a forum for Nordic immunologists to share their research results and to increase collaboration over the borders. In conclusion, the SSI has undoubtedly been and will hopefully continue to be a major strength for Scandinavian immunology.

Acinar adipose tissue infiltration in salivary gland biopsy is associated with kynurenines-Interferon-γ pathway inflammation biomarkers.

OBJECTIVES: Assess if kynurenines metabolites are biomarkers of damage at labial salivary gland biopsy (LSGB). METHODS: This is a cross-sectional study including 99 patients with primary Sjögren's syndrome (AECG 2002 or ACR/EULAR 2017). Kynurenines were measured in plasma using liquid chromatography-tandem mass spectrometry. RESULTS: 95.9% were females, 51±12 years. Most had focal lymphocytic sialadenitis with focus score ≥1 (73.7%, n=73/99). The majority had mild to severe acinar atrophy (70.4%, n=57/81) and adipose infiltration (51.2%, n=39/80). Individuals with adipose infiltration were older (53.49±12.33 vs. 47.51±11.29 years, p=0.016), showed higher frequency of glandular dysfunction and higher kynurenines levels. Schirmer's test ≤ 5 mm/5min was found in 69.2% of individuals with adipose infiltration compared to 41% without (p=0.012) and unstimulated whole salivary flow (UWSF) was found in 87.2% compared to 70% without adipose infiltration (p=0.063). Additionally, individuals with adipose infiltration showed higher kynurenines metabolites compared with those without: quinolinic acid (503.35±193.30 vs. 427.35±285.76 nmol/L, p=0.029), kynurenine (1.99±0.6, 54 vs. 1.61±0.46 µmol/L, p=0.006), kynurenine/tryptophan ratio (KTR) (0.030±0.09 vs. 0.025±0.01, p=0.031) and anthranilic acid (03±4.96 vs. 16.46±5.24 nmol/L, p=0.022). CONCLUSIONS: Kynurenines are biomarkers of greater adipose infiltration in LSGB and glandular dysfunction suggesting that activation of interferon-γ pathway is involved in the salivary and lacrimal glands damage.

Aberrant cell signalling in PBMCs upon IFN-α stimulation in primary Sjögren's syndrome patients associates with type I interferon signature.

Primary Sjögren's syndrome (pSS) is a complex systemic autoimmune disease with heterogeneous disease manifestations. Genetic predisposition, hormonal and environmental factors are all thought to contribute to disease etiology and pathogenesis. A better understanding of the disease pathogenesis is required in order to establish new targeted therapies. We analysed MAPK/ERK and JAK/STAT signalling networks in peripheral blood mononuclear cells (PBMCs) upon stimulation with interferon alpha 2b (IFN-α2b) by flow cytometry to define potentially dysfunctional intracellular signalling pathways involved in disease pathogenesis. Cells derived from pSS patients displayed small but significant increases in basal phosphorylation levels of numerous signalling proteins compared to cells from healthy donors. The phosphorylation profiles following stimulation with IFNα2b differed significantly between pSS patients and healthy donors, especially regarding STAT1 Y701. PCA further grouped patients according to clinical characteristics. Type I IFN induced gene expression was found to negatively correlate with the IFN-α2b induced phosphorylation of STAT3 S727 in T cells and positively with pSTAT1 Y701 in B cells. Increases in pSTAT1 Y701 were associated with the presence of autoantibodies. Our results indicate involvement of both STAT3 S727 and STAT1 Y701 pathways in pSS patients. Therapies targeting these pathways might therefore be beneficial for certain subgroups of patients.

Assessment of major salivary gland ultrasonography in Sjögren's syndrome. A comparison between bedside and post-examination evaluations.

OBJECTIVES: Major salivary gland ultrasonography (SGUS) is a suitable diagnostic tool in Sjögren's syndrome (SS). We aimed to determine the more representative gland, projection and format most applicable for reproducible image analysis. METHODS: One investigator performed SGUS in patients with SS. Parotid and submandibular glands were examined in longitudinal and transverse planes and evaluated bedside using a simplified scoring system (0-3). Longitudinal and transverse images and videos of all glands were stored and later evaluated/graded by three investigators, at two time-points. Agreement was calculated using intraclass correlation coefficient (ICC). RESULTS: The ICC for static image and video scoring compared to bedside evaluation ranged from 0.131 to 0.882. Average ICC for longitudinal/transverse image was 0.667/0.662, and 0.683/0.510 for longitudinal/transverse video. Interobserver reliability was good to excellent (0.81-0.94). Intraobserver reliability scores ranged from fair to excellent (0.46-0.96). The correlation between image and video evaluations of all modalities and examiners was good to excellent (0.614-0.904). The best mean ICC was found for the longitudinal projection of the left parotid gland (0.861) and the lowest mean ICC was for the transverse projection of the left submandibular gland (0.66). CONCLUSIONS: Our study indicates a trend favouring longitudinal video of the parotid gland as preferred projection, gland and storage format.

3-Day monocyte-derived dendritic cells stimulated with a combination of OK432, TLR7/8 ligand, and prostaglandin E2 are a promising alternative for cancer immunotherapy.

Numerous trials using dendritic cell (DC)-based vaccinations for the treatment of cancer are being carried out. However, an improvement of the quality of DC used is highly warranted. We here generated human monocyte-derived dendritic cells using a 3 day protocol and stimulated the cells using a combination of OK432 (Picibanil), TLR7/8 ligand CL097, and reduced amounts of prostaglandin (PG)E2. We analyzed phenotype, migratory, and T-cell stimulatory capacity compared to a cytokine cocktail consisting of IL-1ß, IL-6, TNF, and PGE2. The OK432 cocktail stimulated cells had a similar mature phenotype with upregulated co-stimulatory molecules, HLA-DR and CCR7 as the cytokine cocktail-matured cells and a similar cytokine profile except increased amounts of IL-12p70. Chemotaxis towards CCL19 was reduced compared to the cytokine cocktail, but increased compared to OK432 alone. The T-cell stimulatory capacity was similar to the cytokine cocktail stimulated cells. In conclusion, the OK432 cocktail has the advantage of inducing IL-12p70 production without impairing phenotype or T-cell stimulatory capacity of the cells and might, therefore, be an advantageous alternative to be used in DC-based immunotherapy.

Serum cytokine measurements and biological therapy of psoriasis - Prospects for personalized treatment?

Psoriasis is an immune-mediated disease where the IL-23/Th17 axis as well as TNF comprise main targets of biological therapy. Immune profiling has so far not been embraced as a clinical tool. We aimed to investigate relationships between individual serum cytokine levels in 40 psoriasis patients before and after receiving biological therapy and Psoriasis Area and Severity Index (PASI) and Dermatological Life Quality Index (DLQI). Serum concentration of 25 cytokines was determined by Luminex technology. Mean PASI and DLQI decreased by 71% and 65%, respectively. Increase of IL-2 positively correlated with improvement of PASI and DLQI. Moreover, increase of IL-5, IL-10, IL-12, IL-22 and GM-CSF correlated with treatment effect. Notably, logistic regression revealed four times higher risk of having severe psoriasis when IL-17A increased by 1 pg/mL (OR: 4.06, P < 0.05). Selected serum cytokines might constitute useful biomarkers for monitoring disease activity and optimizing therapeutic strategies in psoriasis patients.

Current concepts on Sjögren's syndrome - classification criteria and biomarkers.

Sjögren's syndrome is a lymphoproliferative disease with autoimmune features characterized by mononuclear cell infiltration of exocrine glands, notably the lacrimal and salivary glands. These lymphoid infiltrations lead to dryness of the eyes (keratoconjunctivitis sicca), dryness of the mouth (xerostomia), and, frequently, dryness of other surfaces connected to exocrine glands. Sjögren's syndrome is associated with the production of autoantibodies because B-cell activation is a consistent immunoregulatory abnormality. The spectrum of the disease extends from an organ-specific autoimmune disorder to a systemic process and is also associated with an increased risk of B-cell lymphoma. Current treatments are mainly symptomatic. As a result of the diverse presentation of the syndrome, a major challenge remains to improve diagnosis and therapy. For this purpose an international set of classification criteria for primary Sjögren's syndrome has recently been developed and validated and seems well suited for enrolment in clinical trials. Salivary gland biopsies have been examined and histopathology standards have been developed, to be used in clinical trials and patient stratification. Finally, ultrasonography and saliva meet the need of non-invasive imaging and sampling methods for discovery and validation of disease biomarkers in Sjögren's syndrome.

The IRF5-TNPO3 association with systemic lupus erythematosus has two components that other autoimmune disorders variably share.

Exploiting genotyping, DNA sequencing, imputation and trans-ancestral mapping, we used Bayesian and frequentist approaches to model the IRF5-TNPO3 locus association, now implicated in two immunotherapies and seven autoimmune diseases. Specifically, in systemic lupus erythematosus (SLE), we resolved separate associations in the IRF5 promoter (all ancestries) and with an extended European haplotype. We captured 3230 IRF5-TNPO3 high-quality, common variants across 5 ethnicities in 8395 SLE cases and 7367 controls. The genetic effect from the IRF5 promoter can be explained by any one of four variants in 5.7 kb (P-valuemeta = 6 × 10(-49); OR = 1.38-1.97). The second genetic effect spanned an 85.5-kb, 24-variant haplotype that included the genes IRF5 and TNPO3 (P-valuesEU = 10(-27)-10(-32), OR = 1.7-1.81). Many variants at the IRF5 locus with previously assigned biological function are not members of either final credible set of potential causal variants identified herein. In addition to the known biologically functional variants, we demonstrated that the risk allele of rs4728142, a variant in the promoter among the lowest frequentist probability and highest Bayesian posterior probability, was correlated with IRF5 expression and differentially binds the transcription factor ZBTB3. Our analytical strategy provides a novel framework for future studies aimed at dissecting etiological genetic effects. Finally, both SLE elements of the statistical model appear to operate in Sjögren's syndrome and systemic sclerosis whereas only the IRF5-TNPO3 gene-spanning haplotype is associated with primary biliary cirrhosis, demonstrating the nuance of similarity and difference in autoimmune disease risk mechanisms at IRF5-TNPO3.

H1N1 vaccination in Sjögren's syndrome triggers polyclonal B cell activation and promotes autoantibody production.

OBJECTIVES: Vaccination of patients with rheumatic disease has been reported to result in lower antibody titres than in healthy individuals. However, studies primarily include patients on immunosuppressive therapy. Here, we investigated the immune response of treatment-naïve patients diagnosed with primary Sjögren's syndrome (pSS) to an H1N1 influenza vaccine. METHODS: Patients with Sjögren's syndrome without immunomodulatory treatment and age-matched and gender-matched healthy controls were immunised with an H1N1 influenza vaccine and monitored for serological and cellular immune responses. Clinical symptoms were monitored with a standardised form. IgG class switch and plasma cell differentiation were induced in vitro in purified naïve B cells of untreated and hydroxychloroquine-treated patients and healthy controls. Gene expression was assessed by NanoString technology. RESULTS: Surprisingly, treatment-naïve patients with Sjögren's syndrome developed higher H1N1 IgG titres of greater avidity than healthy controls on vaccination. Notably, off-target B cells were also triggered resulting in increased anti-EBV and autoantibody titres. Endosomal toll-like receptor activation of naïve B cells in vitro revealed a greater propensity of patient-derived cells to differentiate into plasmablasts and higher production of class switched IgG. The amplified plasma cell differentiation and class switch could be induced in cells from healthy donors by preincubation with type 1 interferon, but was abolished in hydroxychloroquine-treated patients and after in vitro exposure of naïve B cells to chloroquine. CONCLUSIONS: This comprehensive analysis of the immune response in autoimmune patients to exogenous stimulation identifies a mechanistic basis for the B cell hyperactivity in Sjögren's syndrome, and suggests that caution is warranted when considering vaccination in non-treated autoimmune patients.

Standardisation of labial salivary gland histopathology in clinical trials in primary Sjögren's syndrome.

Labial salivary gland (LSG) biopsy is used in the classification of primary Sjögren's syndrome (PSS) and in patient stratification in clinical trials. It may also function as a biomarker. The acquisition of tissue and histological interpretation is variable and needs to be standardised for use in clinical trials. A modified European League Against Rheumatism consensus guideline development strategy was used. The steering committee of the ad hoc working group identified key outstanding points of variability in LSG acquisition and analysis. A 2-day workshop was held to develop consensus where possible and identify points where further discussion/data was needed. These points were reviewed by a subgroup of experts on PSS histopathology and then circulated via an online survey to 50 stakeholder experts consisting of rheumatologists, histopathologists and oral medicine specialists, to assess level of agreement (0-10 scale) and comments. Criteria for agreement were a mean score ≥6/10 and 75% of respondents scoring ≥6/10. Thirty-nine (78%) experts responded and 16 points met criteria for agreement. These points are focused on tissue requirements, identification of the characteristic focal lymphocytic sialadenitis, calculation of the focus score, identification of germinal centres, assessment of the area of leucocyte infiltration, reporting standards and use of prestudy samples for clinical trials. We provide standardised consensus guidance for the use of labial salivary gland histopathology in the classification of PSS and in clinical trials and identify areas where further research is required to achieve evidence-based consensus.

Klinefelter's syndrome (47,XXY) is in excess among men with Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) has a strong female bias. We evaluated an X chromosome dose effect by analyzing 47,XXY (Klinefelter's syndrome, 1 in 500 live male births) among subjects with pSS. 47,XXY was determined by examination of fluorescence intensity of single nucleotide polymorphisms from the X and Y chromosomes. Among 136 pSS men there were 4 with 47,XXY. This was significantly different from healthy controls (1 of 1254 had 47,XXY, p=0.0012 by Fisher's exact test) as well men with rheumatoid arthritis (0 of 363 with 47,XXY), but not different compared to men with systemic lupus erythematosus (SLE) (4 of 136 versus 8 of 306, Fisher's exact test p=NS). These results are consistent with the hypothesis that the number of X chromosomes is critical for the female bias of pSS, a property that may be shared with SLE but not RA.