diagnostics Review Anti-U11/U12 Antibodies as a Rare but Important Biomarker in Patients with Systemic Sclerosis: A Narrative Review Marvin J. Fritzler 1 , Chelsea Bentow 2 , Lorenzo Beretta 3 , Boaz Palterer 4 , Janire Perurena-Prieto 5 , Maria Teresa Sanz-Martínez 5 , Alfredo Guillen-Del-Castillo 6 , Ana Marín 5 , Vicent Fonollosa-Pla 6 , Eduardo Callejas-Moraga 7 , Carmen Pilar Simeón-Aznar 6 and Michael Mahler 2, * 1 Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada 2 Research and Development, Werfen, Autoimmunity Headquarters and Technology Center, San Diego, CA 92131-1638, USA 3 Scleroderma Unit and (Referral) Center for Systemic Autoimmune Diseases, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milan, 20122 Milano, Italy 4 Department of Experimental and Clinical Medicine, University of Florence, 50121 Firenze, Italy 5 Department of Immunology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 6 Unit of Systemic Autoimmune Diseases, Department of Internal Medicine, Hospital Universitari Vall d’Hebron, 08035 Barcelona, Spain 7 Department of Internal Medicine, Hospital Público de Monforte, 27400 Lugo, Spain * Correspondence: mmahler@werfen.com or m.mahler.job@web.de Abstract: Anti-nuclear (ANA) are present in approximately 90% of systemic sclerosis (SSc) patients and are key biomarkers in supporting the diagnosis and determining the prognosis of this disease. In addition to the classification criteria autoantibodies for SSc [i.e., anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA polymerase III], other autoantibodies have been associated with important SSc phenotypes. Among them, anti-U11/U12 ribonucleoprotein (RNP) antibodies, also known as Citation: Fritzler, M.J.; Bentow, C.; anti-RNPC-3, were first reported in a patient with SSc, but very little is known about their association Beretta, L.; Palterer, B.; and clinical utility. The U11/U12 RNP macromolecular complex consists of several proteins involved Perurena-Prieto, J.; Sanz-Martínez, in alternative mRNA splicing. More recent studies demonstrated associations of anti-anti-U11/U12 M.T.; Guillen-Del-Castillo, A.; Marín, antibodies with SSc and severe pulmonary fibrosis as well as with moderate to severe gastrointestinal A.; Fonollosa-Pla, V.; Callejas-Moraga, dysmotility. Lastly, anti-U11/U12 autoantibodies have been strongly associated with malignancy in E.; et al. Anti-U11/U12 Antibodies as SSc patients. Here, we aimed to summarize the knowledge of anti-U11/U12/RNPC-3 antibodies in a Rare but Important Biomarker in Patients with Systemic Sclerosis: A SSc, including their seroclinical associations in a narrative literature review. Narrative Review. Diagnostics 2023, 13, 1257. https://doi.org/10.3390/ Keywords: systemic sclerosis; U11/U12; autoantibodies; interstitial lung disease; SSc diagnostics13071257 Academic Editors: Tania Colasanti and Katia Stefanantoni 1. Introduction Received: 11 February 2023 Systemic sclerosis (SSc, also known as scleroderma) is a chronic autoimmune disease Revised: 8 March 2023 that affects connective tissue and can cause a wide range of symptoms. The main causes Accepted: 9 March 2023 of mortality in SSc are cardiovascular disease, interstitial lung disease (ILD), and renal Published: 27 March 2023 crisis [1]. Additionally, other complications such as pulmonary hypertension, gastrointesti- nal dysfunction, and stroke can also contribute to mortality in individuals suffering from SSc. Anti-nuclear (ANA) or anti-cellular antibodies (ACA) are identified in approximately Copyright: © 2023 by the authors. 90% of SSc patients and are key biomarkers in supporting the diagnosis and determin- Licensee MDPI, Basel, Switzerland. ing the prognosis of SSc [1–3]. As well as the more well-recognized autoantibodies that This article is an open access article are included in the 2013 American College of Rheumatology (ACR)/European League distributed under the terms and Against Rheumatism (EULAR) classification criteria for SSc [3] (i.e., anti-centromere, anti- conditions of the Creative Commons topoisomerase I (Scl-70 or ATA), and anti-RNA polymerase III), other autoantibodies have Attribution (CC BY) license (https:// been associated with important SSc phenotypes (Table 1) [1]. In addition to these well- creativecommons.org/licenses/by/ known autoantibodies, several other antibodies have been specifically associated with 4.0/). SSc, including anti-U3 ribonucleoprotein (RNP) or, more specifically, anti-fibrillarin [4–6], Diagnostics 2023, 13, 1257. https://doi.org/10.3390/diagnostics13071257 https://www.mdpi.com/journal/diagnostics Diagnostics 2023, 13, 1257 2 of 13 anti-Th/To [7–11], anti-eukaryotic initiation factor 2B (eIF2B) [12], anti-RuvBL1/2 [12,13], and anti-TERF-1 antibodies [14,15]. In addition to those SSc-specific antibodies (SSc-SA), a wide range of SSc-associated antibodies have also been reported over the last decades: Anti-U1 RNP, anti-PM–Scl [16–19], anti-Ku [20], anti- Ro52/tripartite motif (TRIM) 21, and anti-human upstream binding factor (hUBF)/anti-NOR-90 antibodies [21]. In myositis, the definition of autoantibody specificities is more established. Antibodies that mostly occur in myositis are referred to as myositis specific antibodies or MSA, while antibodies that occur in myositis, but to a certain extent also in other conditions, are termed myositis associated antibodies (MAA). Here, we aim to introduce a similar nomenclature for SSc, namely SSc-specific (SSc-SA) and SSc-associated antibodies (SSc-AA). While it is well established that the classification criteria markers belong to the group of SSc-AA, for some antibodies, more studies are needed to conclude if they belong to the SSc-SA or SSc-AA group. Anti-U11/U12 RNP (also referred to as RNPC-3) antibodies were first reported in a SSc patient [22] in 1993, but very little is known about their association and util- ity [23]. The U11/U12 RNP macromolecular complex consists of several proteins and is involved in alternative mRNA splicing. Here, we aim to summarize the knowledge of anti-U11/U12/RNPC-3 antibodies in SSc, including their serological and clinical associa- tions in a narrative literature review. Table 1. Overview of autoantibodies in systemic sclerosis. Antigen Prevalence IIF Comments (Main Antigens) Topoisomerase I 20–30% AC-29 DNA topoisomerase I SSc-SA (Scl-70) Included in Centromere [24] 25–50% AC-03 CENP-A, CENP-B ACR/EULAR classification Nucleolar pattern is not reliable in RNA Polymerase III AC-10/ criteria 10–20% detecting RNAP3, often a speckled (RNAP3) AC-04/05 pattern prevails Fibrillarin [4,6] 3–20% Nu (AC-09) U3 RNP Th/To [7–11] 3–10% Nu (AC-08) Rpp25, Rpp38 SSc-SA Double hexamer of RuvBL1 and RuvBL1/2 [8,9,12,13] 1–3% Sp RuvBL2 proteins U1-RNP 70 kDa, U1 RNP-A and U1 RNP 5–15% Sp (AC-05) U1 RNP-C SSc-AA Ku [20] 3–10% Sp (AC-04) 80- and 70-kDa binding dimeric protein PM/Scl [18] 5–10% Nu (AC-08) PM/Scl-100 protein of human exosome Ro52 [25–28] 15–25% ? Ro52/tripartite motif (TRIM) 21 Sp U11/U12 RNP complex of spliceosome. U11/U12 RNP [22,29] 3–5% (AC-02/04/05) RNPC-3 is the 65 kDa main target TERF-1 [14,15] 3–5% TBD Shelterin protein TBD eIF2B [12] 1–3% Cyto Eukaryotic initiation factor 2B NOR 90 [21,30] <5% AC-10 human upstream binding factor (hUBF) B23 [30–32] <5% AC-08 Nucleophosphmin AC = anti-cell pattern by ICAP designation; NOR = nucleolar organizer region; Nu = nucleolar stain- ing pattern; RNAP = RNA polymerase; RNP = ribonucleoprotein; Sp = speckled nuclear staining pattern; SSc-SA = systemic sclerosis specific antibodies; SSc-AA = systemic sclerosis associated antibodies; TBD = to be determined; TERF-1 = telomeric repeat-binding factor 1. 2. Methods A search of Medline and Embase up to September 2022 was performed using the medical subject heading terms “anti-U11/U12”, “anti-RNPC-3”, “systemic sclerosis”, “scle- roderma”, “connective tissue disease”, to identify publications. Manual searches of refer- Diagnostics 2023, 13, 1257 3 of 13 ences cited in the retrieved articles were also performed. The eligibility criteria included: (1) studies assessing anti-U11/U12 RNP antibodies in patients with SSc; (2) studies as- sessing anti-U11/U12 RNP antibodies in patients with connective tissue diseases (CTDs); and (3) only peer-reviewed publications written in English and involving human subjects. Abstracts were excluded. No restriction on time was applied. Due to the limited number of retrieved publications, a narrative literature review was conducted. 3. Historical Perspective Although first described almost 30 years ago by Gilliam and Steitz [22], very little is known about the HEp-2 indirect immunofluorescence (IIF) pattern associated with anti- U11/U12 RNP autoantibodies (Figure 1). In the seminal paper, the index patient (Ru) was a 40-year-old Caucasian female who presented with Raynaud phenomenon followed by rapidly progressive cutaneous SSc involving her face, trunk, and extremities accompanied by mild restrictive lung disease and esophageal dysmotility (Table 2). The HEp-2 IIF result was reported as a titer of 1:650 with a nuclear speckled pattern “resembling” the IIF pattern “associated with anti-Sm antibodies”. ANA analysis showed an “anti-extractable nuclear antigen” in the absence of antibodies directed to double-stranded DNA, Scl-70, SS-A/Ro, or SS-B/La by ELISA. Using immunoprecipitation (IP) assays, the patient’s serum was found to have antibodies directed to U11/U12 RNP. More recently, the screening of large cohorts of patients revealed that anti-U11/U12 RNP antibodies were present at low frequency (3.2–8.0%), but with high disease specificity for SSc. Among the components of the U11/U12 RNP complex, the 65 kDa RNA binding domain containing 3 (RNPC-3) protein was defined as an immunodominant target of human autoantibodies [22,29,33,34]. RNPC-3 is part of the minor spliceosome complex responsible for removing U12-type introns from pre-messenger RNA and is comprised of two RNA recognition motifs that likely contact one of the small nuclear RNAs [35]. Diagnostics 2022, 12, x FOR PEER REVIEW 4 of 14 Diagnostics 2023, 13, 1257 4 of 13 History of Figure 1. History of anti-U11/U12 anti-U11/U12RNP RNPautoantibodies. autoantibodies. The Thehistory history of of anti-U11/U12 anti-U11/U12RNPRNPantibodies antibodiesstarted startedin in1993 1993with withthe thediscovery discovery of of the the antibodies antibodies by by Gilliam Gilliam and Steitz [22]. During the following years, several studies focused on the molecular aspects and and cellular cellular function function of of the the U11/U12 U11/U12 RNP complex. It It was was not until 2009 when Fertig et al. [36] added to the knowledge of clinical associations of anti-U11/U12 anti-U11/U12 RNP. RNP. Later Later on, Shah Shah et al. al. first first reported reported on on aa potential potential association association with cancer cancer and and McMahan McMahan et et al. al. [37] [37] with gastrointestinal involvement. with gastrointestinal involvement. Other Other key key studies studies on on anti-U11/U12 anti-U11/U12 RNP RNP antibodies antibodies are are included included [29,33,34,38,39]. [29,33,34,38,39]. Diagnostics 2023, 13, 1257 5 of 13 4. Detection Methods In accord with the limited number of studies published on anti-U11/U12 antibodies, a narrow range of methods are available for their detection. However, in the US, some specialized laboratories may offer anti-U11/U12 antibody testing under the umbrella of a laboratory developed test (LDT) [40]. 4.1. Immunoprecipitation As referenced above, anti-U11/U12 antibodies can be detected by IP. Early studies used cell extracts for IP while most recent studies relied on IP of RNPC-3 using in-vitro translated products of the cognate cDNA. Using cellular extracts can be challenging since many proteins have a molecular mass approximating that of RNPC-3 producing many bands of approximately 65 kDa. Therefore, it has been important to develop a solid phase immunoassay using the specific antigenic target of anti-U11/U1 RNP antibodies. 4.2. Indirect Immunofluorescence According to Gilliam and Steitz, anti-U11/U12 RNP antibodies were associated with an IIF pattern that resembled the coarse speckled nuclear pattern (RNP like) without staining of the chromatin region [22]. In order to better characterize the characteristics of anti-U11/U12 RNP antibodies, in a previous study we identified monospecific anti- U11/U12 positive serum samples in a SSc cohort that used a particle-based multi-analyte technology (PMAT) for the detection of anti-U11/U12 antibodies. Two serum samples with clear monospecific reactivity to U11/U12 were selected and subsequently used for IIF testing (Figure 2). We assessed the commutability of four different HEp-2 IIF substrates (BioRad, Inova Diagnostics Inc., ImmunoConcepts, Binding Site) in detecting anti-U11/U12 RNP (RNPC-3) antibodies in human sera. The IIF pattern was a fine discrete nuclear speckled staining that had features similar to ICAP AC-02 (dense fine speckles), AC-04 (fine speckles), or AC-05 (large coarse speckles). However, the anti-U11/12 exhibited distinctive IIF staining features such as somewhat larger and more rounded, discrete nuclear speckles (compared to AC-02 and AC-04) that were smaller and more clearly defined than those typically associated with anti-U1 RNP and anti-Sm (i.e., AC-05), a finding in keeping with previous reports [22,39,41]. Depending on the commercial source of the HEp-2 substrate, variable staining of the nucleolar region, intense staining of the chromatin and/or perichromatin region of metaphase cells, and faint cytoplasmic speckled staining was also observed. When serial serum dilutions were used to analyze the end point titers, all four kits used were within one end-point dilution (i.e., 1:640–1:1280). In a preliminary study, co-localization by IIF of human anti-U11/U12 (RNPC-3) with rabbit anti-RNPC-3 (rabbit polyclonal) showed significant overlap between the structures targeted by the two antibody populations [39]. Further experiments are warranted to understand the co-localization with other autoantigens including but not limed to [PML, coilin/p80 and Survival of Motor Neuron (SMN) Complex]. ICAP [1] and others [19] recommend reflex testing of samples with AC-04 or AC-05 IIF patterns to assays containing known autoantigens, including RNP/Sm, Ro60/SSA, and La/SS-B. However, based on our observation, inclusion of anti-U11/U12 (RNPC-3) in reflex testing algorithms should be considered when HEp-2 IIF patterns resembling the AC-02, AC-04, or AC-05 are observed and clinical suspicion of SSc is present, especially when no antibodies to RNP/Sm, Ro60/SSA, and La/SS-B can be detected. In addition, since the HEp-2 IIF pattern is not identical to AC-04 or AC-05, the pattern associated with anti-RNPC-3 antibodies might be considered in future efforts of ICAP to define new clinically relevant patterns (expert level). Whether this is applicable to clinical practice is unlikely, considering the possibility of mixed patterns and the rarity of these antibodies. This is important as the HEp-2 IIF is still used as the first line screening test for patients suspected of having SSc [13]. Reflexing strategies taking into consideration both the clinical suspicion and the IIF pattern might be necessary in order to appropriately implement testing for anti-U11/U12 (RNPC-3) antibodies in the clinical routine. Landon-Cardinal of having SSc [13]. Reflexing strategies taking into consideration both the clinical suspi- Diagnostics 2023, 13, 1257 cion and the IIF pattern might be necessary in order to appropriately implement testing 6 of 13 for anti-U11/U12 (RNPC-3) antibodies in the clinical routine. Landon-Cardinal et al. in a cohort of patients with scleromyositis, recognized three novel clinico-serological subsets, including et al. in aa cohort groupofofpatients patients with with a speckled IIF scleromyositis, pattern three recognized [42]. novel clinico-serological subsets, including a group of patients with a speckled IIF pattern [42]. Figure 2. Anti-U11/U12 (RNPC-3) antibodies on HEp-2 substrates by indirect immunofluores- Figure cence2.(IIF) Anti-U11/U12 (RNPC-3) assay. Images antibodies of patterns on HEp-2 derived from HEp-2substrates by indirect substrates from immunofluorescence different manufacturers (IIF)[(a) assay. Images Bio Rad; of patterns (b) Inova derived Diagnostics; from HEp-2 substrates (c) ImmunoConcepts from HEp-2000; (d) different manufacturers Binding Site] using an index,[(a) Bio Rad;monospecific human anti-U11/U12 (RNPC-3) antibody positive serum display fine discrete speckled mon- (b) Inova Diagnostics; (c) ImmunoConcepts HEp-2000; (d) Binding Site] using an index, ospecific staininghuman anti-U11/U12 of interphase (RNPC-3) nuclei. However, antibody depending positive on the serum HEp-2 slide display fine manufacturer, discrete variable speckled staining staining of interphase nuclei. However, depending on the HEp-2 slide of nucleoli and metaphase chromatin was observed. Original magnification 400×. manufacturer, variable stain- ing of nucleoli and metaphase chromatin was observed. Original magnification 400×. 4.3. Particle-Based Multi-Analyte Technology (PMAT) Recently, a novel 4.3. Particle-Based PMAT system Multi-Analyte has been Technology developed that allows for the detection of (PMAT) antibodies to RNPC-3 as a molecular surrogate for the U11/U12 RNP complex [29,33,34]. Recently, a novel PMAT system has been developed that allows for the detection of The PMAT assay utilizes paramagnetic particles with unique signatures and a digital antibodies to RNPC-3 interpretation system.asMore a molecular surrogate specifically, antigensforarethe U11/U12 coupled RNP complex to paramagnetic [29,33,34]. particles Thethat PMAT carry assay uniqueutilizes paramagnetic signatures particles and are incubated withwith unique diluted signatures patient and a After serum samples. digital in- terpretation system.at 9.5 min incubation 37 ◦ C, More specifically, particles areantigens washed andareincubated coupled to 9.5paramagnetic at 37 ◦ C that min incubationparticles carry withunique signatures anti-human and are incubated IgG conjugated with diluted to phycoerythrin patient (PE) to label serumautoantibodies. the bound samples. After 9.5 minAfter a final wash incubation at 37cycle, fluorescent °C, particles aresignal intensity washed on the particles and incubated is captured 9.5 min using incubation ata37 °C digital imager and analyzed using proprietary algorithms to extract meaningful with anti-human IgG conjugated to phycoerythrin (PE) to label the bound autoantibodies. information for each analyte. After a final wash cycle, fluorescent signal intensity on the particles is captured using a digital imager and 5. Co-Existence of analyzed using proprietary algorithms to extract meaningful infor- Other Antibodies mation Due for each analyte. to the limited number of studies available and the rarity of anti-U11/U12 anti- bodies, the association of anti-U11/U12 antibodies with other SSc-SA or SSc-AA is not 5. Co-Existence of Other well studied. Like Antibodies other SSc-SA, anti-U11/U12 antibodies tend to be exclusive (occurring without Due to other the SSc antibodies). limited number In of a recent study, studies the prevalence available and theand levels rarity of of anti-RNPC-3 anti- anti-U11/U12 antibodies were higher in anti-Scl-70, ACA, and/or anti-RNA Pol III triple bodies, the association of anti-U11/U12 antibodies with other SSc-SA or SSc-AA negative patients is not well [10/106 (9.4%) vs. 6/193 (3.1%), p = 0.03; and median titers 122.0 vs. 107.5, p = 0.08] [39]. Diagnostics 2023, 13, 1257 7 of 13 However, further studies might show coexistence of anti-U11/U12 with ACA, anti-Scl-70, anti-RNA Pol III, or other SSc antibodies. 6. Clinical Associations Anti-U11/U12 (RNPC-3) antibodies have been associated with ILD and pulmonary fibrosis. When compared to other SSc-SA, anti-U11/U12 showed significantly stronger association with pulmonary fibrosis versus all other antibodies studied (anti-centromere, anti-PM/Scl, anti-RNA Pol III, anti-Th/To, anti-Scl-70, anti-U1 RNP, and anti-U3 RNP) with the exception of anti-Ku for which it is likely the number of cases was too low to reach statistical significance [36]. In addition, associations were reported with moderate to severe gastrointestinal (GI) dysmotility and severe Raynaud’s phenomenon [37]. The association with GI involvement is interesting as RNPC-3 deficient mice demonstrate arrested development and impaired gastrointestinal homeostasis [43]. In addition to those clinical associations, in a study of 318 SSc patients with cancer, Shah et al. reported that anti-RNPC-3 antibodies were found in 3.8% of the patients and were associated with a short cancer-scleroderma interval (median 0.9 years). In addition, 33% had an associated myopathy [44]. To further analyze the clinical associations of anti-RNPC-3 antibodies in SSc, sera from 88 SSc patients were studied of which 32 cases had documented cancer. Sera from 64 systemic lupus erythematosus (SLE) patients, 65 idiopathic inflammatory myopathies (IIM), and 20 health individuals (HI) were included as a comparator group [39]. The frequency of autoantibodies to both recombinant RNPC-3 and a synthetic peptide derived from the RNPC-3 sequence tended to be higher in SSc and SLE compared to IIM and HI [39]. When the SSc cancer patients were compared with an unselected SSc cohort, no significant difference was found [39]. This finding appears to be inconsistent with previous findings [44], but might be explained by the different selection process of the samples. Possible explanations likely include the cancer type and the time difference between cancer onset and blood sampling. Further validation studies of large SSc cohorts are needed to determine if anti-U11/U12 RNP antibodies are associated with cancer and/or GI involvement. The most recent study on anti-U11/U12 (RNPC-3) antibodies included a cohort of 447 SSc patients from Barcelona (n = 286) and Milan (n = 161) [29]. All samples were tested using the PMAT assay using recombinant RNPC-3 as the antigen. Anti-U11/U12 (RNPC-3) positive and negative patients were compared in terms of clinical presentations. Epidemiological, clinical features, and survival were analyzed. End-stage lung disease (ESLD) was defined if the patient had a forced vital capacity < 50% of predicted and needed oxygen therapy or lung transplantation. Event-free survival (EFS) was defined as the period of time free of either ESLD or death. A total of 19 of 447 (4.3%) patients had anti-U11/U12 (RNPC-3) antibodies and ILD was more frequent (11, 57.9% vs. 144, 33.6%, p = 0.030) in individuals with anti-RNPC-3 antibodies. More patients reached ESLD in the positive group (7, 36.8% vs. 74, 17.3%, p = 0.006), and a higher use of non-glucocorticsteroid immunosuppressive drugs was observed (11, 57.9% vs. 130, 30.4%, p = 0.012). Anti- U11/U12 (RNPC-3) positive patients had lower EFS, both in the total cohort (log-rank p = 0.001) as well as in patients with ILD (log-rank p = 0.002). In multivariate Cox regression analysis, diffuse cutaneous subtype, age at onset, the presence of ILD or pulmonary arterial hypertension, and the expression of anti-RNPC-3 positivity or anti-Scl-70 antibodies were independently associated with worse EFS. The presence of anti-U11/U12 (RNPC-3) was associated with higher frequency of ILD and either ESLD or death. These data suggest that anti-RNPC-3 are independently associated with a poor prognosis in SSc, especially if ILD is a co-morbid feature. In summary, evidence is mounting that anti-U11/U12 (RNPC-3) antibodies represent a rare but important antibody specificity as they often occur in the absence of other SSc-related antibodies and are detected in patients with ILD [36]. Recently, using a bead-based antigen array, high levels of anti-U11/U12 antibodies were described in patients hospitalized with COVID-19 [45]. Although the findings are Diagnostics 2023, 13, 1257 8 of 13 worth mentioning in the context of this review, further studies are required to interpret the data. No validation data for anti-U11/U12 antibodies on the method used for the study by Chang et al. [45] was available. Table 2. Overview of studies on anti-U11/U12 antibodies. References No. Positives Method/Antigen Comments Initial description of anti-U11/U12 Gilliam & Steitz 1993 [22] 1 patient IP with HeLa extract antibodies. Patient Ru Association with GI involvement 33 U11/U12 positive Fertig et al. 2009 [36] IP, RT-PCR and SB (p = 0.01), pulmonary fibrosis 15/462 (3.2%) in SSc (p = 0.0001) and mortality (p = 0.01) An RNP-like IP pattern and coarsely 1/863 (0.1%) of SSc speckled ANA IIF, without any Mierau et al. 2011 [46] patients defined as IP + IIF U1-RNP signals in line assay and ID; anti-U11/U12 positive U11-RNP specificity detected by C Will and R Lührmann, Marburg, Germany 4/16 (25%) CTP-negative IP Detection of antibodies to other Xu et al. 2016 [38] SSc patients with cancer PhIP-Seq components of the U11/U12 complex positive for anti-U11/U12 PLATO-BC Shah et al. 2017 [44] 12/318 (3.8%) in SSc IP with RNPC-3 NOTE: all of the SSc had cancer Prevalence of anti-RNPC-3 antibodies in an Italian and Spanish 19/613 (3.1%) SSc patients Beretta et al. 2018 [33] PMAT cohort of SSc patients positive for anti-U11/U12 Strong association with ILD 13/19 CTP-negative 3/90 (3.3%) SSc patients Epitope mapping identified several Mahler et al. 2018 [34] PMAT/SPPA positive for anti-U11/U12 linear epitopes on RNPC-3 10/106 (9.4%) CTP-negative Mahler et al. 2020 [39] SSc patients positive for PMAT No association with cancer observed anti-U11/U12 Association with male gender and African American decent McMahan et al. 2018 [37] 6 anti-RNP-C positive IP with RNPC-3 Association with moderate-to-severe GI disease and ILD Callejas-Moraga et al. 2021 [29] 19/447 (4.3%) in SSc PMAT RNPC-3 Association with severe ILD COVID-19 patients Individual COVID-19 patients with Chang et al. [45] Multiplex (no cut-off provided) high levels of anit-U11/U12 antibodies ILD = interstitial lung disease; IP = immunoprecipitation; GI = gastrointestinal; PMAT = particle-based multi-analyte technology; SB = southern blotting; RT-PCR = reverse transcription-polymerase chain reaction; SPPA = Solid phase peptide arrays. 7. Biochemical Aspects and Immunogenicity The U11/U12 complex consists of at least eight U11/U12 RNP specific proteins and is involved in alternative splicing [35,47]. Members of the complex include hPrp43 (DHX15), RNPC-3, PDCD7, snRNP48, snRNP35, ZCRB1, snRNP25, and ZMAT5 (see Table 3). In addition, Sm proteins and the SF3b complex join the U11/U12 specific proteins in the complex. The U11/U12 snRNP 65 kDa protein (or RNPC-3) acts as a molecular bridge, binding the U12 snRNA and U11-59 kDa protein [48]. Although RNPC-3 has been defined as a major antigenic target of the complex, systematic analyses have not been performed, especially with newer approaches and technologies. However, in a recent study, antibodies to other components of the U11/U12 complex were identified [38]. Bacteriophage Immuno- precipitation Sequencing (PhIP-Seq) identified SNRNP48 and PDCD7 as antigenic targets and barcoded parallel analysis of translated open reading frames (PLATO-BC) detected immune reactivity to SNRNP25, SNRNP35 in addition to RNPC-3. Diagnostics 2023, 13, 1257 9 of 13 Table 3. Proteins of the U11/U12 complex [32] ordered according to the molecular weight. Protein ID Molecular Weight ID Alternative Names/Protein Function hPrp43, DHX15 90 kDa O43143 ATP-dependent RNA helicase 46, DHX15 RNA-Binding Region-Containing Protein 3 RNPC-3 65 kDa Q96LT9 RNA-Binding Protein 40 U11/U12 Small Nuclear Ribonucleoprotein 65 kDa Protein Programmed Cell Death Protein 7 PDCD7 59 kDa Q8N8D1 U11/U12 SnRNP 59K 2 3 snRNP48 48 kDa Q6IEG0 snRNP35 35 kDa Q16560 Zinc Finger CCHC-Type And RNA-Binding Motif-Containing Protein 1 ZCRB1 31 kDa Q8TBF4 U11/U12 Small Nuclear Ribonucleoprotein 31 kDa Protein snRNP25 25 kDa Q9BV90 Minus-99 Protein Zinc Finger Matrin-Type Protein 5 ZMAT5 20 kDa Q9UDW3 U11/U12 Small Nuclear Ribonucleoprotein 20 kDa Protein SF3b heptameric N/A N/A SF3b1, SF3b2, SF3b3, SF3b4, SF3b5, SF3b6 and SFb7 proteins. complex Sm proteins N/A N/A n/A = not applicable; ID = UniProtKB/Swiss-Prot. 8. Epitope Mapping of RNPC-3 The epitope distribution of anti-U11/U12 antibodies was analyzed in two studies [34,38]. In 2016, Xu et al. used phage-immunoprecipitation sequencing to study the antibody response in SSc patients with cancer and observed both intra-and inter-molecular epitope spreading [38]. In 2018, the second study utilizing solid phase peptides revealed several linear epitopes across the entire protein [34]. The identified candidate epitopes were subsequently utilized to synthesize synthetic, biotinylated, and soluble peptides for testing using the novel PMAT described above. The reactivity to recombinant RNPC-3 (rRNPC-3) and to the RNPC-3 derived peptide (pRNPC-3) was correlated in two independent cohorts. In the first study using samples from SSc, IIM, and SLE patients and healthy individuals, a high level of correlation was observed (Spearman’s rho = 0.64, 95% Confidence interval 0.58–0.69; p < 0.0001) [34]. In the second study of 299 patients, the correlation was less pronounced but was still highly significant (Spearman = 0.32, p = 0.0001; chi-squared p = 0.0002) [39]. Immunoadsorption showed significant inhibition using the RNPC-3 derived peptide, but not with a control peptide [39]. Taken together, these data indicate that the identified epitope represents a major determinant of the B-cell immune response towards RNPC-3. Future studies are warranted to evaluate if the identified peptides representing the key epitopes might be used as molecular surrogates for the detection of anti-U11/U12 antibodies or if they identify more specific subsets of patients. 9. Implication for New Treatment Strategies In contrast to traditional treatment strategies, modern approaches are targeting specific clinical presentations and organ involvement [1]. During the last years, special focus was placed on lung disease in SSc patients. Consequently, biomarkers such as anti-U11/U12 antibodies might be useful to identify patients with rapidly progressing ILD [1,49], es- pecially since the classification criteria marker anti-Scl-70, ACA and anti-RNA Pol III do not effectively allow for stratification, albeit still being useful f identify patients at risk for SSc-ILD [49–51]. Similarly, it must be determined to what extent these antibodies, in conjunction with other risk factors for SSc-ILD, such as the diffuse cutaneous subset or early disease onset, may identify patients that warrant close monitoring or early and aggressive intervention. In this context, a proper stratification of patients with SSc-ILD is relevant considering most recent data on nintedanib in patients with SSc-associated ILD [52]. Nintedanib effectively slowed progression (decline of forced vital capacity) after initial and long-term use [53], yet without heterogeneity across serological or clinical sub- Diagnostics 2023, 13, 1257 10 of 13 groups [54]. Autoantibodies, such as anti-U11/U12 RNP, anti-Th/To, and others, should be tested on cohorts such as the SENSCIS trial in order to explore their potential to reduce the heterogenicity of SSc-related ILD. This is especially warranted since (a.) the effect of nintedanib was more pronounced in the anti-Scl-70 negative group [54], and (b.) anti- U11/U12 antibodies are more prevalent in CTP negative patients [39]. However, this is not only limited to nintedanib, but applies to other novel and alternative treatment approaches in SSc-ILD, including agents with strong-supporting evidence, such as tocilizumab and rituximab [55,56], or others with biological or promising compoundssuch as abatacept, lenabasum, romilkimab, belimumab, bermekimab, brodalumab, tofacitinib, or even ritux- imab [reviewed in [1,57]]. 10. Other Considerations No data has been published to date on the fluctuation of anti-U11/U12 antibodies over time. Historically, it is established that autoantibody specificities in SSc are rather stable. However, a recent case report on anti-Th/To antibodies has shown significant fluctuations in antibody levels and also seroconversion (switch in autoantibody profile) [58]. The stability of anti-U11/U12 antibody levels might have significant implications on the interpretation of previous data including their association with cancer. Longitudinal studies of large SSc cohorts are required to analyze this aspect of anti-U11/U12 antibodies. 11. Conclusions Although very limited literature is available on anti-U11/U12 autoantibodies, existing evidence strongly indicates that these antibodies are specific for SSc and associated with a severe phenotype with a poor prognosis, mostly characterized by the presence of severe ILD. The reported associations with other disease manifestations, GI involvement, and cancer deserves further studies. Author Contributions: Conceptualization, M.J.F. and M.M.; methodology, M.J.F. and M.M.; formal analysis, M.J.F., C.B., L.B., B.P., M.M., J.P.-P., M.T.S.-M., A.G.-D.-C., A.M., V.F.-P., E.C.-M. and C.P.S.-A.; resources, M.J.F., C.B., L.B., B.P., M.M., J.P.-P., M.T.S.-M., A.G.-D.-C., A.M., V.F.-P., E.C.-M. and C.P.S.-A.; writing—original draft preparation, M.M. and M.J.F.; writing—review and editing, M.J.F., C.B., L.B., B.P., M.M., J.P.-P., M.T.S.-M., A.G.-D.-C., A.M., V.F.-P., E.C.-M. and C.P.S.-A. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: Michael Mahler and Chelsea Bentow are employed at Werfen, a company that sells autoimmune diagnostic tests. Marvin Fritzler has been a consultant to Werfen and is Medical Director of Mitogen Diagnostic Corporation. The rest of the authors declare no conflict of interest. Abbreviations AC = anti-cell staining pattern from ICAP nomenclature; ACA = anti-centromere; CENP = cen- tromere protein; CTP-negatives = centromere, Topoisomerase and RNA Polymerase negatives; EFS = event-free survival; ESLD = end-stage lung disease; GI = gastrointestinal; HI = health individu- als; ICAP = International Consensus on Autoantibody Patterns; IIF = indirect immunofluorescence; ILD = interstitial lung disease; IIM = idiopathic inflammatory myopathies; IP = immunoprecipitation; kDa = kilodalton; MFI = Median Fluorescent Intensity; PE = phycoerythrin; PMAT = particle-based multi-analyte technology; RNAP = RNA polymerase; RNP = ribonucleoproteins; SLE = systemic lupus erythematosus; SSc = systemic sclerosis; SSc-AA = systemic sclerosis associated autoantibodies; SSc-RA = systemic sclerosis related autoantibodies; TERF-1 = telomeric repeat-binding factor 1. Diagnostics 2023, 13, 1257 11 of 13 References 1. 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