Histopathological comparison of Sjögren-related features between paired labial and parotid salivary gland biopsies of sicca patients.

OBJECTIVES: To compare focus score and other histopathological features between paired labial and parotid salivary gland biopsies in a diagnostic cohort of suspected Sjögren's disease (SjD) patients. METHODS: Labial and parotid salivary gland biopsies were simultaneously obtained from patients with sicca complaints, suspected of having SjD. Biopsies were formalin fixed and paraffin embedded. Sections were stained with haematoxylin & eosin, and for CD3, CD20, CD45, cytokeratin, CD21, Bcl6, activation-induced deaminase (AID) and IgA/IgG. Focus score and other histopathological features characteristic for SjD were analysed. RESULTS: Based on the expert opinion of three experienced rheumatologists, 36 patients were diagnosed as SjD and 63 as non-SjD sicca patients. When taking all patients together, absolute agreement of various histopathological features between labial and parotid biopsies was high and varied between 80% (focus score) and 93% [(pre-)lymphoepithelial lesions (LELs)]. More labial gland biopsies had a focus score ≥1 compared with their parotid counterpart. Accordingly, the area of infiltrate was larger in labial gland biopsies. When considering only SjD patients, labial glands contained significantly fewer B-lymphocytes and germinal centres/mm2, and less severe LELs compared with parotid glands. CONCLUSION: Labial and parotid glands from SjD patients contain similar histopathological key features, and thus both glands can be used for diagnosis and classification of SjD. However, parotid salivary glands reveal more evident B-lymphocyte-related features, while labial glands exhibit more inflammation, which may be partially unrelated to SjD.

Increased Interferon Signaling in Vaginal Tissue of Patients With Primary Sjögren Syndrome.

OBJECTIVE: Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS. METHODS: Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs]). RESULTS: The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway (P < 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher (P < 0.01 for both pathways). Conversely, transforming growth factor-ß signaling and angiogenesis pathway scores were lower in pSS (P = 0.02 and P = 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity. CONCLUSION: Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness).

Lymphoma-associated mutations in autoreactive memory B cells of patients with Sjögren's syndrome.

We recently demonstrated that normal memory B lymphocytes carry a substantial number of de novo mutations in the genome. Here, we performed exome-wide somatic mutation analyses of bona fide autoreactive rheumatoid factor (RF)-expressing memory B cells retrieved from patients with SjÓ§gren's syndrome (SS). The amount and repertoire of the de novo exome mutations of RF B cells were found to be essentially different from those detected in healthy donor memory B cells. In contrast to the mutation spectra of normal B cells, which appeared random and non-selected, the mutations of the RF B cells were greater in number and enriched for mutations in genes also found mutated in B-cell non-Hodgkin lymphomas. During the study, one of the SS patients developed a diffuse large B-cell lymphoma (DLBCL) out of an RF clone that was identified 2 years earlier in an inflamed salivary gland biopsy. The successive oncogenic events in the RF precursor clone and the DLBCL were assessed. In conclusion, our findings of enhanced and selected genomic damage in growth-regulating genes in RF memory B cells of SS patients together with the documented transformation of an RF-precursor clone into DLBCL provide unique novel insight into the earliest stages of B-cell derailment and lymphomagenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Rheumatoid factor autoantibody repertoire profiling reveals distinct binding epitopes in health and autoimmunity.

BACKGROUND: Rheumatoid factors (RF) are one of the hallmark autoantibodies characteristic of rheumatoid arthritis (RA), and are frequently observed in other diseases and in healthy individuals. RFs comprise multiple subtypes with different specificities towards the constant region of human IgG. Studies indicate that these patterns differ between naturally occurring RFs and RFs associated with disease. However, individual specificities characteristic of either have not been clearly defined. METHODS: In this study, we developed an extended set of engineered IgG-fragment crystallisable (Fc) targets with preferential RF binding to specific (conformational) epitopes, which was subsequently used for profiling of RF binding patterns in a compiled exploration cohort, consisting of sera from healthy donors with measurable RF and patients with RA, primary Sjögren's syndrome (pSS) and seropositive arthralgia. RESULTS: We identified an epitope that is strongly associated with RA, which was targeted by both IgM-RF and IgA-RF. We also identified an epitope that is preferentially targeted by healthy donor (IgM) RFs. IgM-RFs derived from healthy donors and patients with RA and pSS all target distinct regions on the IgG-Fc, whereas overall, the IgA-RF repertoire is largely restricted to pathology-associated specificities. Using monoclonal RFs with different specificities, we furthermore demonstrate that the capacity to activate complement or even inhibit IgG-mediated complement activation varies according to the epitopes to which RFs bind. CONCLUSIONS: Our results demonstrate both the need and feasibility to redefine 'RF' into pathological and physiological autoantibody subtypes.

Cytokine producing B-cells and their capability to polarize macrophages in giant cell arteritis.

OBJECTIVE: The lack of disease-specific autoantibodies in giant cell arteritis (GCA) suggests an alternative role for B-cells readily detected in the inflamed arteries. Here we study the cytokine profile of tissue infiltrated and peripheral blood B-cells of patients with GCA. Moreover, we investigate the macrophage skewing capability of B-cell-derived cytokines. METHODS: The presence of various cytokines in B-cell areas in temporal artery (n = 11) and aorta (n = 10) was identified by immunohistochemistry. PBMCs of patients with GCA (n = 11) and polymyalgia rheumatica (n = 10), and 14 age- and sex-matched healthy controls (HC) were stimulated, followed by flow cytometry for cytokine expression in B-cells. The skewing potential of B-cell-derived cytokines (n = 6 for GCA and HC) on macrophages was studied in vitro. RESULTS: The presence of IL-6, GM-CSF, TNFα, IFNγ, LTß and IL-10 was documented in B-cells and B-cell rich areas of GCA arteries. In vitro, B-cell-derived cytokines (from both GCA and HC) skewed macrophages towards a pro-inflammatory phenotype with enhanced expression of IL-6, IL-1ß, TNFα, IL-23, YKL-40 and MMP-9. In vitro stimulated peripheral blood B-cells from treatment-naïve GCA patients showed an enhanced frequency of IL-6+ and TNFα+IL-6+ B-cells compared to HCs. This difference was no longer detected in treatment-induced remission. Erythrocyte sedimentation rate positively correlated with IL-6+TNFα+ B-cells. CONCLUSION: B-cells are capable of producing cytokines and steering macrophages towards a pro-inflammatory phenotype. Although the capacity of B-cells in skewing macrophages is not GCA specific, these data support a cytokine-mediated role for B-cells in GCA and provide grounds for B-cell targeted therapy in GCA.

Lymphoepithelial lesions in the salivary glands of primary Sjögren's syndrome patients: the perfect storm?

In patients with primary Sjögren's syndrome (pSS), inflamed salivary gland (SG) tissue may contain lymphoepithelial lesions (LELs). LELs are histopathological phenomena whereby B cells are present in hyperplastic ductal epithelium of the SG. Despite the potential role of LELs in pSS pathogenesis, studies on their formation, detection, and prevalence in benign lesions (not complicated with lymphoma) are scarce. Recent evidence however shows that LELs are present in approximately half of the patients with pSS, both in minor and major SGs. Migration of a small number of B cells into the epithelium appears to be a critical initial step in LEL formation. These intra-epithelial B cells are proliferative, exhibit an innate-like phenotype, and may be linked to MALT lymphoma development. Alongside intra-epithelial B cells, the hyperplastic epithelial partner in LELs also engages in the local immune reaction. Epithelial cells are a source of cytokines and chemokines, with CXCL10 in particular playing a potential role in LEL formation. Importantly, LELs also have a negative impact on the maintenance of SG homeostasis by SG progenitor cell (SGPC) populations, likely due to dysregulation of SGPC lineage commitment or induction of plasticity. In conclusion, LEL formation mirrors a perfect storm of B and epithelial cell interaction culminating in increased risk of B cell derailment and SGPC dysregulation in pSS patients. We therefore argue that attenuation of LEL formation is an important treatment goal to preserve SG function and prevent B cell derailment in pSS.

Fatigue in primary Sjögren's syndrome is associated with an objective decline in physical performance, pain and depression.

OBJECTIVES: Fatigue is a major complaint in primary Sjögren's syndrome (pSS). To acquire a better understanding of fatigue in pSS, we investigated objective measures of performance decline (performance fatigability). Furthermore, we evaluated the relationship of self-reported fatigue with performance fatigability and factors modulating perceptions of fatigability (perceived fatigability). METHODS: Thirty-nine pSS patients and 27 healthy controls were included. To assess performance fatigability, force decline was measured during a sustained (124s) maximal voluntary contraction (MVC) with the index finger abductor muscle, and voluntary muscle activation was indexed using peripheral nerve stimulation. Self-reported fatigue was quantified using the Fatigue Severity Scale (FSS) and Modified Fatigue Impact Scale (MFIS). Pain, depression, and anxiety assessed using questionnaires and inflammatory biomarkers measured in blood were used as factors relating to perceived fatigability. RESULTS: Voluntary muscle activation was reduced in pSS (p=0.030), but force decline during the sustained MVC did not differ between groups. Self-reported fatigue was significantly higher in pSS than in controls (FSS: 4.4 vs. 2.6, p<0.001). Multivariable linear regression showed that both performance fatigability (force decline) and perceived fatigability (pain and depression) were associated with the MFIS physical domain in pSS (total explained variance of 47%). Negative associations with fatigue were observed for two interferon-associated proteins: MxA and CXCL10. CONCLUSIONS: This study demonstrates that performance fatigability in pSS was compromised by a reduced capacity of the central nervous system to drive the muscle. Furthermore, self-reported fatigue is a multifactorial symptom associated with both performance fatigability and perceived fatigability in patients with pSS.

Immunisation with Coronavirus-2 vaccines induces potent antibody responses and does not aggravate the lymphocyte compartment of primary Sjögren's syndrome patients.

OBJECTIVES: The peripheral lymphocyte compartment of patients with primary Sjögren's syndrome (pSS) differs strongly from healthy individuals. Whether this altered lymphocyte composition also changes abnormally during immune reactions, especially by novel CoV-2-vaccines, is unknown. METHODS: Peripheral blood mononuclear cells (PBMC) from 26 pSS patients and 6 healthy controls were compared before Coronavirus-2 (CoV-2) vaccination (Pfizer/BNT162b2, Moderna/mRNA-1273, AstraZeneca/AZD122 ChAdOx1 nCoV-19) and 7 days after secondary vaccination. Spike 1 (S1)-receptor binding domain (RBD)-specific IgG antibodies were measured in serum samples. Among PBMCs, B and T cell subpopulations were phenotypically analysed and RBD-specific B and plasma cells were evaluated. RESULTS: Immunisation induced CoV-2 specific serum antibodies in all pSS patients and healthy participants. When analysing pSS patients and controls together, frequencies of circulating IgG+ RBD-specific antibody-secreting cells (ASC) and anti-RBD serum titres correlated (r=0.42, p=0.022). Previously described alterations of peripheral B cells in pSS patients (e.g. reduced memory B cells, increased naive and transitional B cells and higher maturity of ASCs) remained stable during vaccination. The subset distribution of CD4+ and CD8+ T cells also stayed largely unchanged. However, frequencies of CD4+CXCR5-PD-1+ circulating peripheral helper T (cTPH)-like cells increased in pSS patients comparing pre- and post-vaccination (p=0.020), while circulating CD4+CXCR5+PD-1+ follicular helper T (cTFH)-like cells declined (p=0.024). CONCLUSIONS: An immune reaction induced by vaccination with the novel CoV-2 vaccines yields adequate antibody production and vaccine specific lymphocytes in pSS patients and controls. Aberrant lymphocyte subset distribution in pSS patients persisted after vaccination and no major changes were induced despite small changes in cTPH and cTFH cells.

Selection of study endpoints and patients for clinical trials in primary Sjögren's syndrome.

In the last decade, many randomised controlled trials (RCTs) with biological DMARDs (bDMARDs) have been performed in patients with primary Sjögren's syndrome (pSS). Unfortunately, no bDMARD has yet been approved for systemic treatment of pSS. The heterogeneity of disease manifestations raises two essential questions: 1) which outcome measure is valid, reliable and responsive to demonstrate treatment efficacy and should be used as primary study endpoint? and 2) which pSS patients should be included in clinical trials? Both the selection of the primary study endpoint and the selection of patients are crucial and evolving issues in clinical trial design in pSS. This article summarises the history and comments the selection of primary study endpoints including the novel development of composite endpoints. Furthermore, this article gives an overview of inclusion criteria used for phase II and III trials, and illustrates by data-analysis based on two prospective observational cohorts that each additional selection criterion will (largely) decrease the number of eligible patients in daily clinical practice.

Patient Acceptable Symptom State (PASS) in patients with primary Sjögren's syndrome in daily clinical practice.

OBJECTIVES: To explore Patient Acceptable Symptom State (PASS) in a standard of care cohort of patients with primary Sjögren's syndrome (pSS) and to compare patient characteristics including EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) between PASS and non-PASS groups. METHODS: All pSS patients fulfilling ACR/EULAR classification criteria from the Registry of Sjögren's Syndrome LongiTudinal (RESULT) cohort, who had available PASS data at baseline were included. Patient-reported outcomes included the PASS question: "Considering all the different ways your disease is affecting you, if you were to stay in this state for the next few months, do you consider your current state satisfactory?"; yes: PASS / no: non-PASS. RESULTS: Of the 278 included pSS patients, 199 (72%) had an acceptable symptom state according to the PASS question, and median ESSPRI was 6 (IQR 4-7). In the PASS group, 118 (59%) patients had an unacceptable symptom state according to ESSPRI (score ≥5). In multivariable regression analyses, ESSPRI and disease duration were independently associated with presence of PASS. The accuracy of ESSPRI to predict PASS was fair (AUC of 0.78). The cut-off point of ESSPRI for presence of PASS with the highest Youden's index was 7.2 (sensitivity 85%, specificity 56%), followed by 5.2 (sensitivity 48%, specificity 90%). CONCLUSIONS: The majority of pSS patients reported being in an acceptable symptom state according to the PASS question, despite high ESSPRI scores. In our standard of care cohort, the optimal cut-off point of ESSPRI to predict PASS is different when focusing on sensitivity (±7) or specificity (±5).

Decreased BAFF Receptor Expression and Unaltered B Cell Receptor Signaling in Circulating B Cells from Primary Sjögren's Syndrome Patients at Diagnosis.

Animal models of autoimmunity and human genetic association studies indicate that the dysregulation of B-cell receptor (BCR) signaling is an important driver of autoimmunity. We previously showed that in circulating B cells from primary Sjögren's syndrome (pSS) patients with high systemic disease activity, protein expression of the BCR signaling molecule Bruton's tyrosine kinase (BTK) was increased and correlated with T-cell infiltration in the target organ. We hypothesized that these alterations could be driven by increased B-cell activating factor (BAFF) levels in pSS. Here, we investigated whether altered BCR signaling was already present at diagnosis and distinguished pSS from non-SS sicca patients. Using (phospho-)flow cytometry, we quantified the phosphorylation of BCR signaling molecules, and investigated BTK and BAFF receptor (BAFFR) expression in circulating B cell subsets in an inception cohort of non-SS sicca and pSS patients, as well as healthy controls (HCs). We found that both BTK protein levels and BCR signaling activity were comparable among groups. Interestingly, BAFFR expression was significantly downregulated in pSS, but not in non-SS sicca patients, compared with HCs, and correlated with pSS-associated alterations in B cell subsets. These data indicate reduced BAFFR expression as a possible sign of early B cell involvement and a diagnostic marker for pSS.

T cells in primary Sjögren's syndrome: targets for early intervention.

A histologic hallmark of primary SS (pSS) is lymphocytic infiltration of the salivary and lacrimal glands, in particular by CD4+ T and B cells. In the early stages of the disease, infiltrates are dominated by CD4+ T cells, while B cell accumulation occurs at later stages. Activated T cells contribute to pathogenesis by producing pro-inflammatory cytokines and by inducing B cell activation, which results in the establishment of a positive feedback loop. In the inflamed glandular tissues, many different CD4+ effector subsets are present, including IFN-γ-producing Th1 cells, IL-17-producing Th17 cells and IL-21-producing T follicular helper cells. In blood from pSS patients, frequently observed abnormalities of the T cell compartment are CD4+ T cell lymphopenia and enrichment of circulating follicular helper T (Tfh) cells. Tfh cells are critical mediators of T cell-dependent B cell hyperactivity and these cells can be targeted by immunotherapy. Inhibition of T cell activation, preferably early in the disease process, can mitigate B cell activity and may be a promising treatment approach in this disease.

Clinical determinants of vaginal dryness in patients with primary Sjögren's syndrome.

OBJECTIVES: The majority of women with primary Sjögren's syndrome (pSS) suffer from vaginal dryness, which negatively impacts daily and sexual activities. As little is known about the aetiology and clinical context of this complaint, this study investigated the relationship between vaginal dryness and other clinical parameters associated with pSS. METHODS: Female participants of the REgistry of Sjögren syndrome at UMCG - LongiTudinal (RESULT) cohort who fulfilled ACR-EULAR and/or AECG classification criteria for pSS were included, using baseline data for analyses. Patient-reported vaginal dryness (range 0-10) was correlated with demographic characteristics, systemic disease activity (i.e., ESSDAI), Sjögren's Syndrome Disease Damage Index, salivary and lacrimal gland function, patient-reported outcomes (ESSPRI, MFI), serology and quality of life (SF-36, EQ-5D). Significantly associated parameters (p<0.05) were corrected for potential confounders. RESULTS: This cross-sectional study included 199 women with pSS; mean age was 52±14 years, 53% were postmenopausal, and median vaginal dryness score was 5 (IQR 2-7). Vaginal dryness was significantly associated with older age, postmenopausal status, peripheral neuropathy, oral and ocular dryness, ESSPRI and SF-36 mental and general health. After correction for age, menopausal status and medication use, peripheral neuropathy (B=1.632), oral dryness (B=0.302), and ocular dryness (B=0.230) were independently associated with vaginal dryness. CONCLUSIONS: The independent association of vaginal dryness with oral and ocular dryness might imply that the aetiology of these symptoms is partly shared. Of all extraglandular features, only peripheral neuropathy was independently associated with vaginal dryness, suggesting that peripheral neuropathy plays a significant role in the pathology of vaginal dryness in pSS.

Differences in presentation between paediatric- and adult-onset primary Sjögren's syndrome patients.

OBJECTIVES: Primary Sjögren's syndrome (pSS) is a rare disease in paediatric patients. Presenting symptoms differ from those in adult patients. The aim of this study was to evaluate presenting symptoms, classification criteria and clinical assessments, including salivary gland ultrasonography (SGUS), at disease onset in paediatric and adult patients with pSS. METHODS: Data of 23 paediatric- and 33 adult-onset patients with pSS were obtained from our standardised multidisciplinary REpSULT and RESULT cohorts, respectively. Clinical, patient-reported, serological, functional, biopsy and SGUS parameters were compared. RESULTS: In paediatric-onset pSS (pedSS) patients, recurrent parotid gland swelling (91% vs. 49%, p<0.001) and fever (30% vs. 3%, p=0.006) were more often present than in adult-onset patients. In contrast, sicca symptoms of mouth (52% vs. 79%, p=0.046) and eyes (26% vs. 73%, p<0.001) were less common in pedSS patients. In paediatric patients, the entry criteria of the ACR/EULAR classification were most often met due to activity in the glandular domain of the ESSDAI. When applying the ACR/EULAR classification criteria, only 78% of pedSS fulfilled these criteria compared to 100% of adult patients. Abnormal glandular function tests had a greater contribution to fulfilling the criteria in adults, while the biopsy had a greater contribution in paediatric patients. Anti-SSA/Ro serology had similar contribution for both cohorts. SGUS Hocevar score was significantly higher in paediatric compared to adult patients (median 25 vs. 18, p=0.004). CONCLUSION: PedSS has a different presentation than adult-onset pSS. Recurrent parotid gland swelling in paediatric patients should alert clinicians to the potential presence of pSS.

B Cell Involvement in the Pathogenesis of Ankylosing Spondylitis.

Extensive research into ankylosing spondylitis (AS) has suggested the major role of genetics, immune reactions, and the joint-gut axis in its etiology, although an ultimate consensus does not yet exist. The available evidence indicates that both autoinflammation and T-cell-mediated autoimmune processes are actively involved in the disease process of AS. So far, B cells have received relatively little attention in AS pathogenesis; this is largely due to a lack of conventional disease-defining autoantibodies. However, against prevailing dogma, there is a growing body of evidence suggestive of B cell involvement. This is illustrated by disturbances in circulating B cell populations and the formation of auto-reactive and non-autoreactive antibodies, along with B cell infiltrates within the axial skeleton of AS patients. Furthermore, the depletion of B cells, using rituximab, displayed beneficial results in a subgroup of patients with AS. This review provides an overview of our current knowledge of B cells in AS, and discusses their potential role in its pathogenesis. An overarching picture portrays increased B cell activation in AS, although it is unclear whether B cells directly affect pathogenesis, or are merely bystanders in the disease process.

Gene expression profiling of epithelium-associated FcRL4+ B cells in primary Sjögren's syndrome reveals a pathogenic signature.

In primary Sjögren's syndrome (pSS), FcRL4+ B cells are present in inflamed salivary gland tissue, within or in close proximity to ductal epithelium. FcRL4 is also expressed by nearly all pSS-related mucosa-associated lymphoid tissue (MALT) B cell lymphomas, linking FcRL4 expression to lymphomagenesis. Whether glandular FcRL4+ B cells are pathogenic, how these cells originate, and how they functionally differ from FcRL4- B cells in pSS is unclear. This study aimed to investigate the phenotype and function of FcRL4+ B cells in the periphery and parotid gland tissue of patients with pSS. First, circulating FcRL4+ B cells from 44 pSS and 54 non-SS-sicca patients were analyzed by flow cytometry. Additionally, RNA sequencing of FcRL4+ B cells sorted from parotid gland cell suspensions of 6 pSS patients was performed. B cells were sorted from cell suspensions as mini bulk (5 cells/well) based on the following definitions: CD19+CD27-FcRL4- ('naive'), CD19+CD27+FcRL4- ('memory'), and CD19+FcRL4+ B cells. We found that, although FcRL4+ B cells were not enriched in blood in pSS compared with non-SS sicca patients, these cells generally exhibited a pro-inflammatory phenotype. Genes coding for CD11c (ITGAX), T-bet (TBX21), TACI (TNFRSF13B), Src tyrosine kinases and NF-κB pathway-related genes were, among others, significantly upregulated in glandular FcRL4+ B cells versus FcRL4- B cells. Pathway analysis showed upregulation of B cell activation, cell cycle and metabolic pathways. Thus, FcRL4+ B cells in pSS exhibit many characteristics of chronically activated, pro-inflammatory B cells and their gene expression profile suggests increased risk of lymphomagenesis. We postulate that these cells contribute significantly to the epithelial damage seen in the glandular tissue and that FcRL4+ B cells are an important treatment target in pSS.

Vaginal dryness in primary Sjögren's syndrome: a histopathological case-control study.

OBJECTIVE: The aim was to study clinical, histopathological and immunological changes in the vagina and cervix of women with primary SS, which might explain vaginal dryness. METHODS: We included 10 pre-menopausal female primary SS patients with vaginal dryness and 10 pre-menopausal controls undergoing a laparoscopic procedure. The vaginal health index was recorded. Multiplex immunoassays and flow cytometry were performed on endocervical swab and cervicovaginal lavage samples to evaluate cellular and soluble immune markers. Mid-vaginal and endocervical biopsies were taken and stained for various leucocyte markers, caldesmon (smooth muscle cells), avian V-ets erythroblastosis virus E26 oncogene homologue (ERG; endothelial cells) and anti-podoplanin (lymphatic endothelium). The number of positive pixels per square micrometre was calculated. RESULTS: One patient was excluded because of Clamydia trachomatis, and two controls were excluded because of endometriosis observed during their laparoscopy. Vaginal health was impaired in primary SS. CD45+ cells were increased in vaginal biopsies of women with primary SS compared with controls. Infiltrates were predominantly located in the peri-epithelial region, and mostly consisted of CD3+ lymphocytes. In the endocervix, CD45+ infiltrates were present in patients and in controls, but a higher number of B lymphocytes was seen in primary SS. Vascular smooth muscle cells were decreased in the vagina of primary SS patients. No differences were found in leucocyte subsets in the vaginal and endocervical lumen. CXCL10 was increased in endocervical swab samples of primary SS patients. CONCLUSION: Women with primary SS show impaired vaginal health and increased lymphocytic infiltration in the vagina compared with controls. Vaginal dryness in primary SS might be caused by vascular dysfunction, possibly induced by IFN-mediated pathways.

Blocking T cell co-stimulation in primary Sjögren's syndrome: rationale, clinical efficacy and modulation of peripheral and salivary gland biomarkers.

There is accumulating evidence that patients with primary Sjögren's syndrome (pSS) display aberrant CD4+ T cell responses, both in the peripheral compartment and in the inflamed salivary glands. CD4+ T cell abnormalities are also critically associated with B cell hyper activation, one of the hallmarks of disease, which is linked with disease severity and evolution to lymphoma. T cell activation and the cross-talk between T and B cells are tightly regulated by the balance between co-stimulatory pathways, such as the interactions between CD80/CD86:CD28, CD40:CD40L and ICOS:ICOSL, and co-inhibitory signals, including the immunoregulatory CTLA-4 protein. Evidence from patients with pSS as well as data from animal models of the disease suggests that these pathways play a critical role in pSS pathogenesis and their targeting could be exploited for therapeutic purposes. In this review, we first summarise the evidence implicating aberrant T cell co-stimulation and co-inhibition in driving the disease before focusing on the results of recent randomised controlled trials (RCTs) with compounds able to block T cell co-stimulation and enhance T cell co-inhibition. Despite a clear biological effect on downstream B cell activation has been observed in patients treated with CTLA-4-Ig (abatacept) and with monoclonal antibodies targeting CD40 and ICOSL, the clinical efficacy of this approach has so far yielded mixed results; while the anti-CD40 monoclonal antibody iscalimab showed significant improvement in systemic disease activity compared to placebo, two large RCTs with abatacept and a phase IIa RCT with an anti-ICOSL monoclonal antibody (prezalumab) failed to reach their primary endpoints. Although the discrepancies between biological and clinical efficacy of targeting T cell co-stimulation on pSS remain unresolved, several factors including drug bioavailability and receptor occupancy, patient stratification based on T-cell related biomarkers and the choice of study outcome are likely to play an important role and form the basis for further work towards the quest for a disease-modifying biologic therapy in pSS.

Presence of intraepithelial B-lymphocytes is associated with the formation of lymphoepithelial lesions in salivary glands of primary Sjögren's syndrome patients.

OBJECTIVES: Lymphoepithelial lesions (LELs) in salivary glands are associated with primary Sjögren's syndrome (pSS). LELs are composed of hyperplastic epithelium infiltrated with lymphocytes. The objective of this study was obtaining insight in the relative roles of intraepithelial B- and T-lymphocytes in the formation of LELs in salivary glands of pSS patients. METHODS: Parotid and labial salivary gland biopsies of pSS patients (n=15), non-SS sicca patients (n=5) and non-sicca controls (n=5) were analysed. Serial sections were stained with H & E and for cytokeratin, CD20 and CD3. Striated ducts with lymphocytes, but without hyperplasia, and striated ducts with LELs were identified in H & E and cytokeratin stained sections. LELs were classified in successive stages of severity based on the amount of hyperplasia (stage1-3). Numbers of B- and T-lymphocytes within striated ducts and LELs were counted in CD20 and CD3 stained sections. RESULTS: Lymphocyte-containing striated ducts of both salivary glands of all pSS and control patients harboured T-lymphocytes, scattered throughout the ductal epithelium. In contrast, B-lymphocytes were exclusively found in a small fraction (21%) of striated ducts without hyperplasia and in nearly all striated ducts with LELs of pSS patients, but not in controls. In striated ducts with LELs B-lymphocytes were mostly located in the areas of proliferating epithelium. Numbers of B-lymphocytes and B/T-ratios increased significantly with higher severity of LELs. This was even more pronounced in the parotid than in the labial gland. CONCLUSIONS: We conclude there is an association between presence of intraepithelial B-lymphocytes and the formation of LELs in salivary glands of pSS patients.