B-cell epitopes of the intracellular autoantigens ro/ssa and la/ssb: tools to study the regulation of the autoimmune response

Routsias JG and Tzioufas AG. Β-λεμφοκυτταρικοί επίτοποι των ενδοκυττάριων
αυτοαντιγόνων Ro/SSA και La/SSB: εργαλεία για την μελέτη της ρύθμισης της
αυτοάνοισης απόκρισης. J Autoimmun. 2010, 35(3):256-64.
intracellular  autoantigens.  Although  their  pathogenesis  is  not  fully  understood,  autoantibodies  are  important  tools  for  establishing  diagnosis,  classification  and  prognosis  of  autoimmune  diseases.  In  Systemic  Lupus  Erythematosus  (SLE)  and  Sjögren's  syndrome  (SS)  autoantibodies  mainly  target  multicomponent  ribonucleoprotein  complex  Ro/La  RNP.  The  last  years,  the  main  characteristics,  the  their  B‐cell  antigenic  determinants  (epitopes)  have  been  addressed.  More  specifically,  the  structural  characteristics  and  clinical  associations  of  epitopes  along  with  their  utility  as  tools  to  investigate  the  autoimmune  response  have  been  investigated  in  detail.  New  insights  for  the  pathogenetic  role  of  regard,  the  role  of  epitope  spreading  in  the  diversification  of  autoimmune  response  and  the  anti‐

Contents lists available at Journal of Autoimmunity B-cell epitopes of the intracellular autoantigens Ro/SSA and La/SSB:Tools to study the regulation of the autoimmune response John G. Routsias, Athanasios G. Department of Pathophysiology, School of Medicine, University of Athens, 75 M Asias st, 11527 Athens, Greece A common serologic finding in systemic autoimmune diseases is the presence of autoantibodies against intracellular autoantigens. Although their pathogenesis is not fully understood, autoantibodies are important tools for establishing diagnosis, classification and prognosis of autoimmune diseases. In Systemic Rheumatic diseases Lupus Erythematosus (SLE) and Sjögren's syndrome (SS) autoantibodies mainly target multicomponent ribonucleoprotein complex Ro/La RNP. The last years, the main characteristics, the clinical significance of Complementary epitopes the anti-Ro/SSA and anti-La/SSB autoantibodies, their biologic function, as well as their B-cell antigenicdeterminants (epitopes) have been addressed. More specifically, the structural characteristics and clinicalassociations of epitopes along with their utility as tools to investigate the autoimmune response have beeninvestigated in detail. New insights for the pathogenetic role of epitopes in initiation, propagation andregulation of systemic autoimmunity have been emerged. In this regard, the role of epitope spreading in thediversification of autoimmune response and the anti-idiotypic antibodies in the regulation of autoanti-bodies (idiotypic) response are addressed.
Ó 2010 Elsevier Ltd. All rights reserved.
2. Major intracellular autoantigens-rationale for thedetection of epitopes In 1991, our laboratory prompted by professor Harry Moutso- poulos, focused on the detection of fine specificity of autoanti- Sera of patients with systemic autoimmune diseases often bodies of intracellular autoantigens. At that time most of the contain autoantibodies directed against intracellular complexes autoantigens had been recently cloned and eventually, their composed of a number of proteins that are non-covalently associ- primary structure had been resolved. The purpose of such a study ated with nucleic acid components Many of these autoanti- was (a) to understand the structures, within the autoantigen, bodies are essential for the clinical evaluation of patients with recognized by autoantibodies and investigate if they share in systemic rheumatic diseases, since: (i) they are included in the common sequences with foreign autoantigens, (b) to investigate diagnostic or classification criteria of certain systemic autoimmune whether certain epitopes are associated with different diseases, disorders , (ii) they are associated with disease activity indices, disease subtypes and individual manifestations or even its activity particularly in SLE and (iii) some of them might be correlated and severity and (c) to develop methods for autoantibody detec- with specific clinical manifestations in the spectrum of a given tion, characterized by high sensitivity and specificity.
systemic disease . In basic research, the most intriguing question After several years of intense research, ours and others labora- is why these particular autoantigens, among thousands of mole- tories realized that the detection and study of B-cell epitopes gave cules expressed in the organism, are selected as targets of the us important insights on the mechanisms involved in the perpet- immune system . To address this question, several investigators uation and regulation of the autoimmune response. In this review tried to define the fine specificity of autoantibodies to intracellular the major advances of B-cell epitopes of intracellular autoantigen, antigens, by identifying the antigenic determinants (or B-cell particularly those directed against Ro/SSA and La/SSB are discussed.
epitopes) recognized most frequently by autoantibodies. Theidentification of B-cell epitopes revealed useful information on themechanisms involved in autoantibody production and their diver-sification in the course of the disease, such as molecular mimicryand epitope spreading . For example, in patients with SLE, at * Corresponding author. Tel.: þ30 210 7462670; fax: þ30 210 7462664.
E-mail address: (A.G. Tzioufas).
least one autoantibody specificity can be detected 1e9 years before 0896-8411/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
J.G. Routsias, A.G. Tzioufas / Journal of Autoimmunity 35 (2010) 256e264 the clinical onset of the disease and its diagnosis. The autoimmune  Quaternary-structure epitopes, which are consisted of amino response is then spreads in an ordered manner to other auto- acids distributed over different subunits within a macromo- antigens and the clinical onset of disease coincides with the lecular complex, forming a structure recognized by the auto- cessation of new autoantibody specificities development The antibody. Such epitopes have been identified in Ro/La RNP earliest autoantibodies detected in the pre-clinical period, as indi- complex as well as in nucleosome subunits, composed of viduals progress toward clinical SLE were antibodies to Ro60 (mean histones and DNA elements.
3.7 years before the disease onset). McClain et al. mapped the  Cryptic epitopes (cryptotopes). These are usually linear epitopes initial, pre-disease target of the anti-Ro60 autoantibody response to hidden within the native structure of the autoantigen. They the region 169e180aa (TKYKQRNGWSHK) of the autoantigen become accessible to antibody binding after disruption of the This region belongs to the previously identified SLE related epitope three-dimensional structure (e.g. by denaturation, proteolytic 169e190aa by Routsias et al. shares sequence homology with degradation or chemical modification of the autoantigen).
Ro orthologs in certain bacteria such as the region KYRQRGGWSHR These epitopes are observed in a number of nuclear auto- from the ribonucleoprotein complex of Mycobacterium smegmatis antigens, such as the Ro/La RNP, where the initial Ro60 epitope and it has been reported to cross-react with a viral peptide (for SLE) is cryptic, masked by the binding of hY RNA.
(GGSGSGPRHRDGVRR) from the EpsteineBarr virus nuclear  Modified epitopes. Amino acids can be post-translationally antigen-1 (EBNA-1) without to exhibit any sequence similarity modified. Examples of these modifications include: (i) Serine, In addition, the characterization of the epitopes of an auto- Threonine, Tyrosine phosphorylation by protein kinases, (ii) antigen with high sensitivity and specificity, allows the develop- Lysine acetylation or ubiquitination, (iii) Cysteine lipidation or ment of immunoassays based on synthetic peptides which can be oxidation (disulphide-bond formation), (iv) Glutamic acid utilized as substrates for autoantibody detection. As example, fil- methylation or g-carboxylation, (v) Glutamine deamidation lagrin in RA is recognized by about 70 percent of the patients. Anti- (vi) Asparagine (N-linked) and Serine/Threonine (O-linked) fillagrin antibodies are targeting mainly post-translationally glycosylation (vii) Arginine citrullination or dimethylation and modified epitopes, containing citrulline . Antibodies to cyclic (viii) Proteolytic cleavage or degradation. In some instances, citrullinated peptides (anti-CCP) are detected in RA patients' sera side chain modifications of specific amino acids, such as cit- long before the onset of the disease and are associated with erosive rullination of arginine residues, are responsible for epitope disease . In diagnostic grounds, when anti-CCP and RF anti- high-affinity binding. Such modified amino acids have been bodies are combined, the specificity for RA is exceeded the 95% reported in a variety of human nuclear proteins, including theSm antigens D1 and D3, and nucleolin. The identification of 3. Structural definition of B-cell epitopes these modified (usually linear) epitopes requires assays thatprovide the amino acid in its modified form. These assays are The B-cell epitopes are diverse in structure and immunoreac- based mainly on synthetic peptides.
tivity and thereof they are classified accordingly. On the basis of the  Neoepitopes. Neoepitopes can be the post-translationally nature of the epitope within the parental protein, they are classified modified epitopes but also epitopes pre-translationally modi- as: (i) linear or continuous, consisting of sequential amino acids in fied, derived by frameshift mutations or alternative splicing of the primary structure of the protein, and (ii) conformational or mRNA. For example, Bachmann et al. , identified a mutated discontinuous epitope, formed by distant regions in the protein La cDNA in patients with SS that contains a frameshift mutation sequence coming together in its tertiary structure. In the majority in a hot spot region. This mutation resulted in premature stop of the epitopes characterized previously as linear, not every amino codon, which is located eleven amino acids downstream of the acid in the sequence is essential for antibody binding. Often, there frameshift mutation. Consequently, only the sequence of the 12 are sequence-positions that can be successfully substituted with all amino acid La peptide (193e204aa: MKKENKIKWKLN, neo- the 20 naturally occurred amino acids, without any loss of immu- epitope) encoded by the patient's La cDNA markedly differed noreactivity. In this regard, linear epitopes larger than 5e6 amino from the corresponding La protein sequence.
acids in length, possess also features of conformational epitopes.
 Apotopes. Apotopes are epitopes that are expressed specifically Moreover, as the autoantigens are organized in large ribonucleo- on the surface of apoptotic cells. This term is not widely protein complexes the term "conformational epitope" can be accepted, since it refers to epitopes that obviously belong to referred either to epitopes comprised by amino acids distributed on one of the above described categories. However, this term has its secondary, tertiary or quaternary structure. Therefore, more been used to describe epitopes on Ro60 that differentiate properly the epitopes can be divided in: Sjögren's syndrome from SLE.
 Mimotopes. Mimotopes are structures that mimic unknown  Primary-structure epitope (or linear epitope), consisting of epitopes. They are usually defined using peptide libraries. Such sequential amino acids. Such epitopes have been identified by mimotopes can either show close homology to an antigenic synthetic peptide mapping the majority of autoantigens sequence of a protein (linear epitope) or, alternatively, are including Ro60, Ro52, La, SmB, SmD, RNP-70 and Scl-70 etc.
structural homologues with a wide variety of different type  Secondary-structure epitope, formed by amino acids distributed epitopes (all the conformational epitope types described in simple three-dimensional structures, such as a-helices or previously) including epitopes belonging to non-protein b-sheets. These epitopes have been identified in PM/Scl-100 molecules such as polysaccharides, lipids or nucleic acids.
autoantigen by a combination of peptide scans and mutationalanalyses. In these studies, epitopes were defined as a local a-helical secondary structure stretch with all amino acids 4. Clinical significance of antibodies to Ro/SSA and LaSSB relevant for antibody binding is located at one side of the helix.
 Tertiary-structure epitopes, are formed by distant regions of the Anti-Ro and anti-La antibodies are found in approximately protein sequence, which are coming together in the tertiary 60e90% and 30e60% of patients with primary Sjögren's syndrome, structure. It has been suggested that such conformational as well as in 30e40% and 10e15% of patients with SLE, respectively epitopes are the main target of some autoantibodies (e.g. anti- , depending on the method used for their detection. A variety of methods have been applied for their detection. Among them RNA J.G. Routsias, A.G. Tzioufas / Journal of Autoimmunity 35 (2010) 256e264 precipitation is considered as the gold standard method by various Ro52-deficient mice demonstrated that in addition to ubiquitinat- authors. However, this method cannot be used in the every-day ing the previously reported targets IRF3 and IRF8, Ro52 is required clinical practice, but it is useful as reference and confirmatory for poly-ubiquitination and degradation of IRF5. Ro52-deficient assay. Agarose gel electrophoresis techniques such as counter- bone marrow-derived macrophages and splenocytes released more immunoelectrophoresis (CIE) and immunodiffusion (ID) are inflammatory cytokines (IL-6, TNF, type I IFN, and IL-23) upon TLR frequently used for the routine evaluation of sera. A small activation. In this regard the ubiquitin ligase Ro52 is induced by IFN subpopulation of patient sera, contain precipitin-negative anti-La/ activation of immune cells, acting as a negative regulator of IFN SS-B antibodies that is believed to possess restricted epitope recognition. These are detected using anti-La ELISA or immunoblot.
The B-cell epitopes of Ro52 have been mapped in various studies Antibodies to Ro/SSA and La/SSB belong in the diagnostic items with different methods (The major immunoreactivity of Ro of primary Sjögren's syndrome (pSS) and their presence in patients 52kD autoantigen was localized, using recombinant Ro52 fusion with suspected pSS supports strongly the diagnosis. Furthermore, proteins, in the middle coiled-coil region of Ro52 . The they are associated with a higher prevalence of extraglandular 190e245aa region of the amino acid sequence was reactive with features, especially vasculitis and higher intensity of the lympho- almost all anti-Ro52 positive sera and was independent of associ- cytic infiltrates in the affected salivary glands However, so far, ated diseases . An epitope spanning the 200e239aa of Ro52, no pathogenetic role has been assigned for the disease. Pregnancy which contains the complete leucine zipper motif, has been also in women with anti-Ro and anti-La antibodies, may be complicated identified in the same region . Autoantibodies against this by the development of neonatal lupus syndrome in the fetus that epitope were associated with Neonatal Lupus and Congenital Heart includes also increased risk for congenital heart block, the most Block. These autoantibodies have the potential to bind on the cell serious manifestation of this disorder . In this rare syndrome, surface of cardiomyocytes in primary cultures and cause a dysre- maternal anti-Ro and anti-La IgG autoantibodies pass through the gulation of the Ca2þ-homeostasis, which is followed by apoptosis placenta to the fetal circulation and cause tissue injury to the heart Anti-Ro52 antibodies are also found in primary biliary and skin. The redistribution of Ro and La autoantigens on the cirrhosis associated with sicca syndrome. The anti-Ro52 antibodies surface of myocardial cells is required in order to become available in this setting are directed against a smaller epitope than in primary for binding of autoantibodies. Such autoantigen translocation can Sjögren's syndrome be induced either by b-estradiol, viral infection or apoptosis .
The Ro60 antigen is found in virtually all vertebrate cells as well as in certain bacteria (e.g. Deinococcus radioreductans) as well as the 5. Epitopes of Ro/SSA and La/SSB nematode Caenorhabditis elegans Its function is related withthe quality control or discard pathway for nascent transcripts 5.1. Structure of the epitopes and clinical significance synthesized by RNA polymerase III (e.g. 5S rRNA precursors). Thus,Ro60 binds misfolded small RNAs (e.g. 5S RNA) leading them to Structurally, human Ro/La RNP is composed of one of the four degradation . Recently, the structure of the Xenopus laevis Ro60, small, uridine rich hY RNAs (human cYtoplasmic RNAs) non-cova- 78% identical to human Ro60, was solved and found to consist of lently associated with at least three proteins, the Ro52, La and Ro60 two distinct domains One domain resembles the von Wille- autoantigens. Additional components of the complex are also the brand Factor A (vWFA) domain, which is found in extracellular proteins calreticulin and nucleolin. The localization of these matrix proteins and proteins that function in cell adhesion. The complexes is mainly cytoplasmic. However, Ro60, Ro52 and La other domain consists of a series of a-helical repeats (HEAT repeats) autoantigens can also be found in the nucleus but they are not that are arranged orbicularly around of an inner hole of 10e15 Å associated with hY RNA. After the assembly of the Ro/La RNP in the ("doughnut"-like structure). This hole most probably holds the 30 nucleus, the complex is rapidly and quantitatively transported to ends of misfolded RNAs while the YRNAs bind to conserved resi- the cytoplasm Under certain circumstances that include stress, dues to the outside of the "doughnut". Another conserved role for UV radiation, apoptosis or viral infection the protein components of the Ro60 in facilitating cell survival after ultraviolet irradiation has Ro/La RNP are on the cell surface.
recently emerged from studies in radiation-resistant eubacterium Ro52, belongs to the tripartite motif (TRIM) or RING-B-box- Deinococcus radiodurans and mammalian cells lacking Ro60 coiled-coil (RBCC) protein family, thus comprising an N-terminal Studies of mice lacking the Ro60kD protein suggest also that RING, followed by a B-box and a coiled-coil region. The RING is the normal function of Ro may be important for the prevention of a cysteine-rich Zn2þ-binding motif of the form C3HC4, which binds autoimmune disease . In these studies, mice lacking Ro were two Zn2þ ions in a tetrahedral manner. The RING is predominantly found to develop autoantibodies and membrano-proliferative a proteineprotein interaction motif, which also acts as a ubiquitin- protein isopeptide (E3) ligase in the ubiquitination pathway Epitopes of Ro60kD have been described by several authors The B-box is the second Zn2þ-binding motif of Ro52 and has the using a variety of epitope mapping procedures (). Initially, form CHC3H2. Ro52 can also homodimerize through its leucine the major antigenic region of Ro60kD was identified within the zipper domain Several different proteomic functions have central part of the molecule (spanning the 181e320aa, been suggested for Ro52, including DNA binding, protein interac- 139e326aa and 155e295aa regions of the sequence, respectively).
tions and Zn2þ-binding. Most probably, overall Ro52 functions as The exact localization of the antigenic determinants was demon- transcription modulator, due to its domain organization. In line strated after the application of epitope mapping with synthetic with many other RING-containing proteins, Ro52 is involved in peptides. Wahren et al. identified a major epitope in synthetic ubiquitination pathway . Recent findings suggest that Ro52 peptide 216e245aa, Scofield and associates, identified numerous autoantigen is a RING-dependent E3 ligase that is overexpressed in epitopes covering the entire length of Ro60 , presumably, due to patients. In this regard, Ro52 may be directly involved in the extended epitope spreading and our group located the antigenic reduced cellular proliferation and increased apoptotic cell death regions of Ro60kD in the 169e190aa and 211e232aa parts of the that is observed in Sjögren's syndrome and SLE . Ro52-deficient antigen In a clinical study it was shown that the epitope mice develop uncontrolled inflammation and systemic autoim- 169e190aa was mainly recognized by anti-Ro positive sera from munity as a consequence of minor tissue injury caused by ear SLE patients and the epitope 211e232aa from patients with SS. The tagging The characterization of immune cells derived from epitope 169e190aa, was found to share conformational and

J.G. Routsias, A.G. Tzioufas / Journal of Autoimmunity 35 (2010) 256e264 Table 1Epitopes mapped on Ro60, Ro52 and La autoantigens.
antigenic similarity with the HLADR3 b-chain, an HLA class II allele, studies suggest also that although the exact Ro epitopes were which has been shown to correlate with the anti-Ro60 response identified as small peptide moieties, their recognition by The same epitope was recently found to be the initial pre- autoantibodies is conformation-dependent and is dramatically disease target of autoantibodies in individuals, who developed SLE enhanced upon interaction with the molecular chaperone calreti- several years later This early recognized epitope has been reported to cross-react directly with a peptide from the latent viral The La antigen is a phosphoprotein binding with a variety of protein EpsteineBarr virus nuclear antigen-1 (EBNA-1) Recent small RNAs, including 5S cellular RNA, tRNA, 7S RNA, and hY RNAs, J.G. Routsias, A.G. Tzioufas / Journal of Autoimmunity 35 (2010) 256e264 all transcribed by RNA polymerase III . In molecular level, it (linker region), (iii) 225e334 aa (RRM2 þ NRE). hY1 RNA model binds a short polyuridylate sequence (poly-U) that exists at the 3- was built on the basis of its known secondary structure by a step- end of almost all nascent pol III transcripts. Moreover, La binds viral wise fashion incorporating stem 1 and its terminal uracils from the RNAs including adenovirus VA, EpsteineBarr EBER, viral and structures of Ro60 and La that were already solved in complex with human RNAs possessing internal ribosomal entry elements (IRES) RNA. It was found that hY1-RNA completely masks epitopes and RNA component of telomerase complex Structurally the 169e190aa of Ro60 and 145e164aa of La that were previously human La contains the La motif in its N-terminal region, a typical associated with SLE but not the SS related epitopes 211e232aa of RNA recognition motif (RRM) in its central part and an unusual Ro60 and 349e364aa of La. ELISA experiments confirmed this RRM, encompassing residues 229e326aa. The latter is followed by prediction and it was found that Ro60ehY1-RNA interaction was a long, flexible polypeptide that contains a short basic motif (SBM), inhibited by anti-Ro169e190 antibodies, but not by autoantibodies a regulatory phosphorylation site on Ser366 and a nuclear locali- targeting other regions of the molecule as anti-Ro 443e454 or anti- zation signal (NLS). The three-dimensional structure of the La Ro211e232. LaehY1-RNA interaction was inhibited by purified motif, the central RRM and the carboxyl-terminal RNA recognition anti-La145e164 antibodies but not by anti-La349e364 and anti- domain of the autoantigen were solved. The La motif folds into La301e32. Furthermore, electrophoric mobility shift assays (EMSA) a winged-helix motif elaborated by the insertion of three helices.
demonstrated that Ro60 and La interactions with hY1 RNA can be The central RRM consists of a four-strand b-sheet attached to inhibited by peptides corresponding to epitopes Ro169e190 and two a-helices while the C-terminal domain folds to generate a five- La145e164. Therefore, the SLE related epitopes on Ro and La stranded, antiparallel b-sheet surface that is terminated by a long autoantigens appear to be cryptotopes in Ro/La RNP three-dimen- a helix. It appears that both the La motif and the adjacent central sional structure, masked by hY1 RNA. On the other hand, SS related RRM are required for high-affinity poly-U RNA binding, and that the epitopes are directly accessible by autoantibodies in Ro/La RNP C-terminal RRM, in conjunction with the SBM downstream, contributes to La interactions with non-poly-U RNA targets such asviral RNAs and TOP (define) mRNAs The specific binding of La 6. Epitope spreading to precursor RNA molecules protects them from exonucleasedigestion thereby regulating their downstream processing. Other The initiation of a vigorous autoimmune response occurs when cellular functions of La/SS-B autoantigen include the retaining of tolerance to self-antigens is broken, a phenomenon that has fasci- precursor RNA molecules in the nucleus, an ATP-dependent heli- nated researchers for over a century. In SLE, the onset and case activity that melts RNAeDNA hybrids, unwinding ability of progression of autoantibody development before the clinical double-stranded RNA, association with telomerase, hence influ- diagnosis has been studied in detail. Arbuckle et al., evaluated encing telomere homeostasis, an RNA chaperone activity per- serum samples obtained from 130 persons before they received formed by transient bipartite (50- and 30-end) binding of nascent a diagnosis of SLE They found that in 88% of the patients with transcripts synthesized by polymerase III (e.g. tRNA precursors) and SLE, at least one SLE autoantibody tested was present before the the induction of cap-independent translation (La binds IRES to diagnosis (up to 9.4 years earlier; mean, 3.3 years). Autoantibodies elements promoting the internal, cap-independent initiation of targeting Ro/La RNP appear earlier than anti-Sm and anti-nuclear translation). La protein is vital for mouse development and the RNP antibodies (a mean of 3.4 years vs. 1.2 years) The propor- establishment of embryonic stem cells. Thus, La/ offspring is not tion of SLE patients with anti-Sm or anti-nuclear RNP antibodies viable and La/ blastocyst outgrowths revealed loss of the inner increases dramatically in the year before diagnosis, indicating that appearance of these autoantibodies herald the clinical onset of the During the last decade, the target B-cell epitopes of anti-La/SS-B disease . In this regard, the rate of appearance of new autoan- autoantibodies have been mapped (Some of the La tibody specificities has been found to gradually increase until the epitopes were found to reside in functional regions of the auto- diagnosis of SLE and to be halted afterwards . The earliest antigen, including the central RNA recognition motif (RRM) and the autoantibodies detected in the pre-clinical period, as individuals ATP binding site. However, the interaction of hY RNA with the RRM progress toward clinical SLE were antibodies to Ro60. McClain et al.
motif did not affect the autoantibody binding in the same region. In mapped the initial, pre-clinical disease target of the anti-Ro60 contrast, the interaction of the ATP binding site with ATP abolished the autoantibody binding at the same part of the protein. Highly (TKYKQRNGWSHK) of the autoantigen . This region belongs to antigenic peptides were identified in the sequences: 147HKAFKGSI154 the previously identified SLE related epitope 169e190aa by Rout- sias et al.
After the initial response against Ro60 autoantigen, the auto- (301e318aa) and 349GSGKGKVQFQGKKTKF364 (349e364aa) . The antibody targets can be expanded to the entire Ro60 molecule by most sensitive and specific epitope in detecting anti-La antibodies a procedure known as epitope spreading. The term epitope was the sequence spanning the sequence 349e364aa, which showed spreading was introduced in the early 1990s to describe the ability a sensitivity and specificity higher than 90%. The presence of anti- of the B and T cell immune response to diversify, at the level of bodies against this epitope was also associated with the HLA- specificity, from a single determinant to many different sites on DQ0501* (Tzioufas Wassmuth, Ann Rheuma Dis). Other epitopes have a given autoantigen . This procedure is not a feature restricted also been identified in other parts of the molecule using recombinant to systemic autoimmune diseases, but is a common characteristic of fragments of La/SS-B or synthetic peptides Their existence is the adaptive immune responses mounted against different patho- believed to be correlated with extended intramolecular spreading of gens. Two types of epitope spreading have been described, (i) the epitopes to the entire La/SS-B molecule.
intramolecular spreading, in which the autoimmune response Recently, we constructed in silico the overall structure of Ro/La spreads in epitopes within the same protein and (ii) the intermo- RNP and studied its structural, antigenic and functional aspects lecular spreading that involves also other protein components, (unpublished observations). Human Ro60 protein was built by physically associated within the same antigenic complex, such as homology modelling from Xenopus Ro (79% similarity), while 81% the spliceosome, and the Ro/La particles In this regard, of human La (spanning amino acids 5e334) was constructed by McClain et al. showed that following immunization of rabbits with assembly of 3 parts: (i) 5e202 aa (La motif þRRM1), (ii) 203e224aa an antigenic peptide of Ro60 autoantigen (274e289aa) led to the

J.G. Routsias, A.G. Tzioufas / Journal of Autoimmunity 35 (2010) 256e264 autoantigens and recently identified as early target of RNP humoralautoimmunity in SLE Thus, besides intramolecular and inter-molecular spreading, the autoimmune response can also diversifyand perpetuate via molecular mimicry of "key driving sequences",common in autoantigens targeted in SLE.
7. Complementary epitopes 7.1. Definition and molecular design In the 1970s Jerne proposed the network hypothesis, in which complementary interactions involving idiotypes and anti-idiotypesof antibodies contributed to the homeostasis of the adaptiveimmune response Antibodies produced against an infectiousagent can elicit anti-idiotypic antibodies that may have the inci-dental property of being antibodies to the host structure. Anti-idiotypic antibodies can either "neutralize" the idiotypic antibodiesor elicit, upon immunization, antibodies with the parental anti-genic specificity. These functions are referred as the idiotypiceanti- Fig. 1. Model showing mechanisms of B-cell epitope spreading. Antigen-presenting idiotypic network serving as intrinsic regulatory mechanism of the cells (APCs) (macrophages or dendritic cells) capture Ro/La complexes (derived from adaptive humoral immune responses. Recently, it has been shown apoptotic or necrotic material) and present La peptides to Th cells, which become that anti-idiotypic antibodies might also act as regulators of the activated and in turn, provide help to autoreactive B cells with anti-La specificity.
Clonal expansion of anti-La B cells occurs, following by their diversi autoimmune response in SLE . Despite the attractive theory, fication to plasma cells, capable of producing anti-La autoantibodies. The same Th cells can activate also B presenting in the opening lines of this section, the detection of anti- cells with anti-Ro specificity, since these cells, acting as antigen-presenting cells, can idiotypic antibodies in clinical specimens is often challenging, since capture Ro/La complexes and present La peptides within their MHCII molecules. This most autoimmune diseases involve polyclonal responses to self results to clonal expansion of anti-Ro B cells, following by diversification to plasma antigen, as a result of the previously mentioned intra- and inter- cells, capable of producing anti-Ro autoantibodies.
molecular spreading. Moreover, some idiotypes are unique to eachpatient, and therefore the performance of general studies and production of antibodies against multiple epitopes of Ro60 and La.
assumptions is often difficult or impossible. The isolation of a non- In addition, antibodies to the common spliceosomal proteins Sm-B0, homogenous population of antibodies and the possibility of cross- Sm-D1, RNP-A and RNP-C were also produced These results reactions, mainly due to polyreactive antibodies .
demonstrated that loss of tolerance to a single antigenic determi- Based on the detailed knowledge of the antigenic structures that nant of the autoantigen can initiate and perpetuate an autoimmune are recognized by autoantibodies, one can design complementary response which virtually recreates the humoral autoimmune epitopes, anticipated to be recognized by anti-idiotypic antibodies, specificity seen in human SLE. However epitope spreading cannot as suggested by the "molecular recognition" theory . According explain how the autoimmune response "jumps" from one particle to this theory, a sense peptide, transcribed and translated from (e.g. hy1 Ro/La RNP) to another (e.g. spliceosome). A clue to the a nucleotide sequence read in the 50 / 30 direction binds to its mechanism involved in the aforementioned production of anti- complementary peptide counterpart, transcribed and translated in bodies to the common spliceosomal proteins has been recently frame with that of its sense peptide from a nucleotide sequence reported by our group and others Since, most of nuclear read in the 50 / 30 direction on the opposite DNA strand. Previous autoantigens in lupus are RNA binding proteins and the major experimental data, utilizing mainly monoclonal antibodies suggest epitopes were previously mapped within their RRM motifs that these interacting complementary peptides have the ability of molecular similarity of conserved sequences within the RRM could generating and eventually detecting interacting pairs of idiotypic be involved in the intermolecular and inter-particle diversification and anti-idiotypic antibodies .
process of autoimmune response. According to this model,a consensus sequence as the RNP motif, conserved in many nuclear, 8. Antibodies to complementary epitopes are anti-idiotypic nucleolar and cytoplasmic antigens, plays the role of a ‘driver' antibodies to autoantibodies epitope. Cross-reactive autoantibodies targeting this epitope havethe potential to spread the autoimmune response to other RNA Studies in our laboratory, using peptides and complementary binding proteins through molecular mimicry. Subsequently, intra- peptides, corresponding to major B-cell epitopes of La/SSB, have molecular spreading to these specific proteins can occur. This demonstrated that in SLE and SS there is an active idiotypiceanti- hypothesis is further supported by the observation that the ‘driving' idiotypic network . The anti-idiotypic antibodies were isolated epitope sequence in RNP motif is a CD4þ T cell epitope in lupus using the complementary epitopes and found to bind anti-La/SSB mice and is often targeted by autoantibodies, very early during the antibodies, competing with La/SSB epitopes for their antigen course of the disease Remarkably, this sequence is present in binding site. In some cases the anti-idiotypic antibodies were components of Ro/La RNP such as Ro60 (aa119e131), La capable of completely masking the anti-La/SSB antibodies, inhibit- (aa146e158) and nucleolin (aa346e358, aa517e529) as well as in ing their anti-La/SSB reactivity. A specific procedure was developed spliceosomal proteins such as RNP-70 (aa139e151) and RNP A with the use of complementary peptides for the release of anti-La/ (aa47e59, aa239e251). Our recent work suggested that the RRM SSB antibodies from their anti-idiotypic counterpart . This region of La/SSB can trigger inter-particle B-cell diversification to procedure applied in anti-La (), anti-Ro/ANA (þ) sera from U1-RNP-70 autoantigen via molecular mimicry confirming the patients with SLE and pSS. Ninety-four percent of pSS sera and 80% above model. The proposed model can be applied also for other of SLE sera were found negative for anti-epitope 349e364 anti- epitopes such as the proline rich region PPPGMRPP that holds bodies in ELISA prior to the treatment with complementary epitope.
a cross-reactive epitope, shared in common by several spliceosomal After unmasking the anti-La antibodies, all SS and SLE sera became

J.G. Routsias, A.G. Tzioufas / Journal of Autoimmunity 35 (2010) 256e264 derives from in vitro and in vivo data, demonstrating that maternalanti-Ro/SSA and/or anti-La/SSB antibodies opsonize fetal apoptoticcardiomyocytes, which in turn induces a proinflammatory/profi-brotic response by phagocytosing macrophages, ultimately leadingto tissue injury. Since, complementary peptides have the potentialto adopt structures that are complementary to B-cell epitopes andmimic the shape of the paratopes of the antibodies recognizingthese epitopes, they can be efficiently used for the detection of anti-idiotypic antibodies. Among the systemic autoimmune diseases,NLS is the ideal model for studying anti-idiotypic antibodies, sincepathogenetic autoantibodies to Ro/SSA and/or La/SSB may bedirectly involved in tissue injury.
In a recent work of our laboratory we evaluated the idiotypic/ anti-idiotypic network of antibodies targeting the dominantepitopes of La/SSB in mothers positive for anti-Ro and/or anti-La/SSB antibodies, aiming to define the role of this network in thedevelopment of NLS To accomplish this task, peptides andcomplementary peptides deduced from the sequences 289e308aaand 349e364aa of La/SSB were synthesized and tested againstmaternal sera. It was found that sera from mothers giving birth toa healthy child and having no history of a child with NLS exhibitedhigher anti-idiotypic antibody activity compared with motherscarrying a child with NLS or mothers giving birth to a healthy child Fig. 2. (A) In neonatal lupus, the presence of anti-idiotypic antibodies to autoanti-bodies against La/SSB may protect the fetus by blocking pathogenic maternal auto- but who previously gave birth to a child with NLS. Sera from antibodies. In this regard, large immunocomplexes are formed that cannot cross the mothers of healthy children, which exhibited no apparent epitope placenta. (B) If anti-Id antibodies do not exist, maternal anti-La IgG autoantibodies pass activity against amino acids 349e364, revealed a significantly through the placenta to the fetal circulation and cause tissue injury to the heart and greater frequency of hidden anti-349e364aa epitope responses, blocked by anti-idiotypic antibodies, as compared with sera from positive for antibodies against the epitope 349e364aa, while none women pregnant with an affected child. Therefore, the presence of of the normal sera exhibited a positive reaction. Animal studies, also anti-idiotypic antibodies to autoantibodies against La/SSB may demonstrated that Balb/c mice immunized with complementary protect the fetus by blocking pathogenic maternal autoantibodies epitopes of La/SSB develop anti-human La/SSB antibodies and testing for these anti-idiotypic responses may be useful suggesting that the complementary epitopes of La/SSB have the in predicting a decreased risk of NLS.
potential of inducing an autoimmune response against La/SSB Type 1 diabetes (T1D) is an autoimmune disease characterized by autoantigen. Recent findings indicate that autoimmunity can be the presence of autoantibodies to multiple islet cell autoantigens.
initiated through an immune response against a peptide that is Autoantibodies to glutamate decarboxylase 65 (GAD65Ab) can be complementary to the autoantigen . Pedergraft III and co- detected in the majority of new-onset T1D patients in patients workers demonstrated that a subset of PR3-ANCA positive patients with latent autoimmune diabetes in adults and in some rare with necrotizing vasculitis harbors also antibodies directed against neurologic diseases, notably Stiff Person Syndrome (SPS) , but the translated protein product of the middle fragment (105e201aa) rarely in the general population. GAD65Ab often herald the onset of of the antisense RNA of PR3, termed complementary PR3 or cPR3 T1D by months or years and are used to predict disease together . These antibodies were not present in patients with vasculitis with other autoantibodies to islet cell In a recent work, Oak and anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA), et al. demonstrated that masked GAD65Ab are present in the healthy patients with SLE, or healthy individuals. It was also demonstrated population and that a lack of particular anti-Ids, rather than that human anti-cPR3 and anti-PR3 antibodies are in fact an idiot- GAD65Ab per se, is a characteristic of T1D . Therefore, anti-Ids ypiceanti-idiotypic pair, since mice immunized with cPR3 develop may play a protective role in the immune response, by preventing both anti-cPR3 and anti-human PR3 antibodies while comple- GAD65Ab to bind to their antigen and potentially modulate T cell mentary PR3 transcripts are present in the peripheral leukocyte responses to GAD65. Lastly, we are very pleased to contribute this RNA from a subset of ANCA patients .
paper to this special issue to recognize the contributions of ProfessorHarry Moutsopoulos, the Chair of our Department as part of the 9. Anti-idiotypic antibodies and disease. The examples special series of distinguished autoimmunologists .
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