High-throughput sequencing of IgH gene in minor salivary glands from Sjögren's syndrome patients reveals dynamic B cell recirculation between ectopic lymphoid structures.

OBJECTIVES: B cells play a central role in Sjögren's syndrome (SS) whereby autoreactive B-cells populate ectopic germinal centres (GC) in SS salivary glands (SG) and undergo somatic hypermutation (SHM) and class-switch recombination of the immunoglobulin genes. However, the capacity of specific B cell clones to seed ectopic GC in different SG and undergo clonal diversification is unclear. To unravel the dynamics of B cell recirculation among minor SG biopsies, we investigated the immunoglobulin heavy chain (IgH) gene usage and the pattern of SHM using a high-throughput sequencing approach. METHODS: We generated ~166,000 reads longer than 350bp and detected 1631 clonotypes across eight samples from four different SS patients, all characterised by the presence of functional ectopic GC as demonstrated by the expression of activation-induced cytidine deaminase. RESULTS: A large number of shared clonotypes were observed among paired mSG biopsies from each patient but not across different patients. Lineage tree analysis revealed significant clonal expansion within the mSG with the identification of shared dominant B cell clones suggestive of extensive recirculation across different SG. Several shared clonotypes with high proliferating capacity displayed IgH-VH gene usage common in autoreactive B cells, including VH1-69, which is typical of rheumatoid factor+ B cells representing potential lymphoma precursors. CONCLUSIONS: The complex dynamic recirculation of B cells that we observed within ectopic GC responses linked with their ability to independently proliferate, undergo ongoing SHM and Ig class-switching within individual glands may explain the difficulty in achieving consistent eradication of ectopic GCs following B cell depleting agents reported in different studies.

H and L Chain Affinity Maturation and/or Fab N-Glycosylation Influence Immunoreactivity toward Neutrophil Extracellular Trap Antigens in Rheumatoid Arthritis Synovial B Cell Clones.

We previously showed that anti-neutrophil extracellular trap (NET) rheumatoid arthritis (RA)-rmAbs derived from CD19+ B cells within RA human synovial tissues frequently react against NETs. In this study, we aimed to characterize the importance of affinity maturation via somatic hypermutation (SHM) within the Ig variable H (VH) and variable L (VL) chains and Fab-N-linked glycosylation in RA synovial B cell clones reactive to NETs and NET-derived Ags such as citrullinated histones. Selected anti-NET RA-rmAbs derived from synovial RA CD19+ B cells were subjected to overlap-PCR to generate germline (GL; VH and VL reverted into GL), hybrid clones (VH/VL region reverted into GL), and N-glycosylation mutants (N→Q) and analyzed for anti-NETs and citrullinated histones (cit-H2B) immunoreactivity. Anti-NET/cit-H2B immunoreactivity of selected RA-rmAbs was abrogated in the VH and VL GL counterpart. In RA B cell hybrid clone RA015/11.88 and RA056/11.23.2, NET and/or cit-H2B immunoreactivity was solely dependent on SHM in the IgVH region whereas RA B cell hybrid clone RA015/11.91 required affinity maturation of both VH and VL for efficient binding to cit-H2B. In 7/80 RA-rmAb, SHM resulted in ex novo N-glycosylation sites in VH and/or VL regions. Removal of Fab-linked glycans in RA056/11.23.2 in the N-mutant counterpart resulted in 90% reduction in immunoreactivity to cit-H2B. Thus, SHM in the IgVH and/or VL regions of RA synovial B cells is necessary for the immunoreactivity to NET-Ags. Fab-N-linked-glycosylation introduction sites are observed in a minority of anti-NET B cell clones but can strongly influence NET-Ag binding.

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.

Characterization of a Synovial B Cell-Derived Recombinant Monoclonal Antibody Targeting Stromal Calreticulin in the Rheumatoid Joints.

Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS+ RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast-like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)+ versus ACPA- RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti-cit-CRT Abs were preferentially detected in ACPA+ versus ACPA- RA sera. We identified a synovial B cell-derived RA-rmAb locally differentiated within the ELS+ RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.

Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs.

OBJECTIVES: Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium. METHODS: Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA. RESULTS: RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a 'citrullinome' multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS-) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-ß, two master regulators of ELS. CONCLUSION: We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.

Autoimmunity to stromal-derived autoantigens in rheumatoid ectopic germinal centers exacerbates arthritis and affects clinical response.

Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.

Dynamic spectrum of ectopic lymphoid B cell activation and hypermutation in the RA synovium characterized by NR4A nuclear receptor expression.

Ectopic lymphoid structures (ELS) can develop in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and selection are not well understood. Here, we identify a synovial B cell population characterized by co-expression of a family of orphan nuclear receptors (NR4A1-3), which is highly enriched in RA synovial tissue. A transcriptomic profile of NR4A synovial B cells significantly overlaps with germinal center light zone B cells and an accrual of somatic hypermutation that correlates with loss of naive B cell state. NR4A B cells co-express lymphotoxins α and ß and IL-6, supporting functions in ELS promotion. Expanded and shared clones between synovial NR4A B cells and plasma cells and the rapid upregulation with BCR stimulation point to in situ differentiation. Together, we identify a dynamic progression of B cell activation in RA synovial ELS, with NR4A transcription factors having an important role in local adaptive immune responses.

Generation of restriction endonucleases barcode map to trace SARS-CoV-2 origin and evolution.

Since the first report of SARS-CoV-2 in China in 2019, there has been a huge debate about the origin. In this work, using a different method we aimed to strengthen the observation that no evidence of genetic manipulation has been found by (1) detecting classical restriction site (RS) sequence in human SARS-CoV-2 genomes and (2) comparing them with other recombinant SARS-CoV-like virus created for experimental purposes. Finally, we propose a novel approach consisting in the generation of a restriction endonucleases site map of SARS-CoV-2 and other related coronavirus genomes to be used as a fingerprint to trace the virus evolution.

The vitamin D receptor agonist elocalcitol inhibits IL-8-dependent benign prostatic hyperplasia stromal cell proliferation and inflammatory response by targeting the RhoA/Rho kinase and NF-kappaB pathways.

BACKGROUND: Benign prostatic hyperplasia (BPH) is characterized by an important inflammatory component. Stimulation of human prostate stromal cells from BPH tissues with proinflammatory cytokines leads to secretion of IL-8, a chemokine involved in BPH pathogenesis. The vitamin D receptor (VDR) agonist elocalcitol can arrest prostate growth in BPH patients, but its mechanism of action in this pathology is still incompletely understood. METHODS: IL-8 levels were measured by real-time RT-PCR and ELISA. NF-kappaB translocation and COX-2 expression were evaluated by confocal microscopy. RhoA and Rho-kinase (ROCK) gene expression and functional activity were studied by real-time RT-PCR, immuno-kinase assays, Western blot analysis, confocal microscopy, and cell invasion. RESULTS: Stimulation of BPH cells with IL-8 activates the calcium-sensitizing RhoA/ROCK pathway, as demonstrated by the increased membrane translocation of RhoA and by phosphorylation of the ROCK substrate myosin phosphatase target subunit 1 (MYPT-1). In agreement with these data, C3 exoenzyme, a selective RhoA inhibitor, inhibits IL-8-induced invasion of BPH cells. The VDR agonist elocalcitol significantly inhibits IL-8 production by BPH cells stimulated with inflammatory cytokines, and IL-8-induced proliferation of BPH cells. In addition, elocalcitol inhibits IL-8-induced membrane translocation of RhoA and MYPT-1 phosphorylation in BPH cells, and inhibits dose-dependently their IL-8-dependent invasion. The inhibition induced by elocalcitol of IL-8 production by BPH cells is accompanied by decreased COX-2 expression and PGE(2) production and by arrest of NF-kappaB p65 nuclear translocation, associated with inhibition of the RhoA/ROCK pathway. CONCLUSIONS: These data provide a mechanistic explanation for the anti-proliferative and anti-inflammatory properties of elocalcitol in BPH cells.

B cells in the formation of tertiary lymphoid organs in autoimmunity, transplantation and tumorigenesis.

Tertiary lymphoid organs named also tertiary lymphoid structures (TLS) often occur at sites of autoimmune inflammation, organ transplantation and cancer. Although the mechanisms for their formation/function are not entirely understood, it is known that TLS can display features of active germinal centres supporting the proliferation and differentiation of (auto)-reactive B cells. In this Review, we discuss current knowledge on TLS-associated B cells with particular reference on how within diseased tissues these structures are linked to either deleterious or protective outcomes in patients and the potential for therapeutic targeting of TLS through novel drugs.

Generation of Recombinant Monoclonal Antibodies from Single B Cells Isolated from Synovial Tissue of Rheumatoid Arthritis Patients.

Ectopic lymphoid structure (ELS) can form in the target tissues of patients with chronic inflammatory autoimmune diseases such as rheumatoid arthritis (RA) and Sjögren's syndrome (SS). Although it is still not clear why ELS form only in a subset of patients, it is well known that these structures can acquire features of ectopic germinal centers and contribute actively to the production of autoantibodies. Here, we describe a method to generate recombinant monoclonal antibodies from single ELS+ synovial tissue B cells obtained from RA patients. This chapter gives a detailed description of the method beginning from the mononuclear cell preparation from RA synovial tissue, single-cell sort of B cells by flow cytometry, amplification of the immunoglobulin (Ig) genes (both heavy- and light-chain genes) by PCR, and subsequent Ig gene expression vector cloning for full recombinant IgG1 monoclonal antibody (rmAb) production in vitro. The recombinant mAbs generated can be then characterized for (1) analysis of the Ig gene repertoires for clonal studies, (2) immunoreactivity profile, and (3) functional studies both in vitro and in vivo.

Ectopic Lymphoid Structures: Powerhouse of Autoimmunity.

Ectopic lymphoid structures (ELS) often develop at sites of inflammation in target tissues of autoimmune diseases, such as rheumatoid arthritis, Sjögren's syndrome, multiple sclerosis, myasthenia gravis, and systemic lupus erythematosus. ELS are characterized by the formation of organized T/B cells aggregates, which can acquire follicular dendritic cells network supporting an ectopic germinal center response. In this review, we shall summarize the mechanisms that regulate the formation of ELS in tertiary lymphoid organs, with particular emphasis on the role of lymphoid chemokines in both formation and maintenance of ELS, the role of emerging positive and negative regulators of ELS development and function, including T follicular helper cells and IL-27, respectively. Finally, we shall discuss the main functions of ELS in supporting the affinity maturation, clonal selection, and differentiation of autoreactive B cells contributing to the maintenance and perpetuation of humoral autoimmunity.

NETosis as Source of Autoantigens in Rheumatoid Arthritis.

In neutrophils (but also in eosinophils and in mast cells), different inflammatory stimuli induce histone deimination, chromatin decondensation, and NET formation. These web-like structures that trap and kill microbes contain DNA, cationic granule proteins, and antimicrobial peptides, but the most abundant proteins are core histones. Histones contained in NETs have been deiminated, and arginines are converted in citrullines. While deimination is a physiological process amplified in inflammatory conditions, only individuals carrying genetic predisposition to develop rheumatoid arthritis (RA) make antibodies to deiminated proteins. These antibodies, collectively identified as anti-citrullinated proteins/peptides antibodies (ACPA), react with different deiminated proteins and display partially overlapping specificities. In this paper, we will summarize current evidence supporting the role of NETosis as critical mechanism in the breach of tolerance to self-antigens and in supporting expansion and differentiation of autoreactive cells. In fact, several lines of evidence connect NETosis with RA: RA unstimulated synovial fluid neutrophils display enhanced NETosis; sera from RA patients with Felty's syndrome bind deiminated H3 and NETs; a high number of RA sera bind deiminated H4 contained in NETs; human monoclonal antibodies generated from RA synovial B cells decorate NETs and bind deiminated histones. In RA, NETs represent on one side an important source of autoantigens bearing posttranslational modifications and fueling the production of ACPA. On the other side, NETs deliver signals that maintain an inflammatory milieu and contribute to the expansion and differentiation of ACPA-producing B cells.

Neutrophil-derived microvesicles enter cartilage and protect the joint in inflammatory arthritis.

Microvesicles (MVs) are emerging as a new mechanism of intercellular communication by transferring cellular lipid and protein components to target cells, yet their function in disease is only now being explored. We found that neutrophil-derived MVs were increased in concentration in synovial fluid from rheumatoid arthritis patients compared to paired plasma. Synovial MVs overexpressed the proresolving, anti-inflammatory protein annexin A1 (AnxA1). Mice deficient in TMEM16F, a lipid scramblase required for microvesiculation, exhibited exacerbated cartilage damage when subjected to inflammatory arthritis. To determine the function of MVs in inflammatory arthritis, toward the possibility of MV-based therapeutics, we examined the role of immune cell-derived MVs in rodent models and in human primary chondrocytes. In vitro, exogenous neutrophil-derived AnxA1(+) MVs activated anabolic gene expression in chondrocytes, leading to extracellular matrix accumulation and cartilage protection through the reduction in stress-adaptive homeostatic mediators interleukin-8 and prostaglandin E2. In vivo, intra-articular injection of AnxA1(+) MV lessened cartilage degradation caused by inflammatory arthritis. Arthritic mice receiving adoptive transfer of whole neutrophils displayed abundant MVs within cartilage matrix and revealed that MVs, but not neutrophils themselves, can penetrate cartilage. Mechanistic studies support a model whereby MV-associated AnxA1 interacts with its receptor FPR2 (formyl peptide receptor 2)/ALX, increasing transforming growth factor-ß production by chondrocytes, ultimately leading to cartilage protection. We envisage that MVs, either directly or loaded with therapeutics, can be harnessed as a unique therapeutic strategy for protection in diseases associated with cartilage degeneration.

Peripheral and synovial mechanisms of humoral autoimmunity in rheumatoid arthritis.

One of the hallmarks of rheumatoid arthritis (RA) is the development of humoral autoimmunity resulting in circulating autoantibodies. The clinical efficacy of B cell-depleting biologic treatments highlighted a key role for autoreactive B cell activation in the pathogenesis of RA. In this review, we discuss the key mechanisms leading to breach of B cell self-tolerance in the peripheral compartment. We also highlight the contribution of synovial ectopic lymphoid structures (ELS) in the development of functional niches of autoreactive B cells promoting humoral autoimmunity in the inflamed RA joints over and above secondary lymphoid organs (SLO).

Accumulation of self-reactive naïve and memory B cell reveals sequential defects in B cell tolerance checkpoints in Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease characterised by breach of self-tolerance towards nuclear antigens resulting in high affinity circulating autoantibodies. Although peripheral B cell disturbances have been described in SS, with predominance of naïve and reduction of memory B cells, the stage at which errors in B cell tolerance checkpoints accumulate in SS is unknown. Here we determined the frequency of self- and poly-reactive B cells in the circulating naïve and memory compartment of SS patients. Single CD27-IgD+ naïve, CD27+IgD+ memory unswitched and CD27+IgD- memory switched B cells were sorted by FACS from the peripheral blood of 7 SS patients. To detect the frequency of polyreactive and autoreactive clones, paired Ig VH and VL genes were amplified, cloned and expressed as recombinant monoclonal antibodies (rmAbs) displaying identical specificity of the original B cells. IgVH and VL gene usage and immunoreactivity of SS rmAbs were compared with those obtained from healthy donors (HD). From a total of 353 VH and 293 VL individual sequences, we obtained 114 rmAbs from circulating naïve (n = 66) and memory (n = 48) B cells of SS patients. Analysis of the Ig V gene repertoire did not show significant differences in SS vs. HD B cells. In SS patients, circulating naïve B cells (with germline VH and VL genes) displayed a significant accumulation of clones autoreactive against Hep-2 cells compared to HD (43.1% vs. 25%). Moreover, we demonstrated a progressive increase in the frequency of circulating anti-nuclear naïve (9.3%), memory unswitched (22.2%) and memory switched (27.3%) B cells in SS patients. Overall, these data provide novel evidence supporting the existence of both early and late defects in B cell tolerance checkpoints in patients with SS resulting in the accumulation of autoreactive naïve and memory B cells.

Role of lymphoid chemokines in the development of functional ectopic lymphoid structures in rheumatic autoimmune diseases.

A sizeable subset of patients with the two most common organ-specific rheumatic autoimmune diseases, rheumatoid arthritis (RA) and Sjögren's syndrome (SS) develop ectopic lymphoid structures (ELS) in the synovial tissue and salivary glands, respectively. These structures are characterized by perivascular (RA) and periductal (SS) clusters of T and B lymphocytes, differentiation of high endothelial venules and networks of stromal follicular dendritic cells (FDC). Accumulated evidence from other and our group demonstrated that the formation and maintenance of ELS in these chronic inflammatory conditions is critically dependent on the ectopic expression of lymphotoxins (LT) and lymphoid chemokines CXCL13, CCL19, CCL21 and CXCL12. In this review we discuss recent advances highlighting the cellular and molecular mechanisms, which regulate the formation of ELS in RA and SS, with particular emphasis on the role of lymphoid chemokines. In particular, we shall focus on the evidence that in the inflammatory microenvironment of the RA synovium and SS salivary glands, several cell types, including resident epithelial, stromal and endothelial cells as well as different subsets of infiltrating immune cells, have been shown to be capable of producing lymphoid chemokines. Finally, we summarize accumulating data supporting the conclusion that ELS in RA and SS represent functional niches for B cells to undergo affinity maturation, clonal selection and differentiation into plasma cells autoreactive against disease-specific antigens, thus contributing to humoral autoimmunity over and above that of secondary lymphoid organs.

Vitamin D receptor agonists as anti-inflammatory agents.

1alpha,25-dihydroxyvitamin D3 (1,25-[OH]2D3), the biologically active form of vitamin D, is a secosteroid hormone that is essential for bone and mineral homeostasis. In addition, this hormone regulates the growth and differentiation of many cell types and has pronounced immunoregulatory and anti-inflammatory properties. Current therapeutic indications include osteoporosis, secondary hyperparathyroidism and psoriasis, but the anti-proliferative, prodifferentiative, antibacterial, immunomodulatory and anti-inflammatory properties of vitamin D receptor agonists could be exploited in a variety of additional clinical conditions. In particular, the pleiotropic anti-inflammatory effects induced by vitamin D receptor agonists could turn out to be beneficial in different pathologies mediated by chronic inflammatory responses.