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ObjectiveAnti-SS-B antibodies are often associated with anti-SS-A in Sjögren’s disease. Compared to anti-SS-A antibody positivity, the significance of the immunological profile anti-SS-B positive/anti-SS-A negative remains controversial. We aimed to evaluate the prevalence and diagnostic significance of isolated anti-SS-B antibodies.MethodsWe conducted a retrospective study across three hospitals of the Assistance Publique—Hôpitaux de Paris. Patients with anti-SS-B positivity were identified using ELISA, addressable laser beam immunoassay (ALBIA) and immunodot assays. They were retained if anti-SS-B was positive in two techniques and anti-SS-A was absent. Clinical, biological and immunological data were extracted and presented in a descriptive analysis.ResultsA total of 80 540 requests for anti-SS-B antibody testing were carried out over a period of 7.9 years. Anti-SS-B positivity was found in 1693 patients. Among them, 335 (19.8%) patients had isolated anti-SS-B in ELISA/ALBIA. Immunodot was performed in 186 of them and confirmed anti-SS-B positivity in 61 patients (3.6% of anti-SS-B positivity). 24 patients (39.3%) presented with a history of various autoimmune or autoinflammatory diseases and only 6 were diagnosed with a new connective tissue disease. After a median follow-up of 26 months, only two new diagnoses were made.ConclusionAnti-SS-B without anti-SS-A is exceedingly rare when accurately identified by a rigorous immunological approach. The initial anti-SS-B positivity does not correlate with a specific condition, both at the time of initial identification and after a 26-month follow-up period. This supports the fact that isolated anti-SS-B has no diagnostic or prognostic value.

Since the first description of nuclear autoantigens in the late 1960s and early 1970s, researchers, including ourselves, have found it difficult to establish monoclonal antibodies (mabs) against nuclear antigens, including the La/SS-B (Sjögrens’ syndrome associated antigen B) autoantigen. To date, only a few anti-La mabs have been derived by conventional hybridoma technology; however, those anti-La mabs were not bona fide autoantibodies as they recognize either human La specific, cryptic, or post-translationally modified epitopes which are not accessible on native mouse La protein. Herein, we present a series of novel murine anti-La mabs including truly autoreactive ones. These mabs were elicited from a human La transgenic animal through adoptive transfer of T cells from non-transgenic mice immunized with human La antigen. Detailed epitope and paratope analyses experimentally confirm the hypothesis that somatic hypermutations that occur during T cell dependent maturation can lead to autoreactivity to the nuclear La/SS-B autoantigen.

Primary Sjögren’s syndrome (pSS) is a complex autoimmune disease characterized by dysfunction of secretory epithelia with only palliative therapy. Patients present with a constellation of symptoms, and the diversity of symptomatic presentation has made it difficult to understand the underlying disease mechanisms. In this study, aggregation of unbiased transcriptome profiling data sets of minor salivary gland biopsies from controls and Sjögren’s syndrome patients identified increased expression of lysosome-associated membrane protein 3 (LAMP3/CD208/DC-LAMP) in a subset of Sjögren’s syndrome cases. Stratification of patients based on their clinical characteristics suggested an association between increased LAMP3 expression and the presence of serum autoantibodies including anti-Ro/SSA, anti-La/SSB, anti-nuclear antibodies. In vitro studies demonstrated that LAMP3 expression induces epithelial cell dysfunction leading to apoptosis. Interestingly, LAMP3 expression resulted in the accumulation and release of intracellular TRIM21 (one component of SSA), La (SSB), and α-fodrin protein, common autoantigens in Sjögren’s syndrome, via extracellular vesicles in an apoptosis-independent mechanism. This study defines a clear role for LAMP3 in the initiation of apoptosis and an independent pathway for the extracellular release of known autoantigens leading to the formation of autoantibodies associated with this disease. ClinicalTrials.gov Identifier: NCT00001196, NCT00001390, NCT02327884.

Decades ago, we and many other groups showed a nucleo-cytoplasmic translocation of La protein in cultured cells. This shuttling of La protein was seen after UV irradiation, virus infections, hydrogen peroxide exposure and the Fenton reaction based on iron or copper ions. All of these conditions are somehow related to oxidative stress. Unfortunately, these harsh conditions could also cause an artificial release of La protein. Even until today, the shuttling and the cytoplasmic function of La/SS-B is controversially discussed. Moreover, the driving mechanism for the shuttling of La protein remains unclear. Recently, we showed that La protein undergoes redox-dependent conformational changes. Moreover, we developed anti-La monoclonal antibodies (anti-La mAbs), which are specific for either the reduced form of La protein or the oxidized form. Using these tools, here we show that redox-dependent conformational changes are the driving force for the shuttling of La protein. Moreover, we show that translocation of La protein to the cytoplasm can be triggered in a ligand/receptor-dependent manner under physiological conditions. We show that ligands of toll-like receptors lead to a redox-dependent shuttling of La protein. The shuttling of La protein depends on the redox status of the respective cell type. Endothelial cells are usually resistant to the shuttling of La protein, while dendritic cells are highly sensitive. However, the deprivation of intracellular reducing agents in endothelial cells makes endothelial cells sensitive to a redox-dependent shuttling of La protein.

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

Objective Primary Sjögren syndrome (pSS) is characterized by T and B cell infiltration of exocrine glands. The cysteine protease cathepsin S (CatS) is crucially involved in MHCII processing and T cell stimulation, and elevated levels have been found in patients with RA, psoriasis and pSS. However, little is known about the functional characteristics and mechanisms of SS-A- and SS-B-specific T cells in pSS patients. We herein investigated the inhibition of CatS activity in different biocompartments of pSS patients including antigen-specific T cell responses. Methods Ex vivo CatS activity was assessed in tears, plasma and saliva of 15 pSS patients and 13 healthy controls (HC) and in the presence or absence of the specific CatS inhibitor RO5459072. In addition, antigen (SS-A (60kD), SS-B, influenza H 3 N 2 , tetanus toxoid and SEB)-specific T cell responses were examined using ex vivo IFN-γ/IL-17 Dual ELISPOT and Bromdesoxyuridin (BrdU) proliferation assays in the presence or absence of RO5459072. Supernatants were analysed for IL-1β, IL-6, IL-10, TNF-α, IL-21, IL-22 and IL-23, using conventional ELISA. Results CatS activity was significantly elevated in tear fluid, but not other biocompartments, was inversely associated with exocrinic function in pSS patients and could significantly be suppressed by RO5459072. Moreover, CatS inhibition by RO5459072 led to strong and dose-dependent suppression of SS-A/SS-B-specific T cell effector functions and cytokine secretion by CD14 + monocytes. However, RO5459072 was incapable of suppressing SS-A/SS-B-induced secretion of cytokines in CD14 + monocytes when T cells were absent, confirming a CatS/MHCII-mediated mechanism of suppression. Conclusion CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. Conversely, CatS inhibition diminishes T cell and associated monokine responses towards relevant autoantigens in pSS. Thus, CatS inhibition may represent a promising novel treatment strategy in pSS.

According to the literature, the autoantigen La is involved in Cap-independent translation. It was proposed that one prerequisite for this function is the formation of a protein dimer. However, structural analyses argue against La protein dimers. Noteworthy to mention, these structural analyses were performed under reducing conditions. Here we describe that La protein can undergo redox-dependent structural changes. The oxidized form of La protein can form dimers, oligomers and even polymers stabilized by disulfide bridges. The primary sequence of La protein contains three cysteine residues. Only after mutation of all three cysteine residues to alanine La protein becomes insensitive to oxidation, indicating that all three cysteines are involved in redox-dependent structural changes. Biophysical analyses of the secondary structure of La protein support the redox-dependent conformational changes. Moreover, we identified monoclonal anti-La antibodies (anti-La mAbs) that react with either the reduced or oxidized form of La protein. Differential reactivities to the reduced and oxidized form of La protein were also found in anti-La sera of autoimmune patients.

Persistent autoantibody production in patients with systemic lupus erythematosus (SLE) suggests the existence of autoreactive humoral memory, but the frequency of self-reactive memory B cells in SLE has not been determined. Here, we report on the reactivity of 200 monoclonal antibodies from single IgG+ memory B cells of four SLE patients. The overall frequency of polyreactive and HEp-2 self-reactive antibodies in this compartment was similar to controls. We found 15% of IgG memory B cell antibodies highly reactive and specific for SLE-associated extractable nuclear antigens (ENA) Ro52 and La in one patient with serum autoantibody titers of the same specificity but not in the other three patients or healthy individuals. The germ-line forms of the ENA antibodies were non-self-reactive or polyreactive with low binding to Ro52, supporting the idea that somatic mutations contributed to autoantibody specificity and reactivity. Heterogeneity in the frequency of memory B cells expressing SLE-associated autoantibodies suggests that this variable may be important in the outcome of therapies that ablate this compartment.

Background Based on the potential involvement of Toll-like receptor (TLR) signalling in the pathogenesis of neonatal lupus (NL), it was hypothesised that fetal exposure to hydroxychloroquine (HCQ), a TLR inhibitor, might reduce the risk of anti-SSA/Ro/SSB/La antibody-associated cardiac manifestations of NL (cardiac-NL). Methods Cardiac-NL children (N=50) and controls (N=151) were drawn from the following overlapping pregnancy studies: Research Registry for NL; PR Interval and Dexamethasone Evaluation in Cardiac-NL; and Predictors of Pregnancy Outcomes: Biomarkers in Antiphospholipid Syndrome and Systemic Lupus Erythematosus (SLE). Pregnancies met the following inclusion criteria: documentation of maternal anti-SSA/Ro/SSB/La antibodies at pregnancy, confirmation of medication use and child's outcome, a diagnosis of SLE before pregnancy and birth by 31 December 2007. Results Seven (14%) of the cardiac-NL children were exposed to HCQ compared with 56 (37%) of the controls (p=0.002; OR 0.28; 95% CI 0.12 to 0.63). Cases and controls were similar with respect to demographic and antibody status. Multivariable analysis adjusting for birth year, maternal race/ethnicity, antibody status, non-fluorinated steroid use and prior cardiac-NL risk yielded an OR associated with HCQ use of 0.46 (95% CI 0.18 to 1.18; p=0.10). Conclusion This case–control study suggests that, in mothers with SLE with anti-SSA/Ro/SSB/La antibodies, exposure to HCQ during pregnancy may decrease the risk of fetal development of cardiac-NL. Prospective studies are needed for confirmation.

Maternal anti-Ro/SSA (Sjögren's syndrome-related antigen A) [± anti-La/SSB (Sjögren's syndrome type B antigen)] antibodies can lead to neonatal lupus, which may present most severely as autoimmune congenital atrioventricular block (CAVB). Although CAVB is uncommon (~ 2% of anti-Ro/SSA-positive pregnancies), once a complete block develops, spontaneous reversal is rare, and many affected infants require permanent pacemaker implantation. Consequently, prevention and early detection are critical components of management, particularly in pregnancies following previous antibody-mediated losses. A 34-year-old woman with primary Sjögren's syndrome, high-titer anti-Ro/SSA and anti-La/SSB, and a history of five prior pregnancy losses-two early (biochemical, 7 weeks) and three midtrimester (16-21 weeks) complicated with severe fetal complete heart block (one demise, two terminations for hydrops)-presented for her sixth pregnancy. A multidisciplinary protocol was implemented, including hydroxychloroquine 400 mg/day, methylprednisolone (initially 6 mg/day, briefly increased to 32 mg/day at 14-16 weeks, then tapered to 8 mg/day), low-dose aspirin 50 mg/day, and fondaparinux 2.5 mg/day from ovulation throughout pregnancy, along with scheduled intravenous immunoglobulin (IVIG; 20 g at 4, 6 + 6, and 8 + 2 weeks; followed by 20 g/day × 3 at 14, 18, and 22weeks). From 14weeks, weekly fetal echocardiography with Doppler atrioventricular (AV)-interval monitoring (16-26weeks) remained normal. At 38 + 2weeks, a cesarean section delivered a female infant weighing 2,710 g and measuring 49 cm, with Apgar scores of 9/10. Neonatal telemetry/ECG showed sinus rhythm at 144 bpm without AV block. Echocardiography revealed a patent ductus arteriosus and a small atrial septal defect, with moderate pulmonary hypertension (SPAP 51 mmHg). Brain MRI and EEG were normal, and there were no cutaneous, hepatic, hematologic, or other features of neonatal lupus. Postpartum, the mother continued methylprednisolone 6 mg/day, hydroxychloroquine 400 mg daily, and enoxaparin 4,000 IU once daily for 4 weeks maintain disease suppression and thromboprophylaxis. In an anti-Ro/SSA-positive pregnancy at extreme risk, a prevention-first, protocol-driven approach-centered on hydroxychloroquine, judicious immunomodulation, and structured AV-interval surveillance-successfully averted CAVB and resulted in a pacemaker-free live birth. Minor cardiac lesions warrant ongoing follow-up; however, the absence of conduction disease underscores the clinical utility of this strategy in carefully selected, extreme-risk pregnancies.