Cellular and molecular diversity in Sjogren's syndrome salivary glands: Towards a better definition of disease subsets.

Primary Sjögren's syndrome (pSS) is a highly heterogeneous disease in terms of clinical presentation ranging from a mild disease localised to the salivary and lacrimal glands, to multiorgan complications of various degrees of severity, finishing with the evolution, in around 5% of pSS patients, to B cell lymphomas most commonly arising in the inflamed salivary glands. Currently, there are poor positive or negative predictors of disease evolution able to guide patient management and treatment at early stages of the diseases. Recent understanding of the pathogenic mechanisms driving immunopathology in pSS, particularly through histological and transcriptomic analysis of minor and parotid salivary gland (SG) biopsies, has highlighted a high degree of cellular and molecular heterogeneity of the inflammatory lesions but also allowed the identification of clusters of patients with similar underlying SG immunopathology. In particular, patients presenting with high degrees of B/T cell infiltration and the formation of ectopic lymphoid structures (ELS) in the SG have been associated, albeit with conflicting results, with higher degree of disease severity and enhanced risk of lymphoma evolution, suggesting that a dysregulated adaptive immune response plays a key role in driving disease manifestations in pSS. Recent data from randomised clinical trials with novel biological therapies in pSS have also highlighted the potential role of SG immunopathology and molecular pathology in stratifying patients for trial inclusion as well as assessing proof of mechanisms in longitudinal SG biopsies before and after treatment. Although significant progress has been made in the understanding of disease pathogenesis and heterogeneity through cellular and molecular SG pathology, further work is needed to validate their clinical utility in routine clinical settings and in randomised clinical trials.

CD8+ tissue-resident memory T cells are expanded in primary Sjögren's disease and can be therapeutically targeted by CD103 blockade.

OBJECTIVE: Tissue-resident memory cells (Trm) are a subset of T cells residing persistently and long-term within specific tissues that contribute to persistent inflammation and tissue damage. We characterised the phenotype and function of Trm and the role of CD103 in primary Sjogren's syndrome (pSS). METHODS: In both pSS and non-pSS sicca syndrome patients, we examined Trm frequency, cytokine production in salivary glands (SG) and peripheral blood (PB). We also analysed Trm-related gene expression in SG biopsies through bulk and single-cell RNA sequencing (scRNAseq). Additionally, we investigated Trm properties in an immunisation-induced animal model of pSS (experimental SS, ESS) mouse model and assessed the effects of Trm inhibition via intraglandular anti-CD103 monoclonal antibody administration. RESULTS: Transcriptomic pSS SG showed an upregulation of genes associated with tissue recruitment and long-term survival of Trm cells, confirmed by a higher frequency of CD8+CD103+CD69+ cells in pSS SG, compared with non-specific sialadenitis (nSS). In SG, CD8+ CD103+ Trm contributed to the secretion of granzyme-B and interferon-γ, CD8+ Trm cells were localised within inflammatory infiltrates, where PD1+CD8+ T cells were also increased compared with nSS and MALT lymphoma. scRNAseq of PB and pSS SG T cells confirmed expression of CD69, ITGAE, GZMB, GZMK and HLA-DRB1 among CD3+CD8+ SG T cells. In the SG of ESS, CD8+CD69+CD103+ Trm producing Granzyme B progressively expanded. However, intraglandular blockade of CD103 in ESS reduced Trm, reduced glandular damage and improved salivary flow. CONCLUSIONS: CD103+CD8+Trm cells are expanded in the SG of pSS and ESS, participate in tissue inflammation and can be therapeutically targeted.

Circulating c-Met-Expressing Memory T Cells Define Cardiac Autoimmunity.

BACKGROUND: Autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. The functional features of cardiac autoimmunity in humans remain undefined because of the challenge of studying immune responses in situ. We previously described a subset of c-mesenchymal epithelial transition factor (c-Met)-expressing (c-Met+) memory T lymphocytes that preferentially migrate to cardiac tissue in mice and humans. METHODS: In-depth phenotyping of peripheral blood T cells, including c-Met+ T cells, was undertaken in groups of patients with inflammatory and noninflammatory cardiomyopathies, patients with noncardiac autoimmunity, and healthy controls. Validation studies were carried out using human cardiac tissue and in an experimental model of cardiac inflammation. RESULTS: We show that c-Met+ T cells are selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met+ T cells are distinct from those of c-Met-negative (c-Met-) T cells, including preferential proliferation to cardiac myosin and coproduction of multiple cytokines (interleukin-4, interleukin-17, and interleukin-22). Furthermore, circulating c-Met+ T cell subpopulations in different heart muscle diseases identify distinct and overlapping mechanisms of heart inflammation. In experimental autoimmune myocarditis, elevations in autoantigen-specific c-Met+ T cells in peripheral blood mark the loss of immune tolerance to the heart. Disease development can be halted by pharmacologic c-Met inhibition, indicating a causative role for c-Met+ T cells. CONCLUSIONS: Our study demonstrates that the detection of circulating c-Met+ T cells may have use in the diagnosis and monitoring of adaptive cardiac inflammation and definition of new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury.

Lymphomas complicating primary Sjögren's syndrome: from autoimmunity to lymphoma.

Lymphoma development is the most serious complication of SS and the main factor impacting on mortality rate in patients with this condition. Lymphomas in SS are most commonly extranodal non-Hodgkin B-cell lymphomas of the mucosa-associated lymphoid tissue and frequently arise in salivary glands that are the target of a chronic inflammatory autoimmune process. Extensive work on lymphomagenesis in SS has established that the progression towards B-cell lymphoma is a multistep process related to local chronic antigenic stimulation of B cells. These neoplastic B cells in SS frequently derived from autoreactive clones, most commonly RF-producing B cells, which undergo uncontrolled proliferation and malignant escape. In this review, we highlight the most important recent findings that have enhanced our understanding of lymphoma development in SS, with particular reference to the close link between autoimmunity and lymphomagenesis. We also discuss how the identification of key factors involved in B-cell malignancies may impact on our ability to identify at early stages patients at increased risk of lymphoma with potential significant repercussions for the clinical management of SS patients. Finally, we identified the most promising areas of current and further research with the potential to provide novel basic and translational discoveries in the field. The questions of finding new biomarkers, developing a validated score for predicting lymphoma occurrence and assessing if a better control of disease activity will decrease the risk of lymphoma in primary SS will be the enthralling questions of the next few years.

Advanced imaging for quantification of abnormalities in the salivary glands of patients with primary Sjögren's syndrome.

OBJECTIVES: To assess non-invasive imaging for detection and quantification of gland structure, inflammation and function in patients with primary Sjogren's syndrome (pSS) using PET-CT with 11C-Methionine (11C-MET; radiolabelled amino acid), and 18F-fluorodeoxyglucose (18F-FDG; glucose uptake marker), to assess protein synthesis and inflammation, respectively; multiparametric MRI evaluated salivary gland structural and physiological changes. METHODS: In this imaging/clinical/histology comparative study (GSK study 203818; NCT02899377) patients with pSS and age- and sex-matched healthy volunteers underwent MRI of the salivary glands and 11C-MET PET-CT. Patients also underwent 18F-FDG PET-CT and labial salivary gland biopsies. Clinical and biomarker assessments were performed. Primary endpoints were semi-quantitative parameters of 11C-MET and 18F-FDG uptake in submandibular and parotid salivary glands and quantitative MRI measures of structure and inflammation. Clinical and minor salivary gland histological parameter correlations were explored. RESULTS: Twelve patients with pSS and 13 healthy volunteers were included. Lower 11C-MET uptake in parotid, submandibular and lacrimal glands, lower submandibular gland volume, higher MRI fat fraction, and lower pure diffusion in parotid and submandibular glands were observed in patients vs healthy volunteer, consistent with reduced synthetic function. Disease duration correlated positively with fat fraction and negatively with 11C-MET and 18F-FDG uptake, consistent with impaired function, inflammation and fatty replacement over time. Lacrimal gland 11C-MET uptake positively correlated with tear flow in patients, and parotid gland 18F-FDG uptake positively correlated with salivary gland CD20+ B-cell infiltration. CONCLUSION: Molecular imaging and MRI may be useful tools to non-invasively assess loss of glandular function, increased glandular inflammation and fat accumulation in pSS.

Tertiary Lymphoid Organs in Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease. RA mainly affects the joints, with inflammation of the synovial membrane, characterized by hyperplasia, neo-angiogenesis, and immune cell infiltration that drives local inflammation and, if untreated, can lead to joint destruction and disability. In parallel to the well-known clinical heterogeneity, the underlying synovitis can also be significantly heterogeneous. In particular, in about 40% of patients with RA, synovitis is characterized by a dense lymphocytic infiltrate that can acquire the features of fully functional tertiary lymphoid organs (TLO). These structures amplify autoimmunity and inflammation locally associated with worse prognosis and potential implications for treatment response. Here, we will review the current knowledge on TLO in RA, with a focus on their pathogenetic and clinical relevance.

Stepwise changes in the murine salivary gland immune response during virally-induced ectopic lymphoid structure formation.

OBJECTIVES: Sjögren's syndrome (SS) is a chronic autoimmune disease characterised by lymphocytic infiltration into the salivary glands (SG) and, in a subset of patients, formation of ectopic lymphoid structures (ELS) in the glands. However, the mechanisms of how ELS form ectopically are not fully elucidated. Here we used a viral inducible murine model of ELS formation in the SG to elucidate the key immunological steps regulating the formation of ELS in the SG. METHODS: We have utilised an inducible murine model of sialadenitis whereby retrograde cannulation of the submandibular SG with a replication-deficient adenovirus 5 leads to the formation of ELS. Flow cytometry, immunofluorescence and gene expression was performed on the SGs at regular time points after cannulation to follow the organisation of ELS. RESULTS: Innate immune cells (neutrophils, eosinophils and monocytes) rapidly infiltrated the SG by 3 days post cannulation (dpc) whereby monocytes started to differentiate into resident macrophages. Myeloid dendritic cells accumulated inside leukocytic aggregates whereas macrophages were excluded from the developing ELS. Meanwhile, CD11b+ cells upregulated Il18, Cxcl13, Ltb, April and other lymphoid genes stimulating the influx of T cells by 12 days and B cells shortly after. Infiltration of T-follicular helper (Tfh) cells correlated with an increase in GL7+ germinal centre B cells, which peaked at 19 dpc. CONCLUSIONS: Immune cell infiltration in virally-infected murine SG follows a highly reproducible step-wise process whereby early innate immune cells reshape the SG myeloid compartment leading to upregulation of genes involved in the ectopic lymphoid neogenesis process. This in turns leads to T and B cell recruitment, differentiation and activation, culminating in the organization of ELS and localised germinal centres responses.

Development and performance of the Clinical Trials ESSDAI (ClinTrialsESSDAI), consisting of frequently active clinical domains, in two randomised controlled trials in primary Sjögren's syndrome.

OBJECTIVES: To develop and evaluate the Clinical Trials EULAR Sjögren's Syndrome Disease Activity Index (ClinTrialsESSDAI), consisting of frequently active clinical domains of the ESSDAI, using two randomised controlled trials in primary Sjögren's syndrome (pSS). METHODS: The ASAP-III trial in abatacept (80 pSS patients) and TRACTISS trial in rituximab (133 pSS patients) were analysed. The most frequently active clinical domains were selected, and ClinTrialsESSDAI total score was calculated using existing weightings of the ClinESSDAI (which also excludes the biological domain). Performance of the ClinTrialsESSDAI was compared to ClinESSDAI and ESSDAI. Responsiveness was assessed using standardised response mean (SRM), and discrimination was assessed using adjusted mean difference. RESULTS: Besides the biological domain, the most frequently active domains were glandular, articular, haematological, constitutional, lymphadenopathy and cutaneous. These domains were selected for the ClinTrialsESSDAI. At primary endpoint visits, SRM values of ClinTrialsESSDAI, ClinESSDAI and ESSDAI were respectively -0.65/-0.59, -0.63/-0.59 and -0.64/-0.61 for abatacept/placebo and -0.33/-0.13, -0.34/-0.12 and -0.41/-0.16 for rituximab/placebo. Adjusted mean differences between active treatment and placebo groups were respectively -1.7, -1.4 and -1.1 for ASAP-III and -1.1, -1.1 and -1.2 for TRACTISS. CONCLUSIONS: The ClinTrialsESSDAI, consisting of six frequently active clinical domains of the ESSDAI, shows closely similar responsiveness and discrimination between treatment groups compared to the ClinESSDAI and ESSDAI. Therefore, this ClinTrialsESSDAI is not preferable to ClinESSDAI and ESSDAI for use as primary endpoint. A composite endpoint combining response at multiple clinically relevant items seems more suitable as primary study endpoint in pSS.

Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjögren's syndrome with ectopic germinal centres and MALT lymphoma.

OBJECTIVES: To explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren's syndrome (SS) patients. METHODS: Salivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production. RESULTS: Transcriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4+CXC-motif chemokine receptor 5 (CXCR5)+programmed cell death protein 1 (PD1)+ICOS+ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4+CD45RO+ICOS+PD1+ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5+CD4+PD1+ICOS+Foxp3- Tfh-cells and a uniquely expanded population of CXCR5-CD4+PD1hiICOS+Foxp3- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α). CONCLUSIONS: Overall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.

CXCL13 as biomarker for histological involvement in Sjögren's syndrome.

OBJECTIVES: SS is an autoimmune condition characterized by systemic B-cell activation, autoantibody production and ectopic germinal centres' formation within the salivary gland (SG). The extent of SG infiltrate has been proposed as a biomarker of disease severity. Plasma levels of CXCL13 correlate with germinal centres' activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B-cell activation. The aim of this study was to evaluate the potential role of CXCL13 as a biomarker of SG pathology in two independent SS cohorts. METHODS: 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n = 60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n = 49). Both sera and matched minor SG biopsy were available. Sicca (n = 57) and healthy subjects' (n = 19) sera were used as control. RESULTS: CXCL13 serum level was higher in SS patients compared with controls. Correlations between its serum levels and a series of histomorphological parameters, including size of the aggregates and the presence germinal centres', were observed. CONCLUSION: Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies.

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.

The use of digital image analysis in the histological assessment of Sjögren's syndrome salivary glands improves inter-rater agreement and facilitates multicentre data harmonisation.

OBJECTIVES: To assess whether the use of digital image analysis (DIA) in primary Sjögren's syndrome (pSS) for the calculation of the total area of the salivary gland (SG), focus score (FS) and SG area occupied by the inflammatory infiltrate (area fraction, AF), was able to generate reproducible readings among different raters, reducing disagreement. METHODS: Haematoxylin and Eosin digital slides from pSS and non-specific chronic sialadenitis (NSCS) patients were analysed blindly by 4 independent raters among 3 centres. Using an open-source software (QuPath) raters were asked to provide the total area of the gland i) using a grid-based method and ii) a software-based area-calculation tool, iii) the number of inflammatory foci and iv) the total area of the inflammatory infiltrate. Collected data was used to calculate the inter-rater agreement. RESULTS: For the calculation of the total SG area, DIA generated higher agreement among raters than grid-based calculation (inter-class correlation coefficient ICC=0.85 vs 0.98). Agreement for calculated total area of the inflammatory infiltrate (ICC=0.94) and for AF (ICC=0.94) was higher than infiltrates count number (ICC=0.54) and FS (ICC=0.56). AF achieved a 30% improvement over the FS at generating consensus among raters when used as a diagnostic cut-off. CONCLUSIONS: A digital approach achieved a far superior inter-rater agreement when calculating the total area compared to a grid-based approach. The calculation of AF proved superior to FS in correctly classifying pSS vs NSCS biopsies. We suggest that digitally calculated AF should be used alongside FS for large multi-centre studies to improve data harmonisation.

One year in review 2020: pathogenesis of primary Sjögren's syndrome.

The pathogenesis of primary Sjögren's syndrome (pSS) remains poorly understood. However, important efforts have been made during the last few months. In this review, following the others of this series we will summarise the most recent literature on pSS pathogenesis focusing in particular on new insights into pSS animal models, genetics and epigenetics, innate and adaptive immune system abnormalities and tertiary lymphoid structures. Hopefully, novel insights into pSS pathogenesis will pave the way to new therapeutic approaches to the disease improving patients' management and prognosis.

B cell depletion with rituximab in the treatment of primary Sjögren's syndrome: what have we learnt?

Despite the well-established role of B cells in the pathogenesis of primary Sjögren's syndrome (pSS), the beneficial role of B-cell depletion therapy with rituximab remains elusive in this condition, contrary to other autoimmune diseases. Although early, small-scale studies showed promising results, two recent large randomised controlled trials did not meet their primary end-points. It is evident from most trials that rituximab has a positive impact on B-cell numbers and activity, both in the peripheral blood and in salivary glands, but clinical outcomes vary among studies. We review here the evidence to date of B-cell depletion in pSS, analysing the underlying causes for the discrepancies in different studies and their limitations. We also discuss the potential use of peripheral and salivary gland biomarkers for patient stratification and targeted patient selection. Overall, rituximab remains a plausible treatment for pSS provided future studies address the shortfalls that emerged from our current knowledge of the use of B-cell depletion in this condition.

Current views on the pathogenesis of Sjögren's syndrome.

PURPOSE OF REVIEW: The purpose of this review is to provide an insight into the pathophysiological mechanisms involved in the pathogenesis of primary Sjögren's Syndrome (pSS), highlighting recent findings with potential therapeutic repercussions. RECENT FINDINGS: In the last 2 years, epigenetic analyses provided new insights into pSS pathogenesis. Characterization of DNA methylation patterns, chromatin structures and microRNA confirmed the importance of aberrant interferon and B-cell responses in the development of the disease. The formation of ectopic B-cell follicles with germinal centers is now a well recognized pathogenic mechanism within salivary glands of pSS. In the context of ectopic germinal centers reaction, T/B-cell interactions, that is regarding T-helper 17 and T-follicular helper cells, and their respective counterparts, T-regulatory and T-follicular regulatory cells, appear particularly relevant in pSS pathogenesis as their imbalance is associated with a dysregulation of B-cell dynamics and the production of autoantibodies. SUMMARY: Advances in the understanding of pSS pathogenesis have paved the way for clinical trials with novel biologic agents targeting immune pathways regulating T/B-cell interactions and downstream B-cell activation. Reverse translation from these studies provides invaluable novel information of the mechanisms sustaining autoimmunity and chronic inflammation in pSS.

A clinical and histopathological analysis of the anti-centromere antibody positive subset of primary Sjögren's syndrome.

OBJECTIVES: ACA-positive/primary Sjögren's syndrome (pSS) represents a distinct overlapping entity with intermediate features in between limited systemic sclerosis (lSSc) and pSS. Few data are available on their general risk for lymphoproliferative complications, specifically regarding adverse predictors at the level of minor salivary gland (MSG) histology. The objectives of this work are: a) to characterise, through a detailed immunohistochemistry study, the organisation of the lymphomonocitic infiltrates in ACA-positive/pSS patient vs. ACA-negative/pSS patients focusing on the presence of GC-like structures in minor salivary gland biopsies; b) to compare the frequency of traditional clinical and serological risk factors for lymphoma between the two subgroups. METHODS: We analysed 28 MSG samples from ACA-positive/pSS patients and 43 consecutive MSGs from ACA-negative/pSS, using sequential IHC staining for CD3, CD20 and CD21 in order to define the T/B cell segregation within the periductal infiltrates and presence of ectopic GC-like on the detection of GC-like structures. Clinical and serological data of all the patients were retrieved and analysed. RESULTS: Ectopic lymphoid structures (ELS) with GC-like structures were observed in 7 out of 28 ACA-positive/pSS patients (25%) and in 13 out of 43 ACA-negative/pSS patients (30.2%). Similarly, no statistical significant difference was found between the two groups as far as the classical pSS risk factors for lymphoproliferative complications was concerned (i.e. salivary gland enlargement, purpura, low C4, leukocytopenia, clonal gammopathy). Finally, the 3 cases of non-Hodgkin's lymphoma observed were equally distributed between the two subsets. CONCLUSIONS: Overall, this study indicates that ACA-positive/and ACA-negative pSS patients apparently present a similar risk for lymphoproliferative complications as suggested indirectly by the analogies between the two groups observed at the histopathology level.

Human liver-resident CD56(bright)/CD16(neg) NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways.

RATIONALE: The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic. OBJECTIVES: We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts. FINDINGS: Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56(bright) lr-NK cells that localize within hepatic sinusoids. CD56(bright) lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. Here, we also show that hepatic sinusoids express CCL3(pos) Kupffer cells, CXCL16(pos) endothelial cells and CCL5(pos) T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56(bright) NK cells that constitutively express CCR5 and CXCR6. CD56(bright) lr-NK cells co-exist with CD56(dim) conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56(dim) c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. CONCLUSION: Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56(bright) lr-NK cells will require modification of hepatic CCR5 and CXCR6.

Dopamine inhibits the effector functions of activated NK cells via the upregulation of the D5 receptor.

Several lines of evidence indicate that dopamine (DA) plays a key role in the cross-talk between the nervous and immune systems. In this study, we disclose a novel immune-regulatory role for DA: inhibition of effector functions of activated NK lymphocytes via the selective upregulation of the D5 dopaminergic receptor in response to prolonged cell stimulation with rIL-2. Indeed, engagement of this D1-like inhibitory receptor following binding with DA suppresses NK cell proliferation and synthesis of IFN-γ. The inhibition of IFN-γ production occurs through blocking the repressor activity of the p50/c-REL dimer of the NF-κB complex. Indeed, the stimulation of the D5 receptor on rIL-2-activated NK cells inhibits the binding of p50 to the microRNA 29a promoter, thus inducing a de novo synthesis of this miRNA. In turn, the increased levels of microRNA 29a were inversely correlated with the ability of NK cells to produce IFN-γ. Taken together, our findings demonstrated that DA switches off activated NK cells, thus representing a checkpoint exerted by the nervous system to control the reactivity of these innate immune effectors in response to activation stimuli and to avoid the establishment of chronic and pathologic inflammatory processes.

Treatment with belimumab restores B cell subsets and their expression of B cell activating factor receptor in patients with primary Sjogren's syndrome.

OBJECTIVE: The aim of this study was to investigate the biological effects of belimumab on B cells in the first phase II open-label trial with belimumab in patients with primary SS (pSS) (BELISS). METHODS: Peripheral blood B cell subsets and their B cell activating factor-receptor (BAFF-R) expression were analysed by multicolour flow cytometry in 10 pSS patients either before or after 24 and 52 weeks of therapy with belimumab. Serum BAFF levels were analysed by ELISA. RESULTS: At baseline, pSS patients showed a significant increase in circulating B cells compared with healthy donors matched for age and sex, with a predominant expansion of transitional and naive B cell subsets. pSS patients also showed higher serum BAFF levels and lower B cell BAFF-R expression. Therapy with belimumab in pSS patients induced a significant reduction in transitional and naive B cell subsets to levels similar to those observed in healthy donors. Furthermore, belimumab normalized BAFF-R expression in all B subsets comprised within the memory compartment. The restoration of B cell frequency and subset composition in response to belimumab was also associated with a decrease in serum levels of Ig, RF, ANAs, and with an increase in the C4 complement fraction. All of these belimumab-mediated effects were observed after 24 weeks of therapy and maintained until the end of the therapeutic protocol. CONCLUSION: Taken together, our findings show that targeting BAFF with belimumab is successful in normalizing B cell frequency, phenotype and functions in pSS. TRIAL REGISTRATION: clinicaltrials.gov; https://clinicaltrials.gov/; NCT01008982.

Expression variability and function of the RET gene in adult peripheral blood mononuclear cells.

RET is a gene playing a key role during embryogenesis and in particular during the enteric nervous system development. High levels of RET gene expression are maintained in different human tissues also in adulthood, although their physiological role remains unclear. In particular, collected evidences of a RET contribution in the development and maintenance of the immune system prompted us to investigate its levels of surface expression on peripheral blood mononuclear cells (PBMCs) from adult healthy donors. Despite variability among samples, RET expression was conserved at similar levels in the different immune cell subsets, with higher correlations in similar lymphocyte populations (i.e. CD4(+) and CD8(+) T cells). Conversely, no correlation was found between the amount of RET receptor, the expression of its putative ligands and co-receptors and the genotypes at the RET locus. Moreover, we investigated the RET-associated inflammatory pathways in PBMCs from healthy donors both in resting conditions and upon glial cell derived neurotrophic factor (GDNF) and GPI-linked co-receptors alpha 1 (GFRα1) mediated RET activation. RET mRNA levels positively correlated with the transcript amount of interleukin-8 (IL-8), a cytokine produced by monocytes and macrophages, though we could not demonstrate its direct effect on RET expression by in vitro experiments on THP1 human monocytic cells. These results imply that RET expression might be influenced by either cis- and/or trans-factors, which together would account for its high variability within the general population, and suggest a putative functional role of the RET gene in modulating immune cell responses during inflammation and carcinogenesis.