Objective. To examine whether the B cell tropic ␥-herpesvirus Epstein-Barr virus (EBV) is aberrantly expressed in its latent and lytic forms within ectopic lymphoid structures (ELS) in the salivary glands of patients with Sjögren's syndrome (SS), and to investigate the relationship between EBV dysregulation, B cell activation, in situ differentiation of autoreactive plasma cells, disease-specific autoantibody production, and cytotoxicity.Methods. Latent and lytic EBV infection in the salivary glands of 28 patients with SS and 38 patients with nonspecific chronic sialadenitis (NSCS), characterized for the presence or absence of ELS, was investigated by reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemical/ immunofluorescence staining. Glandular versus synovial production of anti؊Ro 52, anti-citrullinated protein antibodies (ACPAs), and anti-EBV peptide antibodies was analyzed in situ or in vivo in human SS/SCID and human rheumatoid arthritis/SCID mouse chimeras. Conclusion. Active EBV infection is selectively associated with ELS in the salivary glands of patients with SS and appears to contribute to local growth and differentiation of disease-specific autoreactive B cells. Results. EBV dysregulation within inflammatory infiltrates was observed exclusively in ELS؉ SS salivary gland tissue, as revealed by latent EBV infection and lytic EBV infection in B cells

ObjectivesTo 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.MethodsSalivary 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.ResultsTranscriptomic 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).ConclusionsOverall, 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.

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Ferritin has a key role in Adult-onset Still's disease (AOSD). Its production seems related to macrophage activation of which sCD163 is a major serum marker. Thus, we aimed at evaluating the role of sCD163 in AOSD and its relationship with ferritin. Furthermore, we determined the expression of CD163 and ferritin in a lymph-node from an AOSD patient. sCD163 and serum ferritin were measured in 34 patients with AOSD (21 active, 13 non-active), 18 sepsis and 22 healthy controls (HC). Immunohistology was performed on a lymph-node from an AOSD patient in order to detect CD163 and ferritin. A tonsil from an HC was used as control. Mean sCD163 (8.6 ± 5.4 mg/L) was higher in active AOSD than "non-active" patients (4.6 ± 2.7 mg/L, p = 0.02). The mean sCD163 in AOSD (6.9 ± 4.9 mg/L) and sepsis (7.1 ± 5.6 mg/L) were higher than in HC (2.56 ± 1.17 mg/L, p < 0.001), but no difference between AOSD and sepsis was detected. sCD163 positively correlated with ferritin (p = 0.0045; r = 0.4755) only in AOSD. Serum ferritin (mean 3,640.1 ± 6,896.9 µg/L) was higher in active AOSD than in sepsis (1,720.2 ± 3,882.1 µg/L, p < 0.007). CD163 was equally distributed in the B and T areas of both lymph-node and tonsil. Differently from the tonsil, ferritin was expressed only in the lymph-node B area. sCD163 is a marker of disease activity in AOSD. The correlation with ferritin may lead to hypothesize a macrophage activation related to hyperferritinemia. Ferritin was found expressed only in the B area of the AOSD lymph-node, suggesting a role for this molecule as an antigen in the disease pathogenesis.

A pivotal role for tertiary lymphoid structures (TLSs) in promoting Ag-specific humoral responses during chronic inflammation is emerging in several autoimmune conditions, including rheumatoid arthritis, Sjogren’s syndrome, and autoimmune thyroiditis. However, there is limited evidence on the cellular and molecular mechanisms underlying TLS formation and their contribution to autoimmunity in the pancreas during autoimmune insulitis. In this study, we performed a detailed and comprehensive assessment of the evolution of TLSs during autoimmune insulitis in 126 female NOD mice from 4 to 38 wk of age. We demonstrated that during progression from peri- to intrainsulitis in early diabetic mice, T and B cell infiltration follows a highly regulated process with the formation of lymphoid aggregates characterized by T/B cell segregation, follicular dendritic cell networks, and differentiation of germinal center B cells. This process is preceded by local upregulation of lymphotoxins α/β and lymphoid chemokines CXCL13 and CCL19, and is associated with infiltration of B220+/IgD+/CD23+/CD21− follicular B cells expressing CXCR5. Despite a similar incidence of insulitis, late diabetic mice displayed a significantly reduced incidence of fully organized TLSs and reduced levels of lymphotoxins/lymphoid chemokines. Upon development, TLSs were fully functional in supporting in situ autoreactive B cell differentiation, as demonstrated by the expression of activation-induced cytidine deaminase, the enzyme required for Ig affinity maturation and class switching, and the presence of CD138+ plasma cells displaying anti-insulin reactivity. Overall, our work provides direct evidence that TLSs are of critical relevance in promoting autoimmunity and chronic inflammation during autoimmune insulitis and diabetes in NOD mice.

Background Low-level, chronic viral infections have been suspect in the development of select autoimmune diseases, including primary Sjögren’s syndrome (pSS). Multiple studies have shown stimulation of antiviral response pathways in pSS tissues suggestive of a viral infection. Yet, with this data in hand, a causal link between a viral infection and development of pSS had not been identified. Therefore, a study was designed to further define the viral landscape within pSS-affected salivary gland tissue to identify potential viral-mediated triggers in the pathogenesis of this autoimmune disease. Methods A viral microarray was utilized to measure viral transcripts present in salivary gland tissue from patients diagnosed with pSS compared to healthy controls. Murine models of salivary gland localized HDV antigen expression were developed to evaluate the capacity of a chronic HDV signature to trigger the development of a pSS-like phenotype. Results Through this analysis, two distinct viral profiles were identified, including the increased presence of hepatitis delta virus (HDV) in 50% of pSS patients evaluated. Presence of HDV antigen and sequence were confirmed in minor salivary gland tissue. Patients with elevated HDV levels in salivary gland tissue were negative for detectible hepatitis B virus (HBV) surface antigen and antibodies to HBV or HDV. Expression of HDV antigens in vivo resulted in reduced stimulated saliva flow, increase in focal lymphocytic infiltrates, and development of autoantibodies. Conclusion Identification of HDV in pSS patients and induction of a complete pSS-like phenotype in vivo provides further support of a viral-mediated etiopathology in the development of pSS.

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Rheumatoid Arthritis (RA) is a chronic, inflammatory, autoimmune disease affecting diarthrodial joints and extra-articular tissues; in the absence of an effective treatment, it is characterized by persistent symmetrical and erosive synovitis which leads to structural joint damage and lifelong disability. Several autoantibodies have been associated with RA such as rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA). B cells have been shown to play a crucial role in the pathogenesis of RA by producing autoantibodies and promoting synovial inflammation through antigen presentation, T cells activation and cytokines production [1]. Although biologic agents have notably improved disease outcome and patients' quality of life, currently around 30-40% of subjects do not respond to treatment and the mechanisms leading to resistance are still not known [2]. For this reason, new prognostic biomarkers and predictors of response are needed. We and others have postulated that the development of biomarkers for patients' stratification prior therapeutic intervention may be possible through a better understanding of the different histopathological patterns present both in early and established individual RA patient and the related underlying cellular and molecular mechanisms. To date, Tumor Necrosis Factor (TNF)-α has been shown to be one of the master elements of inflammation in RA; however, even though therapies aimed at blocking this key cytokine have emerged as a major tool in the treatment of RA, a large proportion of patients (approximately 30-40%) do not achieve a meaningful clinical response assessed by either the American College of Rheumatology (ACR) or the European League Against Rheumatism (EULAR) criteria. The same limitation can be applied to the use of rituximab, a chimeric monoclonal antibody directed against CD20, which is uniquely expressed by all B-lymphocytes during the maturation process from late stage pro-B cells to memory cells. The clinical efficacy of rituximab has been proved by several clinical studies [3] but it is highly variable. Currently, NICE guidelines recommend rituximab in patients with inadequate response to a first-line biologic therapy, including at least one anti-TNFα agent independently of their pre-treatment chance to respond. In all cases, whether considering biologics used for several years in RA patients (anti-TNFα or rituximab) or relatively newer biologics in clinical use (i.e. tocilizumab, an anti-interleukin (IL) -6 receptor blocking monoclonal antibody or abatacept, a CTLA4 inhibitor fusion protein designed to target the T cell co-stimulatory signal mediated through the CD28-CD80/86 pathway), no validated biomarkers predictive of clinical response currently exist. Consequently, to date a "trial-and-error" approach is used in the prescription of biologics in RA, which has the obvious disadvantage of potentially exposing patients to drugs that they may not respond, with potential unnecessary side-effects, delaying use of an effective treatment and causing a signi...