Seronegative patients with primary Sjögren's syndrome and non-pSS sicca test positive for anti-SSA/Ro52 and -Ro60 in saliva.

OBJECTIVES: Testing for anti-SSA/Ro antibodies in serum is essential in the diagnostic work-up for primary Sjögren's syndrome (pSS). In this study, we aimed to validate our previous assay for detection of salivary anti-SSA/Ro52, and to develop assays for detection of salivary anti-SSA/Ro60 and for detection of anti-Ro52 and -Ro60 in plasma using the electric field-induced release and measurement (EFIRM) platform. METHODS: Whole saliva samples from two independent Danish cohorts (DN1 and DN2) including 49 patients with pSS, 73 patients with sicca symptoms, but not fulfilling the classification criteria for pSS (non-pSS sicca), and 51 healthy controls (HC), as well as plasma samples from the DN1 cohort were analyzed using EFIRM to detect anti-SSA/Ro52 and -Ro60. RESULTS: In the DN1 cohort, 100 % in the pSS group and 16 % in the non-pSS sicca group were serum anti-SSA/Ro positive by ELISA. EFIRM detected anti-SSA (Ro52 and/or -Ro60) in plasma and saliva in 100 % and 96 % patients with pSS, and 16 % and 29 % with non-pSS sicca. In the DN2 cohort, 80 % patients with pSS and 26 % with non-pSS sicca were serum anti-SSA/Ro positive. Salivary anti-SSA discriminated patients with pSS from HC and non-pSS sicca with an AUC range of 0.74-0.96 in the DN1 and DN2 cohorts. EFIRM discriminated pSS from non-pSS sicca with an AUC of 0.98 in plasma. CONCLUSION: Our findings suggest that salivary anti-SSA/Ro antibodies are potential discriminatory biomarkers for pSS, which may also identify seronegative patients, addressing the unmet clinical need of early detection and stratification of pSS.

Prevalence of anti-Ro52-kDa/SSA (TRIM21) antibodies and associated clinical phenotype in systemic sclerosis: Data from a French cohort, a systematic review and meta-analysis.

OBJECTIVES: Estimate the global prevalence of anti-Ro52-kDa/SSA (TRIM21) autoantibodies in systemic sclerosis (SSc), and describe the associated clinical phenotype, through a systematic review and meta-analysis of published reports and new data from our French cohort. METHODS: Anti-TRIM21 seropositivity and associated SSc characteristics were assessed in a cross-sectional study including 300 patients of Lille University Hospital. A systematic review of the literature was performed in Pubmed and Embase, followed by a meta-analysis, using data on prevalence, clinical/demographical/biological characteristics of SSc patients and the type of assay used for anti-TRIM21 antibodies detection (PROSPERO n° CRD42021223719). FINDINGS: In the cross-sectional study, anti-TRIM21 antibodies prevalence was 26% [95%CI: 21; 31]. Anti-centromere antibodies were the most frequent SSc specific autoantibodies coexisting with anti-TRIM21. Patients with anti-TRIM21 antibodies were more frequently women (91% vs 77%, p = 0.006), more likely to present an associated Sjögren's syndrome (19% vs 7%, p < 0.001), had a higher rate of pulmonary arterial hypertension (PAH) (15% vs 6%, p = 0.017) and a greater frequency of digestive complications such as dysphagia (12% vs 5%, p = 0.038) or nausea/vomiting (10% vs 3%, p = 0.009) than anti-TRIM21 negative patients. Thirty-five articles corresponding to a total of 11,751 SSc patients were included in the meta-analysis. In this population, the overall seroprevalence of anti-TRIM21 antibodies was 23% [95%CI: 21; 27] with a high degree of heterogeneity (I2: 93% Phet: <0.0001), partly explained by the methods of detection. Anti-TRIM21 seropositivity was positively associated with female sex (OR: 1.60 [95%CI: 1.25, 2.06]), limited cutaneous subset (OR: 1.29 [1.04, 1.61]), joint manifestations (OR: 1.33 [1.05, 1.68]), pulmonary hypertension (PH) (OR: 1.82 [1.42, 2.33]), and interstitial lung disease (ILD) (OR: 1.31 [1.07, 1.60]). INTERPRETATION: Anti-TRIM21 antibodies frequently co-exist with usual SSc antibodies, but are independently associated to a higher risk of cardio-pulmonary complications. The presence of these autoantibodies should therefore be considered when assessing the risk of developing PH and ILD, and deserves further studies on appropriate screening and follow-up of patients.

Association of Combined Anti-Ro52/TRIM21 and Anti-Ro60/SSA Antibodies With Increased Sjögren Disease Severity Through Interferon Pathway Activation.

OBJECTIVE: The biologic diagnosis of primary Sjögren disease (SjD) mainly relies on anti-Ro60/SSA antibodies, whereas the significance of anti-Ro52/TRIM21 antibodies currently remains unclear. The aim of this study was to characterize the clinical, serological, biologic, transcriptomic, and interferon profiles of patients with SjD according to their anti-Ro52/TRIM21 antibody status. METHODS: Patients with SjD from the European PRECISESADS (n = 376) and the Brittany Diagnostic Suspicion of primitive Sjögren's Syndrome (DIApSS); (n = 146) cohorts were divided into four groups: double negative (Ro52-/Ro60-), isolated anti-Ro52/TRIM21 positive (Ro52+), isolated anti-Ro60/SSA positive (Ro60+), and double-positive (Ro52+/Ro60+) patients. Clinical information; EULAR Sjögren Syndrome Disease Activity Index, a score representing systemic activity; and biologic markers associated with disease severity were evaluated. Transcriptome data obtained from whole blood by RNA sequencing and type I and II interferon signatures were analyzed for PRECISESADS patients. RESULTS: In the DIApSS cohort, Ro52+/Ro60+ patients showed significantly more parotidomegaly (33.3% vs 0%-11%) along with higher ß2-microglobulin (P = 0.0002), total immunoglobulin (P < 0.0001), and erythrocyte sedimentation rate levels (P = 0.002) as well as rheumatoid factor (RF) positivity (66.2% vs 20.8%-25%) compared to other groups. The PRECISESADS cohort corroborated these observations, with increased arthritis (P = 0.046), inflammation (P = 0.005), hypergammaglobulinemia (P < 0.0001), positive RF (P < 0.0001), leukopenia (P = 0.004), and lymphopenia (P = 0.009) in Ro52+/Ro60+ patients. Cumulative EULAR Sjögren Syndrome Disease Activity Index results further confirmed these disparities (P = 0.002). Transcriptome analysis linked anti-Ro52/TRIM21 antibody positivity to interferon pathway activation as an underlying cause for these clinical correlations. CONCLUSION: These results suggest that the combination of anti-Ro52/TRIM21 and anti-Ro60/SSA antibodies is associated with a clinical, biologic, and transcriptional profile linked to greater disease severity in SjD through the potentiation of the interferon pathway activation by anti-Ro52/TRIM21 antibodies.

Cell surface-expressed Ro52/IgG/HLA-DR complex is targeted by autoantibodies in patients with inflammatory myopathies.

Intracellular proteins are often targeted by autoantibodies in autoimmune diseases; however, the mechanism through which intracellular molecules are targeted remains unknown. We previously found that several intracellular misfolded proteins are transported to the cell surface by HLA class II molecules and are recognized by autoantibodies in some autoimmune diseases, such as rheumatoid arthritis, antiphospholipid syndrome, and microscopic polyangiitis. Ro52 is an intracellular Fc receptor that is a target antigen for myositis-associated autoantibodies. We analyzed the role of HLA class II molecules in the autoantibody recognition of Ro52. Ro52 alone was not transported to the cell surface by HLA class II molecules; however, it was transported to the cell surface in the presence of both IgG heavy chain and HLA class II molecules to form a Ro52/IgG/HLA-DR complex. The Ro52/IgG/HLA-DR complex was specifically recognized by autoantibodies from some patients with inflammatory myopathies. We then evaluated 120 patients with inflammatory myopathies with four types of myositis-specific antibodies and analyzed the autoantibodies against the Ro52/IgG/HLA-DR complex. The specific antibodies against the Ro52/IgG/HLA-DR complex were detected in 90% and 93% of patients who were positive for anti-MDA5 and anti-ARS antibodies, respectively. In individual patients with these two inflammatory myopathies, changes in serum titers of anti-Ro52/IgG/HLA-DR-specific antibodies were correlated with the levels of KL-6 (R = 0.51 in anti-MDA5 antibody-positive DM patients, R = 0.67 in anti-ARS antibody-positive PM/DM patients with respiratory symptoms) and CK (R = 0.63 in anti-ARS antibody-positive PM/DM patients with muscle symptoms) over time. These results suggest that antibodies against Ro52/IgG/HLA-DR expressed on the cell surface could be involved in the pathogenesis of inflammatory myopathy subgroups.

A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment.

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

Strong Association of the Myriad Discrete Speckled Nuclear Pattern With Anti-SS-A/Ro60 Antibodies: Consensus Experience of Four International Expert Centers.

Introduction: The morphological patterns in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) reflect the autoantibodies in the sample. The International Consensus on ANA Patterns (ICAP) classifies 30 relevant patterns (AC-0 to AC-29). AC-4 (fine speckled nuclear pattern) is associated to anti-SS-A/Ro, anti-SS-B/La, and several autoantibodies. Anti-SS-A/Ro samples may contain antibodies to Ro60 and Ro52. A variation of AC-4 (herein designated AC-4a), characterized by myriad discrete nuclear speckles, was reported to be associated with anti-SS-A/Ro. The plain fine speckled pattern (herein designated AC-4b) seldom was associated with anti-SS-A/Ro. This study reports the experience of four expert laboratories on AC-4a and AC-4b. Methods: Anti-Ro60 monoclonal antibody A7 was used to investigate the HEp-2 IFA pattern. Records containing concomitant HEp-2 IFA and SS-A/Ro tests from Durand Laboratory, Argentina (n = 383) and Fleury Laboratory, Brazil (n = 144,471) were analyzed for associations between HEp-2 IFA patterns and disease-associated autoantibodies (DAA): double-stranded DNA, Scl-70, nucleosome, SS-B/La, Sm, and U1-RNP. A total of 381 samples from Dresden Technical University (TU-Dresden), Germany, were assayed for HEp-2 IFA and DAA. Results: Monoclonal A7 recognized Ro60 in Western blot and immunoprecipitation, and yielded the AC-4a pattern on HEp-2 IFA. Analyses from Durand Laboratory and Fleury Laboratory yielded compatible results: AC-4a was less frequent (8.9% and 2.7%, respectively) than AC-4b (26.1% and 24.2%) in HEp-2 IFA-positive samples. Reactivity to SS-A/Ro occurred in 67.6% and 96.3% of AC-4a-pattern samples against 23% and 6.8% of AC-4b pattern samples. Reciprocally, AC-4a occurred in 24% and 47.1% of anti-SS-A/Ro-positive samples, and in 3.8% and 0.1% of anti-SS-A/Ro-negative samples. Data from TU-Dresden show that the AC-4a pattern occurred in 69% of 169 anti-SS-A/Ro-monospecific samples (62% of all anti-SS-A/Ro-positive samples) and in 4% of anti-SS-A/Ro-negative samples, whereas anti-SS-A/Ro occurred in 98.3% of AC-4a samples and in 47.9% of AC-4b samples. In all laboratories, coexistence of anti-SS-B/La, but not other DAA, in anti-SS-A/Ro-positive samples did not disturb the AC-4a pattern. AC-4a was predominantly associated with anti-Ro60 antibodies. Conclusions: This study confirms the association of AC-4a pattern and anti-SS-A/Ro in opposition to the AC-4b pattern. The results of four international expert laboratories support the worldwide applicability of these AC-4 pattern variants and their incorporation into ICAP classification under codes AC-4a and AC-4b, respectively. The AC-4 pattern should be maintained as an umbrella pattern for cases in which one cannot discriminate AC-4a and AC-4b patterns. The acknowledgment of the AC-4a pattern should add value to HEp-2 IFA interpretation.

Current state of technologies and recognition of anti-SSA/Ro antibodies in China: A multi-center study.

BACKGROUND: Previous studies have demonstrated that Ro60 and Ro52 have different clinical implications, and anti-Ro52 antibodies are an independent serum marker of systemic autoimmune diseases, including Sjögren's syndrome. Many different assays have been adopted to detect anti-Sjögren's syndrome antigen A (SSA)/Ro antibodies, while to date no specific approach has been recommended as optimal for anti-SSA/Ro antibody testing. Herein, we performed a multi-center study to explore the current clinical utility of different strategies for anti-SSA/Ro antibody testing in China. METHODS: Twenty-one tertiary care centers were included in this questionnaire-based study. The self-administered questionnaire mainly includes testing methods for anti-SSA/Ro antibodies, reporting system of results, and interpretation of results by clinicians. RESULTS: Six different methods were applied to detect anti-SSA/Ro antibodies in the 21 centers. Line immunoassay (eight different commercial kits) was the most frequently adopted method (21/21, 100%), with different cutoff values and strategies for intensity stratification. There were two reporting systems: One was reported as "anti-SSA antibodies" and "anti-Ro52 antibodies" (12/21, 57%), while the other was "anti-SSA/Ro60 antibodies" and "anti-SSA/Ro52 antibodies" (9/21, 43%). Notably, six centers (29%) considered either positive anti-Ro60 or anti-Ro52 antibodies as positive anti-SSA antibodies, all of which adopted the latter reporting system. CONCLUSION: Significant variabilities existed among anti-SSA/Ro assays. Nearly 30% of centers misinterpreted the definition of positive anti-SSA antibodies, which may be attributed to the confusing reporting systems of line immunoassay. Therefore, we advocate standardization of the nomenclature of anti-SSA/Ro antibodies, changing the "anti-SSA/Ro52" label in favor of the "anti-Ro52" antibodies for a clear designation.

TRIM21/Ro52 - Roles in Innate Immunity and Autoimmune Disease.

TRIM21 (Ro52/SSA1) is an E3 ubiquitin ligase with key roles in immune host defence, signal transduction, and possibly cell cycle regulation. It is also an autoantibody target in Sjögren's syndrome, systemic lupus erythematosus, and other rheumatic autoimmune diseases. Here, we summarise the structure and function of this enzyme, its roles in innate immunity, adaptive immunity and cellular homeostasis, the pathogenesis of autoimmunity against TRIM21, and the potential impacts of autoantibodies to this intracellular protein.

TRIM21 negatively regulates Corynebacterium pseudotuberculosis-induced inflammation and is critical for the survival of C. pseudotuberculosis infected C57BL6 mice.

Corynebacterium pseudotuberculosis, a facultative intracellular bacterium, is an important zoonotic pathogen responsible for chronic inflammatory diseases. TRIM21, an E3 ubiquitin-protein ligase, plays pivotal roles in inflammation regulation. However, its role during C. pseudotuberculosis infection is unclear. Here, we found that TRIM21 expression was significantly increased in C. pseudotuberculosis-infected macrophages. Following infection by C. pseudotuberculosis, we observed a significantly higher number of bacteria and a higher degree of LDH release from Trim21-/- macrophages compared to wild-type (WT) macrophages, suggesting that TRIM21 limits C. pseudotuberculosis replication in macrophages and protects the infected cells from death. Further in vivo experiments showed a significantly higher mortality, higher bacterial load, much more severe abscess formation, and lesions in the organs of C. pseudotuberculosis-infected Trim21-/- mice compared to those of the infected WT mice, suggesting that TRIM21 plays critical roles in protecting against C. pseudotuberculosis infection. Moreover, the secretory levels of IL-1α, IL-1ß, IL-6, and TNF-α were significantly higher in C. pseudotuberculosis-infected Trim21-/- macrophages compared to infected WT macrophages; the levels of these cytokines were also higher in the sera, organs, and ascites of C. pseudotuberculosis-infected Trim21-/- mice compared to infected WT mice. These findings suggest that TRIM21 negatively regulates the secretion of pro-inflammatory cytokines in macrophages, sera, organs, and ascites of mice following C. pseudotuberculosis infection. Collectively, the present study demonstrates that TRIM21 plays a vital role in preventing C. pseudotuberculosis infection, which may be related to the negative regulation of pro-inflammatory cytokines production by TRIM21 during this pathogen infection.

Anti-Ro60 and anti-Ro52/TRIM21: Two distinct autoantibodies in systemic autoimmune diseases.

As iconic and important diagnostic autoantibodies, anti-Ro60 and anti-Ro52/tri-partite motif-containing 21 (TRIM21) make a common appearance in a number of systemic autoimmune disorders such as systemic lupus erythematosus (SLE). These autoantibodies often co-exist together; yet despite their close relationship, there is no evidence that they are physically linked and probably reflect a convergence of separate processes of failed immunological tolerance. Confusingly, they are sometimes classed together as the "SSA" or "Ro" autoantibody system without clear distinction between the two. In this Short Communication, we discuss the diagnostic merits for separate detection and reporting of these two autoantibodies, and discuss avenues for future research. Indeed, further insight into their fascinating origins and pathogenic roles in autoimmunity will surely shed light on how we can prevent and treat devastating autoimmune disorders.

Increased Concentrations of Circulating Soluble MHC Class I-Related Chain A (sMICA) and sMICB and Modulation of Plasma Membrane MICA Expression: Potential Mechanisms and Correlation With Natural Killer Cell Activity in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease of unknown etiology. The major histocompatibility complex (MHC) class I-related chain A (MICA) and B (MICB) are stress-inducible cell surface molecules. MICA and MICB label malfunctioning cells for their recognition by cytotoxic lymphocytes such as natural killer (NK) cells. Alterations in this recognition have been found in SLE. MICA/MICB can be shed from the cell surface, subsequently acting either as a soluble decoy receptor (sMICA/sMICB) or in CD4+ T-cell expansion. Conversely, NK cells are frequently defective in SLE and lower NK cell numbers have been reported in patients with active SLE. However, these cells are also thought to exert regulatory functions and to prevent autoimmunity. We therefore investigated whether, and how, plasma membrane and soluble MICA/B are modulated in SLE and whether they influence NK cell activity, in order to better understand how MICA/B may participate in disease development. We report significantly elevated concentrations of circulating sMICA/B in SLE patients compared with healthy individuals or a control patient group. In SLE patients, sMICA concentrations were significantly higher in patients positive for anti-SSB and anti-RNP autoantibodies. In order to study the mechanism and the potential source of sMICA, we analyzed circulating sMICA concentration in Behcet patients before and after interferon (IFN)-α therapy: no modulation was observed, suggesting that IFN-α is not intrinsically crucial for sMICA release in vivo. We also show that monocytes and neutrophils stimulated in vitro with cytokines or extracellular chromatin up-regulate plasma membrane MICA expression, without releasing sMICA. Importantly, in peripheral blood mononuclear cells from healthy individuals stimulated in vitro by cell-free chromatin, NK cells up-regulate CD69 and CD107 in a monocyte-dependent manner and at least partly via MICA-NKG2D interaction, whereas NK cells were exhausted in SLE patients. In conclusion, sMICA concentrations are elevated in SLE patients, whereas plasma membrane MICA is up-regulated in response to some lupus stimuli and triggers NK cell activation. Those results suggest the requirement for a tight control in vivo and highlight the complex role of the MICA/sMICA system in SLE.

Antigen-driven autoantibody production in lungs of interstitial lung disease with autoimmune disease.

Interstitial lung disease (ILD) sometimes becomes a life-threatening complication of systemic autoimmune diseases; however, little is known about the immune response in lung lesions. We aimed to investigate humoural immunity in ILD associated with rheumatoid arthritis (RA), sjögren's syndrome (SjS), and mixed connective tissue disease (MCTD), using bronchoalveolar fluid (BALF) and serum samples from 15 patients with autoimmune disease associated-ILD. We first showed that BALF contained higher titers of disease-related autoantibodies than serum, suggesting the possibility of autoantibody production in lungs. Next, we produced 326 monoclonal antibodies from antibody-secreting cells in BALF, and the reactivity and their revertants, in which somatic hypermutations were reverted to germline, were analyzed. Among 123 antibodies from RA-ILD, 16 disease-related antibodies (anti-modified protein antibodies and rheumatoid factors) were identified, of which one antibody had both properties. The revertant antibodies changed their target modification in a complicated manner, suggesting that the antibodies were selected against various modifications in lungs. Among 146 antibodies from SjS-ILD and/or MCTD-ILD, seven anti-SSA/Ro60 antibodies and 15 anti-RNP antibodies were identified. Some of the anti-RNP antibodies recognized multiple RNP constituent proteins simultaneously, indicating that epitope spreading may progress in lungs. Our results revealed the existence of an active autoimmunity in the lungs of autoimmune disease associated-ILD.

Presence of anti-TIF-1γ, anti-Ro52, anti-SSA/Ro60 and anti-Su/Ago2 antibodies in breast cancer: a cross-sectional study.

OBJECTIVES: The presence of myositis-specific antibodies (MSA), was recently reported in healthy individuals, cancer patients without myopathy and paraneoplastic rheumatic syndromes. We sought to analyze the frequency of MSA, myositis-associated antibodies (MAA) and autoantibodies related to systemic autoimmune rheumatic diseases (SARD) in breast cancer patients. METHODS: One hundred fifty-two breast cancer patients were enrolled in a cross-sectional study. Clinical information was collected, and autoantibodies tested by immunoprecipitation of an 35S-methionine-labeled K562 cell extract, enzyme-linked immunosorbent assay (ELISA) and Western blot when indicated. All statistical tests were performed using the software statistical package for the social science (SPSS) ver. 19.0 (IBM Inc., NYSE, USA). RESULTS: Autoantibodies associated with SARD: anti-52 kD ribonucleoprotein/tripartite motif-containing 21 (anti-Ro52/TRIM21) was found in 5.9% (9/152), anti-Sjögren syndrome-related antigen A/60 kD ribonucleoprotein antibody (anti-SSA/Ro60) in 3.9% (6/152) and anti-Su antigen/Argonaute 2 antibody (anti-Su/Ago2) in 2.6% (4/152). Meanwhile, anti-transcription intermediary factor-1γ (anti-TIF-1γ, p155/140) antibody was positive in 2 cases and anti-polymyositis/scleroderma antibody was detected in one case. As a whole, 14.47% (22/152) of breast cancer patients showed autoantibodies associated with SARD. These specific autoantibodies were not associated with the presence of rheumatic diseases except one rheumatoid arthritis patient positive for anti-Ro52/TRIM21. CONCLUSIONS: Autoantibodies to TIF-1γ were found in two patients with breast cancer without dermatomyositis (DM). More common specificities were autoantibodies anti-SSA/Ro60, anti-Ro52/TRIM21 and anti-Su/Ago2. More studies are needed in order to establish the biological meaning of the presence of SARD-associated autoantibodies in breast cancer.

Anti-TROVE2 Antibody Determined by Immune-Related Array May Serve as a Predictive Marker for Adalimumab Immunogenicity and Effectiveness in RA.

Anti-drug antibody (ADAb) development is associated with secondary therapeutic failure in biologic-treated rheumatoid arthritis (RA) patients. With a treat-to-target goal, we aimed to identify biomarkers for predicting ADAb development and therapeutic response in adalimumab-treated patients. Three independent cohorts were enrolled. In Cohort-1, 24 plasma samples (6 ADAb-positive and 6 ADAb-negative patients at baseline and week 24 of adalimumab therapy, respectively) were assayed with immune-related microarray containing 1,636 correctly folded functional proteins. Next, we executed statistically powered autoantibody profiling analysis of 50 samples in Cohort-2 (24 ADAb-positive and 26 ADAb-negative patients). Subsequently, immunofluorescence assay was performed on 48 samples in Cohort-3 to correlate with ADAb titers and drug levels. The biomarkers were identified for predicting ADAb development and therapeutic response using the immune-related microarray and machine learning approach. ADAb-positive patients had lower drug levels at week 24 (median = 0.024 µg/ml) compared with ADAb-negative patients (median = 6.38 µg/ml, p < 0.001). ROC analysis based on the ADAb status revealed the top 20 autoantibodies with AUC ≥ 0.7 in differentiating both groups in Cohort-1. Analysis of Cohort-2 dataset identified a panel of 8 biomarkers (TROVE2, SSB, NDE1, ZHX2, SH3GL1, CARD9, PTPN20, and KLHL12) with 80.6% specificity, 77.4% sensitivity, and 79.0% accuracy in discriminating poor from EULAR responders. Immunofluorescence assay validated that anti-TROVE2 antibody could highly predict ADAb development and poor EULAR response (AUC 0.79 and 0.89, respectively). Multivariate regression analysis proved anti-TROVE2 antibody to be an independent predictor for developing ADAb. Immune-related protein microarray and replication analysis identified anti-TROVE2 antibody as a useful biomarker for predicting ADAb development and therapeutic response in adalimumab-treated patients.

NMI Facilitates Influenza A Virus Infection by Promoting Degradation of IRF7 through TRIM21.

Acute respiratory infections caused by influenza A virus (IAV) spread widely and lead to substantial morbidity and mortality. Host cell induction of type I interferon (IFN-I) plays a fundamental role in eliminating the virus during the innate antiviral response. The potential role of N-myc and STAT interactor (NMI) and its underlying mechanisms of action during IAV infection, however, remain elusive. In this study, we found that the expression of NMI increased after IAV infection. Nmi-knockout mice infected with IAV displayed increased survival rate, decreased weight loss, lower viral replication, and attenuated lung inflammation when compared with wild-type mice. Deficiency of NMI promoted the production of IFN-I and IFN-stimulated genes in vivo and in vitro. Reduced levels of NMI also resulted in an increase of the expression of IFN regulator factor (IRF) 7. Further studies have revealed that NMI could interact with IRF7 after IAV infection, and this interaction involved its NID1 and NID2 domain. In addition, NMI facilitated ubiquitination and proteasome-dependent degradation of IRF7 through recruitment of the E3 ubiquitin ligase TRIM21 (tripartite motif-containing 21) to limit the IAV-triggered innate immunity. Our findings reveal a clearer understanding of the role of NMI in regulating the host innate antiviral response and provide a potential therapeutic target for controlling IAV infection.

SS-A52 antigen expression in thymic carcinoma accompanied with Sjögren syndrome: A case report.

RATIONALE: The relationship between thymic tumors and Sjögren syndrome (SjS) is unknown, and surgical resection has not been optimized. Especially, thymic carcinoma with autoimmune disease is rare. Analysis of SS-A52, germinal centers, plasma cells, and Foxp3+ Treg in thymic carcinoma has never been reported, and their pathological roles in causing SjS have not been studied. PATIENT CONCERNS: A 78-year-old man presented with sputum production and xerostomia while asleep. Chest computed tomography showed a homogeneous and hypodense mass in the anterosuperior mediastinum. Serum levels of the antinuclear antibody, antibody to SS-A, and antibody to SS-B were positive. DIAGNOSES: Thymic carcinoma (squamous cell carcinoma) and SjS. INTERVENTIONS: Video-assisted thoracoscopic resection of the mediastinal tumor and postoperative radiation therapy was performed. OUTCOMES: The histological diagnosis was thymic squamous cell carcinoma. Histologically, the squamous carcinomatous cells were arranged in nests and cords in the fibrohyaline stroma with capsular invasion. In the stroma, dense lymphoid tissues containing large reactive germinal centers and many plasma cells were also noted. In the involuted thymus, CD20-positive mature lymphocytes infiltrated, and germinal centers were noted. Double immunohistochemical staining revealed that SS-A52 antigen was positive in both the carcinoma component and CD20-positive mature B cells. Postoperatively, the xerostomia persisted, and serum SS-A and SS-B remained positive. No evidence of carcinoma recurrence with chest computed tomography scan was observed at 18-months follow-up. LESSONS: In the surgical treatment of thymic tumors with SjS, extended thymectomy might be worth considering to stop the progressive destruction of the targets of SjS-specific autoantibodies. However, the postoperative symptoms may not dramatically improve because the target organs might have changed irreversibly, and memory B cells might persist. This is the first report that demonstrated the SS-A52 antigen presentation in a thymic tumor to the best of our knowledge.

T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation.

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.

Significance of anti-Ro/SSA antibodies in the response and retention of abatacept in patients with rheumatoid arthritis: a multicentre cohort study.

Objective: To determine whether the positivity of baseline anti-Ro/Sjögren's syndrome antigen A (SSA) antibodies influences the response to abatacept, we compared therapeutic responses between anti-Ro/SSA antibody-negative and -positive patients with rheumatoid arthritis (RA) using a multicentre RA ultrasonography prospective cohort. Method: We reviewed Japanese patients with RA who started abatacept as the first biological disease-modifying anti-rheumatic drug between June 2013 and April 2018. We assessed 28-joint Disease Activity Score-erythrocyte sedimentation rate (DAS28-ESR) change between baseline and 6 or 12 months after treatment in RA patients treated with abatacept, and European League Against Rheumatism (EULAR) response at 6 and 12 months. The Global OMERACT-EULAR Synovitis Score (GLOESS) was calculated at baseline and at 6 and 12 months. Results: Overall, 51 patients were enrolled and divided into anti-Ro/SSA antibody-negative and -positive groups of 35 and 16, respectively. Median age at baseline was significantly higher in the anti-Ro/SSA antibody-negative group (p = 0.04). The retention rate and percentage of EULAR good responders at 12 months were significantly higher in the anti-Ro/SSA antibody-negative group (both p = 0.02). Anti-Ro/SSA antibody-negative patients exhibited larger decreases in both DAS28-ESR and DAS28-C-reactive protein at 12 months than anti-Ro/SSA antibody-positive patients (p = 0.02 and 0.04, respectively). GLOESS decreased significantly at 6 months in anti-Ro/SSA antibody-negative patients (p = 0.03). Multivariate analyses showed that anti-Ro/SSA antibody positivity was an independent factor associated with change in the DAS28-ESR at 6 months (p < 0.05). Conclusion: Anti-Ro/SSA antibody positivity predicts a poor response to abatacept and low retention rate.

Identification of a unique anti-Ro60 subset with restricted serological and molecular profiles.

Anti-Ro60 is one of the most common and clinically important serum autoantibodies that has a number of diagnostic and predictive capabilities. Most diagnostic laboratories report this simply as a qualitative positive/negative result. The objective of this study was to examine the clinical and serological relevance of a novel subset of anti-Ro60 in patients who display low levels of anti-Ro60 (anti-Ro60low ). We retrospectively identified anti-Ro60 sera during a 12-month period at a major immunopathology diagnostic laboratory in Australia. These all were anti-Ro60-precipitin-positive on the diagnostic gold standard counter-immuno-electrophoresis (CIEP). Lineblot immunoassay was used to stratify patients into either anti-Ro60low or anti-Ro60high subsets. We compared the medical and laboratory parameters associated with each group. Enzyme-linked immunosorbent assay (ELISA) and mass spectrometry techniques were used to analyse the serological and molecular basis behind the two subsets. Anti-Ro60low patients displayed less serological activity than anti-Ro60high patients with less intermolecular spreading, hypergammaglobulinaemia and less tendency to undergo anti-Ro60 isotype-switching than anti-Ro60high patients. Mass spectrometric typing of the anti-Ro60low subset showed restricted variable heavy chain subfamily usage and amino acid point mutations. This subset also displayed clinical relevance, being present in a number of patients with systemic autoimmune rheumatic diseases (SARD). We identify a novel anti-Ro60low patient subset that is distinct from anti-Ro60high patients serologically and molecularly. It is not clear whether they arise from common or separate origins; however, they probably have different developmental pathways to account for the stark difference in immunological maturity. We hence demonstrate significance to anti-Ro60low and justify accurate detection in the diagnostic laboratory.