TYPE Mini Review PUBLISHED 17 October 2022 DOI 10.3389/fimmu.2022.1003054 Not all autoantibodies are OPEN ACCESS clinically relevant. Classic EDITED BY Betty Diamond, Feinstein Institute for Medical and novel autoantibodies Research, United States REVIEWED BY in Sjögren’s syndrome: Athanasios G. Tzioufas, National and Kapodistrian University of Athens, Greece A critical review Astrid Rasmussen, Oklahoma Medical Research Foundation, United States Francisco Vı́lchez-Oya 1, Hector Balastegui Martin 2, *CORRESPONDENCE E. Garcı́a-Martı́nez 2 and Hèctor Corominas 3,4,5* Hèctor Corominas 1 Department of Anaesthesiology, Pain Medicine Section, Hospital Clı´nic de Barcelona, hcorominas@santpau.cat Barcelona, Spain, 2 Department of Immunology, Hospital Universitario Gregorio Marañón, Madrid, SPECIALTY SECTION Spain, 3 Department of Rheumatology and Autoimmune Diseases, Hospital de la Santa Creu i Sant, This article was submitted to Barcelona, Spain, 4 Department of Medicine, Universitat Autònoma de Barcelona (UAB), Barcelona, Autoimmune and Autoinflammatory Spain, 5 Institut d'Investigació Biomèdica Sant Pau (IIB SANT PAU), Barcelona, Spain Disorders: Autoimmune Disorders, a section of the journal Frontiers in Immunology RECEIVED 25 July 2022 ACCEPTED 20 September 2022 Sjögren’s syndrome (SjS) is a heterogeneous systemic disease. The abnormal PUBLISHED 17 October 2022 responses to La/SSB and Ro/SSA of both B-cells and T-cells are implicated as CITATION Vı´lchez-Oya F, Balastegui Martin H, well as others, in the destruction of the epithelium of the exocrine glands, whose Garcı´a-Martı´nez E and Corominas H tissue characteristically shows a peri-epithelial lymphocytic infiltration that can vary (2022) Not all autoantibodies are from sicca syndrome to systemic disease and lymphoma. Despite the appearance clinically relevant. Classic and novel autoantibodies in Sjögren’s of new autoantibodies, anti-Ro/SSA is still the only autoantibody included in the syndrome: A critical review. American College of Rheumatology/European League Against Rheumatism (ACR/ Front. Immunol. 13:1003054. doi: 10.3389/fimmu.2022.1003054 EULAR) classification criteria and is used extensively as a traditional biomarker in clinical practice. The study and findings of new autoantibodies in SjS has risen in COPYRIGHT © 2022 Vı´lchez-Oya, Balastegui Martin, the previous decade, with a central role given to diagnosis and elucidating new Garcı´a-Martı´nez and Corominas. This is aspects of SjS physiopathology, while raising the opportunity to establish clinical an open-access article distributed under the terms of the Creative Commons phenotypes with the goal of predicting long-term complications. In this paper, we Attribution License (CC BY). The use, critically review the classic and the novel autoantibodies in SjS, analyzing the distribution or reproduction in other methods employed for detection, the pathogenic role and the wide spectrum of forums is permitted, provided the original author(s) and the copyright clinical phenotypes. owner(s) are credited and that the original publication in this journal is KEYWORDS cited, in accordance with accepted academic practice. No use, autoimmunity, Sjögren's syndrome, anti-Ro/SSA antibodies, Sjögren, antinuclear distribution or reproduction is antibodies permitted which does not comply with these terms. Frontiers in Immunology 01 frontiersin.org Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 Introduction decreased competition and resulting in the escape of autoreactive B-cells (6). Consistent with this finding, increased Sjögren’s syndrome (SjS) is a systemic autoimmune disease levels of BAFF in serum of SjS patients is correlated with the which characteristically presents organ lymphocytic infiltration presence of anti-Ro/SSA and anti-La/SSB autoantibodies (7). and has a specific predisposition for the exocrine glands. Its Serum autoantibodies are present in most patients with SjS, main consequence is the development of sicca symptoms, and some show a strong association with specific clinical principally in the form of xerostomia and keratoconjunctivitis features, possibly contributing directly to the phenotype of sicca (1). Nonetheless, a third of patients with long-standing SjS individual patients, though SjS classification criteria include suffer from different systemic complications such as just anti-Ro/SSA and anti-La/SSB (8, 9). neurological, pulmonary or nephrological manifestations and Recently, the research seeking novel autoantibodies in SjS nearly 5% of them end up developing lymphoma (2). has increased, opening up a wide range of clinical phenotypes in Multiple factors are involved in the pathogenesis of SjS and it an attempt to predict long-term complications (8). is triggered in individuals with genetic predisposition by In this review, we will analyze different SjS autoantibodies, environmental factors. The core components of the disease including old and newly identified biomarkers and discuss their process are autoimmunity and chronic inflammation utility as diagnostic tools, the pathogenic role and their secondary to the activation of an innate and adaptive immune association with some clinical phenotypes. response (1, 2). It has been proven that the innate immune system plays a paramount role in early SjS through a type I interferon (IFN), Classic autoantibodies in Sjögren’s cytokines such as IL-21 and the B-cell-activating factor (BAFF). syndrome The initial tissue and cell damage caused by different factors results in the production of IFN I and II in a first stage, inducing Anti-nuclear antibodies (ANA) in turn the production of BAFF, which is responsible for the activation of autoreactive B-cells, among other processes. Thus, ANA are antibodies that target antigens in the nucleus of the the TNF cytokine family connects innate immunity with the cell. The gold standard method used for screening to detect ANA autoimmune activation of B-cells. Other cytokines, such as IL- is the indirect immunofluorescence assay (IIFA) on HEp-2 cells, 12, are also central to its pathogenesis, by the activation of the which is sensitive but not specific. The HEp-2 IIFA test provides type II IFN system through the innate immune system (natural further information than just whether or not autoantibodies are killers’ cells), as well as the adaptive one via type 1 T-helper cells. found, including antibody titration and the HEp-2 IIFA pattern In addition, many other molecules are also important in the (10). Higher antibody levels are associated with systemic pathogenesis, such as IL-21, with a role in the regulation of B- autoimmune rheumatic diseases (SARD) and are more likely cells and follicular cells, and the CXCR5-CXCL13 axis, with a to identify a specific autoantigen during patient follow-up key role in lymphocyte recruitment and possibly, in the (10–12). It is of importance that ANAs are widespread in the formation of ectopic germinal centers (GCs) (2). healthy population and are not suitable for screening in B-cells up-regulation is a crucial feature of SjS, reflected by connective tissue diseases, thus a positive result for a specific the broad array of autoantibodies found in the serum of those ANA in the absence of a clinical context or clinical findings is patients and confirmed by the presence of ectopic GCs in the not equivalent to the diagnosis of a connective tissue disease. affected organs tissue (2–4). The development of ectopic GCs on To summarize the classification and descriptions of the the site of inflammation, often within the salivary glands, has specific patterns of HEp-2 IIFA, the international consensus been associated with a higher frequency of local production of on ANA patterns (ICAP) established each pattern and sub- anti-Ro/SSA and anti-La/SSB autoantibodies in SjS patients (5). pattern, which were identified with an anti-cell (AC) pattern Ectopic GCs are functional structures that offer all the suitable code (AC-1 to AC-29) (10, 13). The HEp-2 IIFA antibody machinery necessary for the activation of autoreactive B-cells patterns, their specific antigens and their clinical relevance in and the production of autoantibodies. A complex array of SARD are summarized in Table 1. cytokines and immune cells are thought to be involved in the ANA have been found to be positive in 59 to 85% of SjS formation of these structures. BAFF cytokine, a key molecule in patients in whom a higher prevalence of recurring SjS pathogenesis, is produced by infiltrated immune cells in parotidomegaly and a heightened frequency of extra-glandular salivary glands and regulates B-cells activation, proliferation symptoms are observed (14, 15). and, importantly, B-cells selection through a ligand The naming of the autoantigens is based on their competition mechanism. Contrary to what happens in the biochemical structure or the associated disease, and the bone marrow, BAFF has an effect in B-cells tolerance at the antigens in Sjögren’s syndrome are named SS-A/B. The periphery, with increased levels of circulating BAFF leading to a frequency of elevated ANA titres in SjS is 40-95%. In Frontiers in Immunology 02 frontiersin.org Frontiers in Immunology Vı́lchez-Oya et al. TABLE 1 HEp-2 cell IIFA patterns and their correlated clinical relevance in Systemic Autoimmune Rheumatic Diseases (SARD) (10, 13). Code AC pattern Specific antigens Clinical relevance Nuclear patterns AC-1 Homogeneous Nucleosome (dsDNA, ssDNA, histone) SLE, chronic autoimmune hepatitis, juvenile idiopathic arthritis. AC-2 Dense fine speckled DSF70/LEDGF Healthy individuals or patients without systemic autoimmune rheumatic diseases (SARD). AC-3 Centromere CENP-A/B Limited cutaneous SSc. AC-4 Fine speckled SS-A/B, Mi-2, TIF1gamma, TIF1betta, Ku SjS, SLE, subacute cutaneous lupus erythematosus, neonatal lupus erythematosus, congenital heart block, DM, SSc and SSc-AIM overlap syndrome. AC-5 Large/coarse speckled Sm, U1RNP, RNA polymerase III SLE, SSc, MCTD, SSc-AIM overlap syndrome, UCTD. AC-6 Multiple nuclear dots Sp-100, PML proteins, MJ/NXP-2 PBC, AIM (DM) and other inflammatory conditions. AC-7 Few nuclear dots P80-coilin, SMN Low positive predictive value for any disease. AC-8 Homogeneous PM/Scl-75, PM/Scl-100, Th/To. B23/nucleophosmin, nucleolin, No55/ SSc, SSc-AIM overlap syndrome. nuclear SC65 AC-9 Clumpy nucleolar U3-RNP/fibrilarin SSc. AC-10 Punctate nucleolar RNA polymerase I, NOR-90 SSc, Raynaud’s phenomenon, SjS and cancer. AC-11 Smooth nuclear Lamins A, B, C lamin-associated proteins Autoimmune cytopenias, autoimmune liver diseases, linear scleroderma, APS and other SARDs. envelope AC-12 Punctate nuclear Nuclear pore complex proteins (gp210, p62, LBR, Tpr) PBC, autoimmune liver diseases. envelope 03 AC-13 PCNA-like PCNA SLE (debated). AC-14 CENP-F like CENP-F Neoplastic conditions (breast, lung, colon, lymphoma, ovary, brain). AC-29 TOPOI-like DNA-topoisomerase/SCL70 SSc (particularly with diffuse cutaneous SSc and more aggressive forms). Cytoplasmatic patterns AC-15 Fibrillar linear F-actin, non-muscle myosin AIH type 1, chronic HVC infection, celiac disease. Rare in SARD. AC-16 Fibrillar filamentous Cytokeratin, vimentin, tropomyosin Not typically found in SARD. AC-17 Fibrillar segmental Alpha-actinin, vinculin SLE, AIH type I, chronic inflammatory demyelinating neuropathy. AC-18 Discrete dots GW (182) Neurological symptoms in a variety of diseases. AC-19 Dense fine speckled PL-7, PL-12, ribosomal P proteins SLE, anti-synthetase syndrome, interstitial lung disease, polyarthritis, Raynaud’s phenomenon, mechanic’s hands. AC-20 Fine speckled Jo-1 Anti-synthetase syndrome, interstitial lung disease, polyarthritis, mechanic’s hands. AC-21 Reticular/AMA Mitochondrial structures: PDC-E2/M2, BCOADC-E2, E1alpha, E3BP/ PBC, SSc, PBC-SSc overlap syndrome and PBC-SjS overlap syndrome. 10.3389/fimmu.2022.1003054 protein X AC-22 Polar/Goldi-like Golgi structures: giantin/macrogolgin, golgin-95/GM130, golgin-160, Small number of patients with: SjS, SLE, RA, MCTD, GPA, adult onset Still’s disease, viral infections. golgin-97, golgin-245 AC-23 Rods and rings IMPDH2 HCV patients treated with pegylated interferon-alpha/ribavirin combination therapy. frontiersin.org (Continued) Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 AIM, autoimmune myopathy; AMAs, antimitochondrial antibodies; APS, antiphospholipid syndrome; CENP, centromere-associated protein; DFS, dense fine speckled; DM, dermatomyositis; ENA, extractable nuclear antigens; HCV, hepatitis C virus; IIFA, erythematosus; SMN, survival of motor neuron; SSc, systemic sclerosis; SjS, Sjoügren’s syndrome; TIF, transcription intermediary factor; TRIM, tripartite motif; Tpr, translocated promoter region; UCTD, undifferentiated connective tissue disease; ssDNA, single stranded DNA; dsDNA, double stranded DNA; hUBF, human upstream binding factor; AIH, autoimmune hepatitis; Ago, argonaute protein; CLIP, class II-associated invariant chain peptide; EEA, early endosome antigen; SRP, signal recognition indirect immunofluorescence assay; LAP, lamin-associated polypeptide; LBR, lamin B receptor; LEDGF, lens epithelial derived growth factor; NOR, nucleolus organiser region; NXP, nuclear matrix protein; PBC, primary biliary cholangitis; PCNA, proliferating cell nuclear antigen; PML, promyelocytic leukaemia; PM/Scl, polymyositis-scleroderma; RA, rheumatoid arthritis; RNApol, RNA polymerase; RNP, ribonucleoprotein; SARD, systemic autoimmune rheumatic diseases; SLE, systemic lupus protein; tRNA, transfer ribonucleic acid; Cep, centrosomal protein; DCA, dividing cell antigen; INCENP, inner centromere protein; KIF, kinesin family; MCA, mitotic chromosomal antigen; MKLP, mitotic kinesin-like protein; MPP, M-phase suspected SjS patients, one of the most characteristic patterns is AC-4 (nuclear fine speckled), in which case it would be advisable to have follow-up tests done in search of anti-Ro/SSA and anti- La/SSB antibodies (10). Figure 1 shows different ANA patterns Low positive predictive value for any disease.Very rare: SSc, SLE, Raynaud’s phenomenon and malignancy. of primary SjS patients. Low positive predictive value for any disease.Low percentage in SSc, SLE, Raynaud’s phenomenon. Anti-Ro/SSA Throughout many autoimmune diseases, anti-Ro/SSA antibodies appear to be the most prevalent specificity. Specially among patients with SjS and in the lupus spectrum (SLE, Clinical relevance Low positive predictive value for any disease.SjS and SLE (not specific). phosphoprotein; MSA, mitotic spindle apparatus; NMP, nuclear matrix protein; NuMA, nuclear mitotic apparatus; PCM, pericentriolar material; UCTD, undifferentiated connective tissue disease. cutaneous lupus, congenital heart block and neonatal lupus). The Ro antigen consists of two proteins that weight 52 and 60 kDa. In humans, Ro 60 kDa is a ribonucleoprotein which forms small cytoplasmic complexes with non-coding RNAs (known as Y RNA). Among its functions, it mediates the quality control of misfolded non-coding RNAs and is involved in diverse cellular- stress responses such as survival after ultraviolet radiation damage (16). Ro 52-kDa (or TRIM21) is a E3 ubiquitin ligase implicated in the ubiquitination of many inflammatory related SjS, SLE, UCTD, limited SSc, RA. proteins like IRF transcription factors associated to IFN-I pathway. In addition, acts as cytosolic Fc receptor, being able to bind diverse isotype antibodies and playing a role in Nuclear patterns intracellular antibody-mediated immunity (17, 18). The SSc (difuse). human 60 kDa Ro protein is encoded by a 1.8 kb gene in chromosome 19 which possesses an RNA binding site, whereas the gene responsible for the 52 kDa Ro fraction is located in chromosome 11 and lacks any specific RNA binding domain (19, 20). Although RNA precipitation assay has showed the highest CENP-E, CENP-F, TD60, MSA36, KIF-14, MKLP-1, MPP1/KIF20B. specificity and sensitivity to detect anti-Ro/SSA as well as anti- La/SSB, it can’t be used in routine analysis, in spite of its usefulness as a reference and confirmation assay (19, 20). Therefore, counter-immunoelectrophoresis (CIE) is regarded as the most specific (100%) and sensitive (89%) assay in order Specific antigens to find anti-Ro/SSA antibodies, and better than enzyme-linked immunosorbent assays (ELISA) or the Immunoblotting (IB) assay (19, 21). Pericentrin, ninein, Cep250, Cep110 ELISA is frequently used in order to detect these antibodies, AC-28 Mitotic chromosomal DNA-topoisomerase/SCL70 since they are straightforward to perform and the interpretation of the results is fast. However, ELISA is not more specific than other kind of analyses. Depending on the commercial kit, various types of antigens, such as recombinant, native proteins and synthetic peptide fragments are used for the detection of NuMA HsEg5 SSA/SSB antibodies. Nevertheless, their sensitivity and specificity may vary notably, from 39 to 77% and from 79 to 100% respectively, and most of the ELISAs detecting anti-Ro/ AC-27 Intercellular bridge AC pattern SSA and anti-La/SSB antibodies are generally specific for a single TABLE 1 Continued AC-25 Spindle fibers AC-24 Centrosome target, in spite of a few assays being able to detect several AC-26 NuMA-like antibodies (22). The Immunoblotting (IB) assay showed high specificity for anti-60 and anti-52 kDa (97 and 95% respectively), but lower overall sensitivity (17 and 36% respectively) (19, 20). Code Meanwhile, line immunoassay (LIA), an enzyme-linked Frontiers in Immunology 04 frontiersin.org Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 FIGURE 1 Different ANA patterns of primary SjS patients. (A) ANA IFI: Fine speckled (AC-4) 1/160. Negative specificity study for extractable nuclear antigens (ENAs). (B1, B2) ANA IFI: nuclear dots/nucleolar pattern (AC-7/10) 1/80; it also presents a minor reticular cytoplasmic pattern (AC-21) compatible with anti-mitochondrial antibodies. Organ-specific antibodies: specifity M2/nPCD. (C) Patient with anti-Ro/SSA antibody (Ro-52 kDa) and negative ANA-IFI on HEP2-cells. immunosorbent assay, showed a much better sensitivity (91.4%) Anti-La/SSB and specificity (87%) for detecting anti-Ro/SSA (19, 23). In clinical practice, anti-Ro/SSA antibodies can be associated The La/SSB antigen is a phosphorylated protein which with a wide range of SARDs, such as subacute cutaneous lupus, weighs 48 kDa. It can be found in the nucleus and neonatal lupus, systemic lupus erythematous (SLE), SjS, SjS/SLE the cytoplasm and binds to several RNA molecules (19). overlap syndrome, myositis, rheumatoid arthritis (RA) and It participates in a broad spectrum of RNA metabolism primary biliary cirrhosis (24–28). Anti-La/SSB and anti-52 Ro processes as protecting nascent RNA polymerase III antibodies are both directly implicated in the pathogenesis of transcripts from exonuclease digestion by binding to their neonatal lupus. In this context, fetal complete heart block is due poly(U) termini, processing of 5’ and 3’ ends of pre-tRNA to tissue injury mediated by the expression of Ro (both 52 and 62 precursors and resolving misfolded RNA structures by acting kDa fractions) and La antigens in the cardiac tissue, particularly as RNA chaperone (33). IgG anti-La/SSB antibodies are the located on the surface of myocardial cells’, from the 18th to the major antibody class found in serum and it has been observed 24th week. Although the main autoantibody involved in the its strong association with anti-Ro/SSA antibodies. The latter neonatal pathogenesis is anti-Ro 52 (19, 29). can be found on their own in many patients’ sera, whereas In SjS, anti-Ro/SSA and anti-La/SSB antibodies have been anti-La/SSB are generally found together with anti-Ro/SSA. associated with diagnoses at a younger age, recurring The presence of anti-La/SSB without anti-Ro/SSA antibodies parotidomegaly, more severe dysfunction of the exocrine hasn’t got any significant association with SjS phenotypic glands, a more intense lymphocytic infiltration of the minor characteristics (34). salivary glands and longer disease duration (30, 31). In addition, Anti-Ro/SSA and anti-La/SSB antibodies are commonly some authors suggest a higher prevalence of extra-glandular detected in patients suffering from cryoglobulinemia, manifestations, for instance Raynaud’s phenomenon, vasculitis, hypergammaglobulinemia and in the presence of rheumatoid splenomegaly and lymphadenopathy (30, 32). factor independently of autoimmune disease (19, 35). Frontiers in Immunology 05 frontiersin.org Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 Autoantibodies in Sjögren’s CENP recognition patterns in SjS and SSc are dissimilar. Most SjS sera recognize CENP-C alone (70% of SjS versus 6% of SSc), syndrome associated with whereas most SSc sera recognize CENP-B as well as CENP-C overlap syndromes (83% of SSc versus 0% of SjS) (38, 46). Regarding immunological and clinical features, some Anti-mitochondrial antibodies (AMA) studies have shown that seropositive ACA patients with SjS have a higher disease onset mean age, a higher frequency AMAs are the serologic distinguishing finding of primary of Raynaud’s phenomenon, peripheral neuropathy and biliary cholangitis (PBC). AMAs are found in 95% of patients keratoconjunctivitis sicca compared to seronegative patients with PBC with a specificity of 98% for the disease (36). (38, 47, 48). Moreover, ACA positive SjS patients showed an There are four main autoantigens targeting antimitochondrial inferior prevalence of anti-Ro/SSA and anti-La/SSB antibodies, a antibodies, referred to as an M2 subtype of mitochondrial lower frequency of cytopenia, inferior rates of rheumatoid factor autoantigens and which are identified as the E2 subunits and hypergammaglobulinaemia (38, 49). of the pyruvate dehydrogenase complex, the ketoglutaric The singular features presented in ACA positive SjS patients acid dehydrogenase complex, the branched chain 2-oxo- suggests that they could be a SjS clinical subset with some acid dehydrogenase complex and the dihydrolipoaminade overlapping traits of SjS and SSc (38). Interestingly, a study of dehydrogenase-binding protein (37). When IIFA is used, between SjS patients by capillaroscopy showed that those patients with 1.7 and 13% of SjS patients test positive for AMA. However, the ACA had a scleroderma-like pattern in 10.2%, showing dilated positivity rate is higher (3 to 27%) if Western Blot or ELISA is used capillaries, low capillary density and pericapillary haemorrhages, (38–40). Some SjS patients with a positive AMA test, besides raising the suspicion of an overlapping syndrome between SjS developing more frequently hepatic manifestations, show also a and SSc (50). The percentage of patients progressing to SSc span higher prevalence of hypergammaglobulinaemia, peripheral from 0 to 40% according to different cohorts (38, 49, 51), even neuropathy, Raynaud’s phenomenon and higher titer in the though more accurate and prospective design studies are needed erythrocyte sedimentation rate (ESR) (14, 38). to determine it. The similarity of the histological lesion in PBC and SjS is noteworthy. Both are characterized by lymphocytic infiltrates with a predominance of CD4+ cells that initiate around the ductal epithelium (salivary or bile ducts). Hence, both entities Anti-citrullinated peptide antibodies may share some pathogenetic mechanisms within the context of (ACPAs) some kind of autoimmune epithelitis, in spite of their different autoantibody profiles (38, 41). ACPAs are autoantibodies targeting proteins that have been citrullinated by a calcium-dependent enzyme family of peptidylarginine deiminases. Its specificity in RA is around Anti-centromere antibodies (ACA) 95% and it identifies a phenotype of patients affected by a more serious clinical profile and erosive disease (52). It is ACAs are a heterogeneous group of antibodies targeting widely known that SjS may exist together with other different antigens clustered around the kinetochore plates (42). autoimmune diseases, being autoimmune hypothyroidism the In systemic sclerosis (SSc), ACAs recognize three centromeric most frequent, followed by RA (53). In patients with SjS, the proteins (CENP): CENP-A (19 KDa), CENP-B (89 KDa) and prevalence of ACPAs ranges between 3 and 9.9%, but no CENP-C (140 KDa). Anti-CENP-B autoantibodies seem to have agreement exists regarding its clinical significance (54–58), a greatest relevance for clinical practice among the various ACA, although a small number of authors have described an with a sensitivity of 20 to 30% for SSc (42). Among SARD, ACAs association with non-erosive arthritis (54, 55, 57). are also present in approximately 15% of patients with PBC and Some reports have described a low progression to RA in this association has been described with worsening outcomes cohorts of patients with SjS during a long-term follow-up (59, compared in those seronegative patients (43). 60). Nonetheless, it has been suggested that ACPAs positive SjS In SjS, the prevalence of ACAs ranges from 3.7 to 27% when patients may have a higher risk of progression to RA (57, 61). IIFA is used to detect them, but when ACAs are detected by Thus, ACPAs constitute a valuable tool for the accurate control other methods its prevalence varies between 20 and 27%, of this subgroup of patients in order to ensure their immediate depending on which CENP is used as target antigen (44–46). diagnosis and treatment (62). Frontiers in Immunology 06 frontiersin.org Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 Novel autoantibodies in Sjögren’s In Table 2, we summarize the novel autoantibodies found in SjS patients that may be associated with some clinical features. syndrome As previously commented, classical autoantibodies have been used extensively in the setting of SjS diagnosis and Other relevant antibodies: currently, anti-Ro/SSA is widely accepted and considered Rheumatoid factors and useful for SjS classification criteria as a diagnostic tool (9). cryoglobulins In recent years, several studies have analyzed new autoantibodies, opening up a window of opportunities for the Rheumatoid Factors (RF) and cryoglobulins, require special diagnostic approach, trying to discern the pathologic attention for their important clinical implications in SjS, partly due attributions of disease-associated autoantibodies and providing to their relationship with potentially serious disease complications. the opportunity to identify pre-clinical subjects before SjS onset (8). Despite the fact that most of these antibodies are infrequent Rheumatoid Factors (RF) and, may even be shared with other connective tissue diseases, their study may help to elucidate some of the clinical features of patients Rheumatoid factors (RF) are autoantibodies targeting the with SjS and help to tackle potentially serious complications in those constant part (Fc) portion of other immunoglobulins. The most patients with suspected or confirmed SjS, as well as proving to be distinguished are IgM and IgA binding to the Fc portion of useful in order to reach a diagnosis when other autoantibodies turn IgG (95). out to be negative. Among the antibodies that we will discuss are RF levels are found in 36 to 74% of SjS patients with an anti-alphafodrin, anti-muscarinic type 3 receptor (M3R), anti- increased prevalence during follow-up and have been salivary gland protein 1 (SP1), anti-carbonic anhydrase, anti- unmistakably associated with the number of extra glandular parotid secretory protein (PSP), anti-interferon-inducible protein- features (14, 32). The wide spectrum of clinical manifestations in 16, anti-NA-14, anti-MDM2, anti-stathmin-4, anti-PUF60, anti- patients with RF includes a higher frequency of articular NR2, anti-TRIM38, anti-calponin-3, anti-saccharomyces cerevisiae damage, Raynaud’s phenomenon, parotidomegaly, cutaneous (ASCA), anti-aquaporin (AQ), anti-ganglionic acetylcholine vasculitis, cytopenias, renal involvement and central nervous receptor (gAChR), anti-P-selectin, anti-moesin, anti-carbamylated, system manifestations (32, 96). In addition, RF levels are anti-alpha-enolase, anti-cofilin-1 and anti-Rho GDP-dissociation correlated to an active serological profile with higher presence inhibitor 2 (RGI2) (63–92). of some antibodies, including ANA, anti-Ro/SSA, anti-La/SSB In addition, it is worth highlighting two ANA patterns and cryoglobulins, as well as hypergammaglobulinaemia and mentioned in Table 1 and observed in patients with SjS that may hypocomplementemia (14, 32, 38). RF have been associated with have additional clinical value in the spectrum of clinical diagnoses at a younger age and with lymphocytic infiltration in manifestations. These are the antibodies for nuclear transcription the biopsy of salivary glands. This last is associated with the factor NOR 90/hUBF (anti-NOR 90) and anti-NuMA antibodies. lymphomagenesis process related to the role of monoclonal RF Anti-NOR 90, are antinuclear antibodies that recognize the distal secreted by the B-cells through the chronic stimulation at the ends of the short arms of chromosomes 13, 14, 15, 21 and 22 target organ (95). (nuclear organizer region) on immunofluorescence analysis and 90- kd doublet proteins on immunoblot analysis (93). Fujii T et al. identified that anti-NOR 90 antibodies are rare, although they are Cryoglobulins associated with SjS and overlap syndromes (SjS-RA and SjS-SSc) in Japanese patients. Anti-NuMA antibodies (antinuclear mitotic Cryoglobulinemia is characterized by the precipitation of apparatus), although rare, are mostly associated with SLE and SjS, circulating immunoglobulins at temperatures down 37°C. and could be useful in order to reach a diagnosis when other Serologically, it is classified into three subtypes according to autoantibodies are negative. Those patients’ both clinical and the composition of the immunocomplex. Thus, it is biological profiles were milder, suggesting that these antibodies differentiated as type I cryoglobulinemia (monoclonal may imply a good prognosis marker in both syndromes. SjS patients immunoglobulins), type II (mixed cryoglobulinemia with anti-NuMA antibodies presented fewer ocular sicca syndrome containing monoclonal and polyclonal immunoglobulins) and and dryness complications and anti-Ro/SSA and/or anti-La/SSB type III (mixed cryoglobulinemia containing only polyclonal antibodies were less frequently present. Anti-NuMA positive immunoglobulins). Type II cryoglobulinemia is commonly patients received antimalarial agents less frequently than negative associated with infectious diseases, with special mention to patients, with no difference regarding other treatments (94). hepatotropic viruses, while type III cryoglobulinemia is most Frontiers in Immunology 07 frontiersin.org Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 TABLE 2 Novel autoantibodies in Sjögren’s syndrome. Author Number Autoantibodies Technique Prevalence Sensitivity Clinical features of and patients specificity Hu Q at al. Meta- Anti-alpha fodrin Immunoblot and ELISA 38-42% Sensitivity Moderate accuracy for the diagnosis of (63) analysis 39.3% SjS.Clinical manifestations were not evaluated. Specificity 83% Willeke P 62 ELISA 31-35% Sensitivity 31- Shorter disease duration. Increased prevalence of et al. (64) 35%Specificity recurrent parotid swelling with IgG isotype. unknown Mona M 156 Anti-muscarinic On-Cell-Western assay N/A Sensitivity 75- Correlated with ocular dryness and glandular et al. (65) type 3 receptor 98%Specificity hypofunction and the haematological/biological (M3R) 85% domains of ESSDAI. Useful in SjS diagnosis, especially where clinical assessments are limited. Deng C 956 ELISA N/A Sensitivity 4- Potential diagnostic biomarker for SjS.Clinical et al. (66) 98%Specificity features were not evaluated. 58-100% Shen L 123 Anti-salivary gland ELISA 19% isolated34% N/A Associated with anti-Ro/SSA and anti-La/SSB. No et al. (67) protein 1 (SP1) associated to other distinct clinical manifestations were identified in autoantibodies. patients expressing anti-SP1. Xuan J 134 Western-blot 40% N/A Higher levels during earlier stages of the disease. et al. (68) Karakus S 136 N/A 27 % N/A Dry eye. Correlated with having a Schirmer test ≤ et al. (69) 5 mm. Karakus S 136 Anti-carbonic N/A 27% (CA6)54% (PSP) N/A Anti-CA6 was associated with severe ocular et al. (69) anhydrase 6 (CA6) surface staining (corneal and conjunctival). Anti- and anti-parotid CA6 may indicate early stages of SjS.Anti-PSP secretory protein was the only autoantibody that correlated with (PSP) primary SjS. Pertovaara 74 Carbonic ELISA N/A N/A CA-II, CA-VI and CA XIII (associated with renal M et al. anhydrase auto- manifestations).CA-VII and CA-XIII (correlated (70) antibodies (CA) to b2 microglobulin)CA-I (oral dryness and associated with interstitial lung disease in other connective tissue diseases). Alunno A 30 Anti-Interferon- ELISA 33% N/A Pathogenesis of glandular inflammation. et al. (71) Inducible Protein- Baer AN 133 16 ELISA 29% N/A Severe disease: greater prevalence of abnormal et al. (72) Schirmer’s test, ANA >1:320 and germinal center-like structures in the labial salivary gland lymphocytic infiltrates. Focus scores were significantly higher. Alunno A 67 ELISA 34% N/A Pathogenesis of glandular inflammation. et al. (73) Uomori K 72 Anti-NA-14 ELISA 11.1% N/A Elevation of IgA levels. Low prevalence of ANA et al. (74) positive patients. Disease duration tended to be shorter (although the difference did not reach statistical significance). Liu Y et al. 100 Anti-MDM2 ELISA 21% N/A Longer disease duration and more lymphocytes (75) focal gathering in labial gland. Higher prevalence of anemia, thrombocytopenia and anti-Ro/SSA. Duda S 72 Anti-stathmin-4 ELISA 33% (pSS with PNP) N/A Polyneuropathy. et al. (76) 7.8% (pSS without PNP)15% in sSS (Continued) Frontiers in Immunology 08 frontiersin.org Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 TABLE 2 Continued Author Number Autoantibodies Technique Prevalence Sensitivity Clinical features of and patients specificity Zhang YM 79 Anti-PUF60 ELISA and 10.1% N/A Overlap syndrome with myositis. et al. (77) immunoblotting Fiorentino 84 ELISA 30% Specificity May be more associated with Asian and African- DF et al. 29% American ethnicity, hypergammaglobulinemia, (78) anti-Ro/SSA, anti-La/SSB and rheumatoid factor. Lauvsnes 66 Anti-NR2 ELISA in serum and 20% N/A Cognitive disturbances and mood disorders. MB et al. electrochemiluminescence (79) in CSF Lauvsnes 50 Electrochemiluminescence 12% N/A Loss of hippocampal gray matter. MB et al. in CSF (80) Tjensvoll 71 Electrochemiluminescence N/A N/A Cognitive impairment. AB et al. in CSF (81) Wolska N 235 Anti-TRIM38 TNT Quick coupled 10.21% N/A Higher severity of disease: severe sialadenitis, et al. (82) transcription/translation higher van Bijsterved scores and lower Schirmer’s system and test scores. immunoprecipitation assay Birbaum J 209 Anti-calponin-3 ELISA 11% N/A Neuropathy. et al. (83) Alunno A 104 Anti- Immunodot test 4.8% Very low Patients displayed a triple combination of et al. (84) saccharomyces sensitivity, circulating anti-Ro60/SSA, anti-Ro/52/SSA and cerevisiae (ASCA) 100% anti-La/SSB antibodies associated with low specificity complement and cutaneous involvement. Birnbaum 109 Anti-aquaporin Fluorescence-activated cell 10% N/A Neuromyelitis optica spectrum disorder. J et al. (85) (AQ) sorting (FACS) assay Alam J 112 Indirect 76.8% Sensitivity Low resting salivary flow. et al. (86) immunofluorescence assay 73%Specificity 68% Tzartos JS 34 ELISA verified by 38.2% N/A Severe xeropthalmia, suggesting a potential et al. (87) radioimmunoassay, pathogenic role. western blot and AQP- transfected cells. Mukaino 39 Anti-ganglionic LIPS assay 23.1% N/A Autonomic symptoms. et al. (88) acetylcholine receptor (gAChR) Hu YH 70 Anti-P-selectin ELISA 40.6% (SjS patients N/A May lead to platelet destruction and endothelial et al. (89) with injury. Possible role in the pathogenesis of thrombocytopenia) thrombocytopenia. and 7.8% (SjS patients without thrombocytopenia). Zhang Y 50 Anti-moesin ELISA 42% N/A N/A et al. (90) Bergum B 78 Anti-carbamylated ELISA 27% N/A Increased focal lymphocytic infiltration, et al. (91) formation of ectopic GC-like structures in minor salivary glands and diminished salivary gland function. (Continued) Frontiers in Immunology 09 frontiersin.org Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 TABLE 2 Continued Author Number Autoantibodies Technique Prevalence Sensitivity Clinical features of and patients specificity Cui L et al. 70 Anti-cofilin-1 ELISA in saliva samples N/A Sensitivity May predict progression to MALT lymphoma (92) 80%Specificity 90% Cui L. 70 Anti-alpha-enolase ELISA in saliva samples N/A Sensitivity May predict progression to MALT lymphoma et al. (92) 90%Specificity 84% Cui L. 70 Anti-Rho GDP- ELISA in saliva samples N/A Sensitivity May predict progression to MALT lymphoma et al. (92) dissociation 90%Specificity inhibitor 2 (RGI2) 80% ELISA, enzyme-linked immunosorbent assay; SjS, Sjögren’s Syndrome; ESSDAI, EULAR Sjögren’s syndrome disease activity index; ANA, antinuclear antibodies; anti-NA-14, nuclear autoantigen of 14 KDa; anti-MDM2, human homologue of mouse double minute 2; pSS, primary Sjögren ‘s Syndrome; sSS, secondary Sjögren’s Syndrome; anti-PUF60, poly(U)-binding- splicing factor 60 KDa; anti-NR2, N-methyl-D-aspartic acid receptor 2; CSF, cerebrospinal fluid; anti-TRIM38, tripartite motif-containing protein 38; LIPS assay, luciferase immunoprecipitation system assay; MALT, mucosa-associated lymphoid tissue; N/A, not available. commonly associated with autoimmune connective tissue 10-15% of patients with SLE, respectively, but anti-Ro/SSA and diseases (95). anti-La/SSB autoantibodies are not the only ones that can be Cryoglobulins differentiate a subgroup of SjS patients with a linked to SjS or SLE. To data, these autoantibodies do not worse prognosis, as they have been associated with an increased correlate with the etiopathogenesis of the disease, although in risk of lymphoproliferative disease (96). The wide spectrum of pregnancy, it may increase the risk of developing neonatal lupus clinical manifestations in patients with cryoglobulinemia (97). The detection of ANAs has been considered one of the includes a higher frequency of Raynaud’s phenomenon, most sensitive techniques in the screening of autoimmune parotidomegaly, vasculitis, cytopenias, renal involvement and diseases, but may appear related to other conditions such as peripheral neuropathy (32, 96). Cryoglobulinemia is also infectious diseases, malignancy or in healthy population. There commonly associated with the presence of other are numerous shared autoantigens between SjS and SLE, and it immunological markers such as hypocomplementemia, RF and seems that the differential pathogenic mechanism between them anti-Ro/SSA (32, 96). may be the inflammatory tropism at the glands in primary Sjögren’s Syndrome, although the molecular explanation of that is still unknown (2). For all these reasons, despite that Discussion autoantibodies are important tools in the diagnosis of autoimmune diseases, they may be analyzed in a specific The pathogenesis of SjS is still elusive, although the role of clinical context to evaluate their diagnostic value. abnormal autoreactive B-cells, which lead to the production of Antinuclear antibodies are serological biomarkers with a crucial autoantibodies and the formation of immune complexes, seems to role in the classification of systemic autoimmune rheumatic be fundamental in developing the syndrome. The presence of diseases. There is a continuous needed for harmonization of the these autoantibodies is one of the hallmarks of SjS. Amid them, methods for autoantibody determinations and in the research the most common are antibodies against Ro/SSA and La/SSB setting for the identification of novel autoantibodies, where ribonucleoprotein complexes, which are included in the several assay methods have become available in the last decades. classification criteria of the American College of Rheumatology/ The HEp-2 indirect immunofluorescence assay (IIFA) is European League Against Rheumatism (ACR/EULAR). Both broadly used in order to detect ANA, and its outcome is anti-Ro/SSA and anti-La/SSB positive SjS patients are diagnosed included in both diagnostic and classification criteria when a at a younger age, have a graver glandular dysfunction and a higher systemic autoimmune disease is suspected. We agree with frequency of extraglandular features. Damoiseaux J et al. that the HEp-2 IIFA test reveals a lot, One of the diagnostic criteria for the majority of considering the fluorescence pattern to be an important autoimmune diseases is the presence of autoantibodies, whose diagnostic tool which reveals clinically suitable information. It detection may be overlapping in the spectrum of other is important to reach a consensus in order to standardize the autoimmune diseases. As commented throughout this review, current diagnostic tools and achieve greater accuracy in our daily there are no specific autoantibodies for SjS. Anti-Ro/SSA and clinical practice, as well as aligning pattern descriptions across anti-La/SSB constitute a diagnostic marker for the disease, laboratories to homogenize the same criterion to help clinicians. however, these autoantibodies are found in up to 30-40% and We consider that the initiative by the International Consensus Frontiers in Immunology 10 frontiersin.org Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 on ANA Patterns (ICAP) eases to be more accurate and emerge number of obstacles when standardizing these new tools in daily as an important tool in the diagnostic work-up, promoting the clinical practice: issues in the design and optimization of all targets understanding of HEp-2 IIFA staining pattern nomenclature, as in a single array, the lack of available normalized standards well as optimizing usage in patient care by providing new protocols and the lack of batch-to-batch reproducibility and inter- guidelines. Despite this, ANA test results still need to answer laboratory comparison (99). On the other hand, given the some questions, methodologic consensus statements and importance of inflammatory cytokines in the pathogenesis of SjS, semantic issues. techniques with a more functional approach based on the Previous SjS reviews have focused on traditional measurement of cell expression of different immune-related autoantibodies, and then on some of the novel autoantibodies molecules such as IFN, and its correlation with the presence of described in the recent years. Thus, there are two main works certain auto-antibodies, are being studied in other rheumatic that analyze the usefulness of the different antibodies related to autoimmune diseases with interesting results related to their Sjögren’s syndrome. Firstly, Shen L et al. in 2017, discussed possible role in the autoimmunity response in those patients (100). autoantibodies including the traditional ones, those identified Our work aims to combine the information collected in initially from mouse models and those associated with other recent years regarding classic and novel autoantibodies in SjS, autoimmune diseases, examining their detection methods and with a special emphasis on the role of ANA and the HEp-2 IIFA their prevalence. Secondly, in 2019, Martı́n-Naresa E et al. staining pattern, trying to define the usefulness of “organ- mainly discussed their usefulness in order to identify a specific” biomarkers in the creation of new patient phenotypes clinical subtype. that may step up the diagnosis of SjS in the future. In this way, as The advance in biological techniques for antigen new biomarkers are identified, more subtypes of patients may be determination have resulted in the knowledge of more auto- established, with their own intrinsic features, helping other antigen specificities associated with autoimmune diseases, physicians in the clinical suspicion of SjS and additionally including SjS. Due to this heterogeneity in autoantibody guiding the clinician in the face of possible potentially serious responses and in clinical phenotypes, a broad-spectrum of complications. In this sense, we also emphasize the role of anti- diagnostic tools as antigen microarrays are increasingly Ro/SSA antibodies, review the antibodies shared with other demanded, targeting multiple specificities. autoimmune diseases and analyze the novel antibodies that Conventional techniques used in the majority of diagnostic could provide valuable information for understanding the laboratories bring different non-specific initial screening methods pathophysiology of the disease, predicting new clinical profiles based on the gold standard immunofluorescence assay of ANA in the future and probably helping in the development of new autoantibodies or ELISAs, that use antigen mixtures. Although therapeutic targets. Likewise, understanding and validating this useful for its high sensitivity, these techniques would have to be new spectrum of autoantibodies may make it possible to carry evaluated for its efficacy in detecting patients with new autoantibody out a diagnostic approach in those patients with negative anti- targets as positive. Some assays of ANA-ELISA have resolved this Ro/SSA antibodies with specific clinical phenotypes that do not issue by adding a number of extra purified recombinant antigens, as meet the American College of Rheumatology/European League is the case of some of the classical epitopes of Sjogren’s syndrome Against Rheumatism (ACR/EULAR) classification criteria. (Ro/SSA and La/SSB) and also in other rheumatic autoimmune To sum up, detecting serum antibodies is practical in order diseases (98). To further identify the specificity of auto-antibodies, to determine both diagnosis and prognosis in autoimmune the initial screening is usually followed by a number of assays based diseases. The identification of new autoantibodies in SjS opens on different technologies (i.e., line immunoassays, immunoblots or up a window of opportunity to gain a better understanding of the multiplex color-coded bead assays as Luminex®) that allow the SjS pathophysiology, to ascertain clinical phenotypes and to study of only a reduced number of auto-antibody specificities. With foresee long-term associated complications. the appearance of autoantibody arrays assays, the ability to measure The characterization of autoantibodies and their target a large number of autoantibodies with a small sample volume on a autoantigens in patients with primary SjS is helping to unravel single platform matrix, opened the possibility of reducing the time more information regarding this common systemic autoimmune and cost of performing several tests that included only small subsets disease. Any positive serological result with clinically defined of the most classical antigens. Thus, with the increasingly sicca symptoms or other extraglandular features, should raise knowledge of novel auto-antibody targets, antigen microarrays red flags in the follow up of those patients. Some of the may become an established tool for routine diagnostic procedures autoantibodies discussed in this review were identified in a in the future. Also, these new technologies provide the opportunity research environment, but in the near future they may also be of defining new autoantibody profiles and explore the relevance of used as helpful tools in common clinical practice. Many of the different isotypes, also identifying patient subsets that could autoantibodies mentioned in this review are found in correlate with different prognostic outcomes or be candidates to subpopulations of SjS patients without any specificity and their novel personalized treatments. However, currently there are a real usefulness should be balanced and powered in later studies. Frontiers in Immunology 11 frontiersin.org Vı́lchez-Oya et al. 10.3389/fimmu.2022.1003054 Author contributions Conflict of interest All authors listed have made a substantial, direct, and The authors declare that the research was conducted in the intellectual contribution to the work and approved it absence of any commercial or financial relationships that could for publication. be construed as a potential conflict of interest. Publisher’s note Acknowledgments All claims expressed in this article are solely those of the We are grateful to Dr. David Cañadas Bustos for his authors and do not necessarily represent those of their affiliated comments that greatly improved the manuscript. We also organizations, or those of the publisher, the editors and the thank to the autoimmunity section of the clinical laboratory of reviewers. 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