Submitted Manuscript: Confidential template updated: February 28 2012 Title: Functional clusters of autoantibodies targeting TLR and SMAD pathways stratify novel subgroups of Systemic Lupus Erythematosus Authors: M. J. Lewis1, M. B. McAndrew2, C. Wheeler2, N. Workman2, P. Agashe2, J. Koopmann2, E. Uddin2, L. Zou1, R. Stark2, J. Anson2, A. P. Cope3, T. J. Vyse4* Affiliations: 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK. 2Oxford Gene Technology, Unit 15, Oxford Industrial Park, Yarnton, Oxfordshire, OX5 1QU, UK. 3Academic Department of Rheumatology, Centre for Molecular and Cellular Biology of Inflammation, King’s College London, London, SE1 9RT, UK. 4Department of Medical and Molecular Genetics, King’s College London, London, SE1 9RT, UK. *To whom correspondence should be addressed: [email protected] Tel. +44 20 7188 8431 Fax +44 20 7188 2585 One Sentence Summary: Using an advanced design of protein microarray, we reveal the identity of over 100 proteins targeted by autoantibodies in the autoimmune disease Systemic Lupus Erythematosus, and show that these novel autoantigens cluster into functionally-related groups involved in toll-like receptor pathways and SMAD signaling. Abstract: The molecular targets of the vast majority of autoantibodies in systemic lupus erythematosus (SLE) are unknown. Using a baculovirus-insect cell expression system to create an advanced protein microarray with improved protein folding and epitope conservation, we assayed sera from 277 SLE individuals and 280 age, gender and ethnicity matched controls. Here, we identified 103 novel autoantigens in SLE sera and show that SLE autoantigens are distinctly clustered into four functionally related groups. The original SLE autoantigens Ro60, La, and SMN1/Sm complex formed the first distinct antigen cluster (SLE1a). A further set of antigens (SLE1b) extended this group with other proteins involved in RNA, DNA and chromatin processing. The largest two subgroups of novel autoantigens involved interconnected genes involved in receptor-regulated SMAD signalling (SLE2) and toll-like receptor pathways and lymphocyte development (SLE3). SLE individuals clustered into four subgroups matching the four functional clusters of autoantigens, which suggests that autoantibody profiles of SLE patients identify subgroups with different underlying pathogenic immune mechanisms. The microarray revealed a significant number of ethnicity-sensitive autoantibodies in keeping with the strong influence of ethnicity on SLE prevalence, severity and organ involvement. Multiple recent failures of new SLE therapies in clinical trials demonstrates that there is a need for improved classification of SLE and related connective tissue diseases. The novel autoantibody clusters identified in this study represent a major advance in SLE diagnostics, and enable identification of new subgroups of SLE, each of which may require different therapeutic strategies. 2 [Main Text: ] Introduction Although first described in 1957, anti-nuclear antibodies (ANA) and anti-double-stranded DNA (dsDNA) antibodies remain the main diagnostic tests for systemic lupus erythematosus (SLE) (1, 2). Following the development of assays for extractable nuclear antigens Ro, La, Sm and U1- RNP, there have been no significant improvements in diagnostic assays for SLE for many years (3). In contrast, the identification of citrullinated proteins as autoantigen epitopes in rheumatoid arthritis (RA) led to a marked improvement in RA diagnostic tests with the development of anti- cyclic citrullinated peptide (CCP) assays. Although numerous SLE-associated autoantibodies have been described (4), they have not significantly improved upon the diagnostic and biomarker abilities of conventional SLE tests, and all too often the true molecular target remains undefined. Protein microarrays using spots of autoantigens applied to glass slides for detecting autoantibodies in sera of SLE patients were first reported in 2002 by Robinson et al (5). This initial study was largely based on existing autoantigens, as were subsequent similar studies, which identified several glomerular antigens and serum factors including B cell-activating factor (BAFF) as autoantibody targets in SLE individuals (6-10). Microarrays utilising large scale de novo synthesis of thousands of proteins have detected autoantibodies in cancer and other diseases (11, 12), but were only able to identify one novel SLE autoantigen, a chloride intracellular channel, CLIC2 (13). Older, first generation protein microarrays may have failed to identify autoantibodies for a number of reasons: bacterial and yeast protein expression systems employed in older studies are prone to generate proteins of poor conformation due to misfolding and lack of higher species post-translational modifications; furthermore, early protein microarrays were not designed to preserve protein epitopes in their native conformation. In this study, we describe a next generation protein microarray utilising 1545 distinct proteins chosen from multiple functional and disease pathways, to probe for the presence of autoantibodies to novel autoantigens in SLE. Our overall aim being to identify previously undiscovered autoantibodies that in combination might act as SLE biomarkers that will substantially improve the diagnostic (and potentially prognostic) performance over existing clinical assays. The protein microarray platform used in this study has been designed to display full-length, correctly folded proteins. Proteins bound to the array are expressed with a biotin carboxyl carrier protein (BCCP) folding tag using a baculovirus insect cell expression system and screened for correct folding before being arrayed in a specific, oriented manner designed to conserve native, discontinuous epitopes (Fig. 1A) (14, 15). This newer design of protein microarray has never before been applied to autoimmune disease. In this study, we set out to validate this novel protein microarray as a platform for improving antibody-based diagnostic tests and to identify the underlying nature of autoantigens in SLE. Results Identification of novel SLE-associated autoantigens During the discovery phase of the study, serum samples from 86 SLE patients and 90 age, gender and ethnicity matched healthy controls were analysed for IgG autoantibody levels against 1545 correctly folded, full-length human proteins using the Discovery array v.3.0 protein array (Oxford Gene Technology, UK) (Fig. 1A). Samples were assayed by ELISA for ANA and anti- dsDNA antibodies for comparison. Candidate autoantigens were identified for validation in two 3 further cohorts of UK SLE patients and tested against a confounding autoimmune disease cohort including individuals with the following conditions: rheumatoid arthritis (n=68), systemic sclerosis (n=12) and primary Sjögren’s syndrome (n=6), polymyositis (n=3) and mixed connective tissue disease (n=3). After correction for multiple testing, the array identified a total of 116 autoantibodies (Figure 1B, Table 1), with higher antibody levels in SLE patients compared to controls, at a false discovery rate (FDR) of <0.01. The well-known SLE autoantigens TROVE2 (Ro60) and SSB (La) showed the most significant difference between SLE and control groups. The array validated a further 11 previously reported SLE autoantigens (Fig. 1C): PABPC1 (16), ANXA1 (Annexin A1) (17), HMGB2 (18), HNRNPA2B1 (19, 20), PSME3 (also known as Ki) (21), ARAF (22), DEK (23), MAP3K14 (NIK) (23), APEX1 (24), MAGEB1 and MAGEB2 (25), RPL10 (26) and PSIP1 (27). A total of 103 novel autoantigens were identified by the microarray, with the most statistically significant six novel autoantibodies shown in Fig. 1D. Ten of the autoantigens have been shown to be implicated in SLE pathogenesis through immunological studies, but were not previously known to be autoantigens: CREB1, ZAP70, VAV1, PPP2CB, TEK (Tie2 receptor), IRF4, IRF5, EGR2, PPP2R5A and LYN (28-33). Five novel autoantigens are the products of SLE susceptibility genes including the well known genes IRF5 and LYN, as well as PIK3C3, NFKBIA and DNAJA1 (34-38). In summary, 26 of the 116 autoantigens have a previously identified link to SLE, either as known autoantibody targets or directly implicated in SLE pathogenesis. In a secondary analysis of all three cohorts pooled, autoantibodies from the array were ranked by positivity in SLE patients, defined as autoantibody levels >2 SD of the control population, and tested for statistical significance using Fisher’s exact test, corrected for multiple testing. The most prevalent autoantibodies were the known SLE autoantigens Ro60 (37.5%), SSB/La (35.4%), HNRNPA2B1 (29.6%) and PSME3/Ki (23.8%) (Fig. 2A). The most prevalent novel autoantibodies were LIN28A (22.4%), IGF2BP3 (21.7%) and HNRNPUL1 (21.3%). SLE patients tended to be simultaneously positive for multiple autoantibodies in contrast to the control group (Kruskal-Wallis test with post-hoc Nemenyi test, P<2 x 10-16) and confounding disease group (P=6.7 x 10-9), with some individuals producing antibodies against over 60 antigens (Fig. 2B). SLE autoantibodies cluster into distinct subgroups Since we observed that groups of autoantibodies showed strong cross-correlation, we performed unsupervised hierarchical clustering of autoantibody levels in SLE individuals (n=277). Four distinct clusters of autoantibodies (Clusters 1a, 1b, 2 and 3) could be observed