Candidate Autoantigen Research Articles

Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA-DRB1*0401-positive humanized mice and rheumatoid arthritis patients

Antibodies toward the citrullinated form of the synovial antigen vimentin are specific for rheumatoid arthritis (RA) and are associated with HLA-DRB1*0401. This suggests that T cells specific for peptides derived from citrullinated vimentin presented in the context of HLA-DRB1*0401 may contribute to the etiopathogenesis of RA. The aim of this study was to identify immunodominant epitopes from citrullinated vimentin presented by HLA-DRB1*0401 and to characterize the resulting T cell responses. We first predicted an HLA-binding T cell epitope from citrullinated vimentin based on the binding motif of HLA-DRB1*0401 and then confirmed its affinity. A class II major histocompatibility complex (MHC) tetramer loaded with the citrullinated form of vimentin aa 59-78 (cit-vimentin aa 59-78) was constructed and used to screen for specific T cells in HLA-DRB1*0401-transgenic mice, patients with RA, and healthy control subjects. Additionally, the cytokine output following cit-vimentin aa 59-78 challenge was analyzed in patients and healthy control subjects by multicolor flow cytometry and Luminex-based analysis. The citrullinated form of vimentin aa 59-78 bound to HLA-DRB1*0401, but the native form could not. Subsequently, cit-vimentin aa 59-78-specific T cells were detected in immunized mice and in the periphery of both HLA-DR*0401-positive healthy control subjects and HLA-DR*0401-positive patients with RA, using class II MHC tetramers, CD154 up-regulation, and intracellular cytokine measurements. As demonstrated in cell culture supernatants, the production of cytokines (predominantly interferon-γ) in response to cit-vimentin aa 59-78 was significantly higher in patients compared with controls. Here, we describe a posttranslational modification of an RA candidate autoantigen toward which HLA-DRB1*0401-restricted T cells can be detected in both patients with RA and healthy controls but for which a proinflammatory response is observed uniquely in patients with RA.

Charge isomers of myelin basic protein: structure and interactions with membranes, nucleotide analogues, and calmodulin.

As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids - a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.

Antigen-specific B cells can acquire and present the rheumatoid arthritis candidate autoantigen aggrecan from a non-internalisable surface

Background and objectivesRecent experiments propose an essential role for B cells in the development of several autoimmune diseases either as autoantibody-producing cells or an antigen presenting cells (APC). However, the...

The HLA‐DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85‐99, with high affinity

Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype carrying the strongest genetic association with MS. In contrast with HLA-DR and -DQ molecules, HLA-DP molecules are poorly characterized with respect to the binding of self-peptides. We show here that HLA-DP2 binds MBP85-99 with high affinity, and that the amino acid residues in position MBP91, MBP92 and MBP93 are influencing the binding, as shown by alanine scans. We further used a series of truncated peptides to identify the core of the binding. Moving the frame along the peptide from residues 87-97 to 89-99 progressively decreased the binding affinity for HLA-DP2, while moving further towards the C-terminal completely abrogated the binding of peptides to HLA-DP2. The data suggest that the docking of the MBP85-99 peptide into the HLA-DP2 groove is dependent on MBP88V and MBP89V and may use either of them as primary anchor for the p1 position. HLA-DP2 might thus present the MBP85-99 peptide in the same register as the HLA-DRB1*1501, where the MBP89V is preferred as the p1 anchor. Notably, full-length MBP was able to compete for peptide binding with an affinity similar to that seen for the high-affinity binding peptides, DRα170-83 and IIP53-65. In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS, MBP85-99, possibly in more than one register.

Cutting Edge Issues in Polymyositis

Skeletal muscle is the target tissue of immunoflogistic processes in patients affected with idiopathic inflammatory myopathies (IIM). IIM are classified into three major forms: polymyositis (PM), dermatomyositis (DM), and inclusion body myositis. Recent data suggest that, in the major subsets of myositis, antigens in muscles drive a B-cell antigen-specific immune response. Moreover, some non-immunological mechanisms have been advocated. In this regard, an increased expression of Jo-1 and Mi-2 in muscle biopsies from PM and DM patients compared to normal muscle has been demonstrated; these candidate autoantigens in myositis are expressed at high levels in regenerating muscle cells rather than in mature myotubes. Myositis autoantigen upregulation has also been observed in neoplastic tissues, thus representing a potential link between cancer and autoimmunity in myositis. Myositis-specific autoantibodies (MSA) are disease markers and target intracellular proteins involved in key processes such as translocation and nuclear transcription. Myositis target antigens encompass aminoacyl-tRNA synthetases, the Mi-2 helicase/histone deacetylase protein complex, the signal recognition particle ribonucleoprotein, together with novel target antigens including p155/140, CADM-140, and SAE. Despite their high specificity for autoimmune myositis, MSA target non-muscle restricted proteins ubiquitary to all cell types, making the specific muscle involvement difficult to explain. Non-immunological mechanisms also seem to contribute to the pathogenesis of IIM; activation of endoplasmic reticulum stress response due to muscle regeneration and inflammation but independent to MHC-1 up-regulation has been recently reported in patients with myositis.

Identification of a novel autoantigen in inflammatory bowel disease by protein microarray

Patients with inflammatory bowel disease (IBD) display immunoreactivity to self-antigens and microbial antigens. We used a protein microarray approach to identify novel autoantigens in IBD. ProtoArray Human Protein Microarray v4.0 containing 8268 human proteins from Invitrogen (La Jolla, CA) was used. Twenty-five IBD patients and five healthy controls were screened for candidate autoantigens. For 256 antigens, IBD patients had a higher seroreactivity than controls. Twenty antigens were selected for further evaluation in a larger cohort (60 ulcerative colitis [UC] patients, 60 Crohn's disease [CD] patients, 60 healthy controls, and 60 gastrointestinal-diseased controls) by means of a customized protein microarray. Out of these 20 antigens, one antigen, family with sequence similarity 84 member A (FAM84A), was identified as a target antigen in IBD. Antibodies to FAM84A were significantly more prevalent in IBD patients (19%) than in gastrointestinal-diseased controls (1.7%) (P = 0.0008) and healthy controls (5%) (P = 0.01). Anti-FAM84A antibodies were found in 26.6% of UC patients and in 11.7% of CD patients. FAM84A was confirmed as target antigen in IBD by means of Western blotting in a large independent cohort (100 UC patients, 106 CD patients, 102 healthy controls, and 100 gastrointestinal-diseased controls). Antibodies to FAM84A were significantly more prevalent in IBD patients (20%) than in gastrointestinal-diseased controls (5%) (P = 0.0004) and healthy controls (0%) (P < 0.0001). Anti-FAM84A antibodies were found in 18% of UC patients and in 22% of CD patients. We identified FAM84A as a novel autoantigen in IBD. (Inflamm Bowel Dis 2011;).

Identification of Hnrph3 as an autoantigen for acute anterior uveitis

Acute anterior uveitis (AAU) is the most common form of autoimmune uveitis in the eye with few known autoantigens. Identification of autoantigens will improve our understanding of the molecular mechanisms and capability for disease diagnosis. Phage display is a powerful technology for autoantigen identification. However, because of uncontrollable reading frames, phage display with conventional cDNA libraries identifies high percentage of non-open reading frames (non-ORFs) with minimal implications for autoantigen identification. We recently developed ORF phage display technology with minimal reading frame problem. Herein we used ORF phage display to identify 18 patient-specific clones, including 16 ORFs encoding endogenous proteins as candidate autoantigens for AAU. One of the identified antigens was heterogeneous nuclear ribonucleoprotein H3 (Hnrph3) that was further characterized for AAU relevance and independently verified by Western blot. These results demonstrate that ORF phage display is a valuable approach for identification of unknown autoantigens.

Thiopalmitoylated Peptides from the Peripheral Nervous System Myelin P0 Protein: Synthesis, Characterization, and Neuritogenic Properties

Thiopalmitoylation (i.e., the covalent attachment of palmitic acid via a thioester linkage to cysteine residues in the polypeptide backbone) is a common post-translational modification of proteins. Several proteins that have been identified as putative autoantigens in a variety of T-cell mediated autoimmune diseases are thiopalmitoylated, and thus, we have hypothesized that endogenous thiopalmitoylated peptides released during tissue breakdown may play a role in the development and chronicity of autoimmune diseases. To investigate this, we have studied the effect of thiopalmitoylation on the immunogenic and neuritogenic properties of P0, the major peripheral nervous system (PNS) myelin protein, which is thiopalmitoylated at cysteine 153, and described as a candidate autoantigen in Guillain-Barre syndrome (GBS), a human inflammatory demyelinating disease of the PNS. This paper describes the synthesis of palmitoylated peptide P0(180-199) and P0(152-171) by on-resin acylation using specific cysteine side-chain protecting groups: Mmt (labile in diluted acid) and StBu (labile in the presence of tributylphosphine). Our results show that the thiol protecting group had to be adjusted to the peptide sequence: Mmt was efficiently used for P0(180-199) thioacylation, but it was not suitable for thiopalmitoylation of P0(152-171) because of a premature deprotection of the Boc protecting group on the epsilon-NH(2) Lys in the presence of 2% TFA, leading to dipalmitoylation. Palmitoylated P0(152-171) was successfully obtained by using StBu as the thiol protecting group. We could show by circular dichroism that palmitoylation has no influence on the structuration of the peptide in solution but palmitoylation increased the stability of the peptide in the presence of serum. Using EAN (experimental autoimmune neuritis), the rat model of GBS, we have compared the immunological properties of palm and non-palm P0 peptides and showed that thiopalmitoylation has indeed a great influence on their neuritogenic and immunogenic properties. This study provides further support for our hypothesis concerning the role of thiopalmitoylation in the development and chronicity of inflammatory demyelinating diseases and confirms that thiopalmitoylation of peptides may provide a simple means to increase MHC class II restricted responses.

MS analysis of rheumatoid arthritic synovial tissue identifies specific citrullination sites on fibrinogen

Citrullination is a post-translational modification of arginine residues to citrulline catalyzed by peptidyl arginine deiminases. Induced expression of citrullinated proteins are frequently detected in various inflammatory states including arthritis; however, direct detection of citrullination in arthritic samples has not been successfully performed in the past. Citrullination of human fibrinogen, a candidate autoantigen in arthritis, was studied. Accurate identification of citrullinated fibrinogen peptides from rheumatoid arthritis synovial tissue specimens was performed using accurate mass and retention time analysis. A peptide with the sequence ESSSHHPGIAEFPSRGK corresponding to amino acids 559-575 of fibrinogen α-chain was identified to be citrullinated with an occupancy rate between 1.4 and 2.5%. Citrullination of the peptide KREEAPSLRPAPPPISGGGYRARPAK corresponding to amino acids 52-77 of the fibrinogen β-chain was identified with an occupancy rate of 1.2%. We report a proof of principle study for the identification of citrullinated proteins and within them, identification of citrullination sites and quantification of their occupancies in synovial tissue from rheumatoid arthritis patients using high-resolution MS. Detailed studies on which molecules are citrullinated in arthritis can provide information about their role in immune regulation and serve as novel biomarkers and potentially even as therapeutic targets.

The antibody response to oligodendrocyte specific protein in multiple sclerosis

The role of autoantibody responses in pathogenesis or progression of multiple sclerosis (MS) remains controversial. This is partly because the methods that can distinctly identify pathogenic antibody reactivities targeting native membrane proteins from the reactivities that originate as an epiphenomenon in such disease are just emerging. Oligodendrocyte specific protein (OSP or claudin-11) is a candidate autoantigen in MS and CSF reactivity towards OSP has been reported in MS patients. We characterized the autoantibody response to OSP using sensitive cell based assays. In line with a previous report, higher antibody response to OSP 114–120 peptide and denatured protein was observed. However applying assays based on native OSP we did not observe any specific OSP response in MS patients. Our results demonstrate that anti-OSP antibodies do not recognise native glial OSP and may therefore rather represent an epiphenomenon in MS.

The Endoplasmic Reticulum Stress-Inducible Protein, Herp, Is a Potential Triggering Antigen for Anti-DNA Response

Anti-dsDNA Abs are highly specific indicators of systemic lupus erythematosus (SLE) and play a pathogenic role in lupus nephritis. Human anti-dsDNA Abs are most likely generated by an Ag-driven mechanism. However, the Ag responsible for triggering anti-dsDNA Ab production has not been identified. To search for proteins that are cross-reactive with anti-dsDNA Abs, we screened a cDNA library from a patient with SLE with single-chain Fv of O-81 human anti-ss/dsDNA mAb by using a two-hybrid system. Homocysteine-induced ER protein (Herp), an endoplasmic reticulum (ER) stress-inducible ER membrane protein, was identified and shown to bind to original O-81 Ab and human lupus anti-dsDNA Abs. Some IgG purified from patients with active SLE by Herp-immobilized affinity chromatography bound to dsDNA. BALB/c mice immunized with Herp showed IgG anti-dsDNA Abs, IgG anti-nucleosome Abs, and glomerular IgG deposition. Herp reactivity was strongly positive in a proportion of PBLs from patients with active SLE, but undetectable in those from healthy controls. Moreover, activation of caspases was observed in the Herp-positive cells, implying that ER stress-induced apoptosis likely occurs in patients with active SLE. Herp is exposed on blebs of ER stress-induced apoptotic cells, suggesting that Herp can be recognized by immune cells. These results indicate that Herp mimics structural determinants of DNA immunologically and can be immunogenic in vivo. Thus, Herp represents a candidate autoantigen for anti-DNA Abs. This study may help explain how common environmental factors induce the production of anti-DNA Abs and contribute the development of SLE.

Immune therapy for type 1 diabetes mellitus—what is unique about anti-CD3 antibodies?

Type 1 diabetes mellitus (T1DM) is a prototypic organ-specific autoimmune disease that results from selective destruction of insulin-secreting beta-cells by immune-mediated inflammation (insulitis), that is, the infiltration of pancreatic islets by autoreactive CD4(+) and CD8(+) T lymphocytes. Current treatment is substitutive-chronic use of exogenous insulin-which, in spite of considerable advances, is still associated with constraints and lack of effectiveness over the long-term in relation to the prevention of vascular and neurological complications. Finding a cure for T1DM is an important medical health challenge, as the disease's incidence is steadily increasing in industrialized countries and projections of future prevalence are alarming. Crucially, as T1DM mainly affects children and young adults, any candidate immune therapy must be safe and avoid chronic use of immunosuppressants that promote sustained depression of immune responses. The ideal approach would, therefore, involve induction or, in the case of established T1DM, restoration of immune tolerance to target autoantigens. This Review presents, in particular, two strategies that are still in clinical development but hold great promise. These strategies are focused on the use of candidate autoantigens and anti-CD3 monoclonal antibodies.

Identification of complement C3 as an autoantigen in inflammatory bowel disease

Autoantibodies against goblet cells in the gastrointestinal mucosa have been described in patients with inflammatory bowel disease (IBD) but a corresponding autoantigen has not yet been identified. The aim of this study was to identify such an antigen. First, 10 candidate autoantigens were discarded based on double stainings of appendiceal sections and a mucin-producing cell line (HT29-mtx). Second, an appendiceal cDNA library was immunoscreened with IBD sera. Three out of 48 positive clones were identified as complement C3. Using immunoprecipitation of in vitro transcribed and translated C3, seven of 17 primary sclerosing cholangitis patient sera, 15 of 65 IBD sera, and none out of 54 sera from healthy blood donors showed C3 immunoreactivity. The results were confirmed using western blot and an enzyme-linked immunosorbent assay with alternative sources of C3 protein. In conclusion, we have identified complement C3 as a potential autoantigen in IBD and primary sclerosing cholangitis.

Identification of autoantigens specific for systemic lupus erythematosus with central nervous system involvement

Using proteomic analysis, we identified candidate autoantigens specific for central nervous system (CNS) involvement in systemic lupus erythematosus (SLE). Proteins, extracted from cultured human neuroblastoma cells, were separated both by SDS-PAGE (1-DE) and two-dimensional electrophoresis (2-DE), and transferred to membranes. Western blot analysis was performed using serum samples from 30 SLE patients with CNS involvement (CNS-Lupus) and from 30 SLE patients without CNS involvement (non-CNS-SLE). The detected autoantigens were identified using MALDI-TOF/TOF MS. On the 1-DE Western blot, we detected 32 antigenic bands in the serum samples from the CNS-Lupus patients. Among them, four bands were detected significantly more frequently in the CNS-Lupus patients than in the non-CNS-SLE patients. Three bands were detected in four or more of the CNS-Lupus patients but in only one or none of the non-CNS-SLE patients. We thus selected these seven bands for the next investigations. Next, we detected protein spots corresponding to the selected seven bands by 2-DE Western blot and identified four proteins. They are peroxiredoxin-4, ubiquitin carboxyl-terminal hydrolase isozyme L1, splicing factor arginine/serine-rich 3, and histone H2A type 1. These four candidate autoantigens for the anti-neuronal cell antibodies would be a useful marker for CNS-Lupus.

Annexin A2 on lung epithelial cell surface is recognized by severe acute respiratory syndrome-associated coronavirus spike domain 2 antibodies

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection causes lung failure characterized by atypical pneumonia. We previously showed that antibodies against SARS-CoV spike domain 2 (S2) in the patient sera can cross-react with human lung epithelial cells; however, the autoantigen is not yet identified. In this study, we performed proteomic studies and identified several candidate autoantigens recognized by SARS patient sera in human lung type II epithelial cell A549. Among the candidate proteins, annexin A2, which was identified by mass spectrometry analysis and had the highest score by Mascot data search, was further characterized and investigated for its role as an autoantigen. By confocal microscopic observation, SARS patient sera and anti-S2 antibodies were co-localized on A549 cells and both of them were co-localized with anti-annexin A2 antibodies. Anti-annexin A2 antibodies bound to purified S2 proteins, and anti-S2 bound to immunoprecipitated annexin A2 from A549 cell lysate in a dose-dependent manner. Furthermore, an increased surface expression and raft-structure distribution of annexin A2 was present in A549 cells after stimulation with SARS-induced cytokines interleukin-6 and interferon-γ. Cytokine stimulation increased the binding capability of anti-S2 antibodies to human lung epithelial cells. Together, the upregulated expression of annexin A2 by SARS-associated cytokines and the cross-reactivity of anti-SARS-CoV S2 antibodies to annexin A2 may have implications in SARS disease pathogenesis.

Autoimmunity against Fibrinogen Mediates Inflammatory Arthritis in Mice

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the presence of anticitrullinated protein Abs, although the exact targets and role of anticitrullinated protein autoimmunity in the pathogenesis of RA remain to be defined. Fibrinogen, which can be citrullinated, has recently emerged as a candidate autoantigen. To determine whether autoimmunity against fibrinogen can mediate inflammatory arthritis, we immunized a variety of common mouse strains with fibrinogen and found that DBA/1 and SJL mice developed an inflammatory and erosive arthritis. Mice with fibrinogen-induced arthritis (FIA) possess fibrinogen-reactive T cells that produce the proinflammatory cytokines IL-6, IL-17, TNF-alpha, and IFN-gamma. FIA can be adoptively transferred with either plasma or fibrinogen-specific T cells from diseased mice. Mice with FIA possess rheumatoid factor, circulating immune complexes, and anticyclic citrullinated peptide Abs, all of which are characteristic of human RA. These observations demonstrate that fibrinogen is arthritogenic in mice and that the pathogenesis of FIA is mediated by both autoantibodies and fibrinogen-reactive T cells.

High-Throughput Analysis of Antigen Recognition by the B Cell Receptors of Malignant Lymphomas with High-Density Protein Microarrays.

Abstract 2945Poster Board II-921The etiology of indolent B cell lymphomas (iNHL) is largely unknown. However, systemic autoimmune diseases are associated with certain iNHL. Organ-restricted chronic inflammation, i.e. H. pylori-associated gastritis, Sjogren's syndrome, and others, plays an etiological role in extranodal marginal zone lymphoma (MZL). Finally, shared structural features of the B cell receptor (BCR) suggest antigen recognition in iNHL etiology, particularly in CLL. Indeed, recognition of common autoantigens has been shown for CLL BCR. However, there has been no unbiased comparative assessment of antigen binding by iNHL BCR.We measured binding of lymphoma BCR simultaneously to 8000 human proteins displayed on high-density microarrays. BCR from 45 lymphomas, including 13 mantle cell lymphomas (MCL), 10 CLL, 5 nodal MZL, 5 diffuse large B-cell lymphomas (DLBCL), 4 follicular lymphomas (FL), 3 myelomas, 2 splenic MZL, and 2 LPL, were expressed as recombinant Fab fragments in E. coli. The most abundant among the 19 represented different VH segments were VH 4-34 (n=10), 1-69 (6), 3-30 (4), 3-21 (3), and 1-8 (3). Bound Fab was detected by 647AlexaFluor-goat-anti-human IgG. Z-scores and Z-factors (Zhang et al., 1999) were calculated for each Fab-protein interaction. Fab binding was defined as either Z-score >1.65 and Z-factor >0, or Z-score >1 and Z-factor >0.5. 108 robust Fab-Protein interactions were identified that involved 48 different proteins. 21 NHL BCR, derived from all lymphoma types, did not bind any protein. 12 BCR recognized one protein only. 9 BCR were highly polyreactive as defined by binding to ≥5 proteins (3 VH4-34-utilizing Fabs, 3 VH1-69 Fabs, 1 VH1-8 Fab, 1 VH3-21 Fab, and the only VH2-26 Fab). All Fab-protein interactions were analyzed by biclustering after adaptation of the Bimax algorithm used in gene expression studies to the protein microarray platform. 28 biclusters involving 11 Fabs and 21 proteins were identifed. 11 of the bicluster proteins have a nuclear localization; the cellular localization of 6 proteins is unknown. Subsequent hierarchical clustering of the biclusters distinguished three separate clusters. The DILIMOT algorithm identifed GxAxSxA as a potential consensus protein motif among 8 proteins clustering together (Scons=5.29×10-23, p=3.83×10-8). Although the arrays used here carried the most comprehensive assembly of proteins available, they represent only a small fraction of the human proteome. Therefore, homologues to recognized proteins were identified by BLAST search and included, among others, the paraneoplastic neuronal autoantigen Ma1 as being potentially recognized by 4 lymphomas, including a primary CNS DLBCL, and 2 cell wall proteins of pathogenic bacteria. There was no preponderance of any particular NHL entity within the biclusters, except a group of 3 MZL Fabs. Each of these MZL BCR utilized VH1-69 and Vk3-20, and all 3 were classified as polyreactive with very similar protein recognition patterns, including calcium binding and coiled domain 1 and the autoantigens cardiolipin and Ro-60/SS-A.This study of a broad selection of lymphoma types and BCR structures establishes protein microarrays as a novel and valuable platform to study antigen recognition by lymphoma cells in an unbiased and quantitative fashion, and thereby to deduct a comprehensive view of antigen stimulation in iNHL development. The currently available array generations permit unbiased identification of directly recognized candidate autoantigens and potentially bound homologues in appr. 60% of cases. The remaining cases may recognize carbohydrate and/or microbial antigens not represented on the array. In addition, the development of appropriate bioinformatic tools within this study permitted to define recurrent oligo- and polyreactivity patterns of antigen recognition by lymphoma BCR. With the possible exception of MZL, these patterns appear to operate across various lymphoma entities, generally suggesting two different components in lymphoma development: A requirement for BCR-mediated stimulation in the majority of cases which may occur during various scenarios and may be specific or follow definable patterns of cross-reactivity, and full malignant transformation of the stimulated cell by genetic alterations. The latter step would be expected to define the lymphoma type through the nature of the oncogenic event and the maturation stage of the cell of origin. Disclosures:No relevant conflicts of interest to declare.

Using biology of disease to understand and guide therapy of JIA

Juvenile idiopathic arthritis (JIA) is a heterogeneous, multi-factorial disease. The ability to distinguish various subtypes of JIA has enabled more directed and uniform clinical care. Possible triggers of the disease include genetic and environmental factors such as infections and vaccinations. Autoreactive T cells responding to unknown antigens are among the mechanisms that perpetuate the cycle of autoimmune inflammation once established. There are two groups of candidate auto-antigens that are thought to play a role in induction or modulation of T-cell responses. The first group is derived from cartilage and other joint-related tissue and the second group is derived from stress proteins. Several molecules have been implicated in the quest to restore immune homeostasis and tolerance, such as regulatory T cells, FoxP3, heat shock proteins, cytokines, chemokines and other key mediators of inflammation. Treatment strategies might focus on the induction of regulation in a more specific fashion.

A broad screen for targets of immune complexes decorating arthritic joints highlights deposition of nucleosomes in rheumatoid arthritis

Deposits of Ig and complement are abundant in affected joints of patients with rheumatoid arthritis (RA) and in animal models of RA in which antibodies are demonstrably pathogenic. To identify molecular targets of the Igs deposited in arthritic joints, which may activate local inflammation, we used a combination of mass spectrometry (MS) and protein microarrays. Immune complexes were affinity-purified from surgically removed joint tissues of 26 RA and osteoarthritis (OA) patients. Proteins complexed with IgG were identified by proteomic analysis using tandem MS. A striking diversity of components of the extracellular matrix, and some intracellular components, copurified specifically with IgG from RA and OA tissues. A smaller set of autoantigens was observed only in RA eluates. In complementary experiments, IgG fractions purified from joint immune complexes were tested on protein microarrays against a range of candidate autoantigens. These Igs bound a diverse subset of proteins and peptides from synovium and cartilage, different from that bound by normal serum Ig. One type of intracellular protein detected specifically in RA joints (histones H2A/B) was validated by immunohistology and found to be deposited on the cartilage surface of RA but not OA joints. Thus, autoantibodies to many determinants (whether deposited as "neoantigens" or normal constituents of the extracellular matrix) have the potential to contribute to arthritic inflammation.

Novel Autoimmune Hepatitis-Specific Autoantigens Identified Using Protein Microarray Technology

Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the liver with a poorly understood etiology. Detection of nonorgan-specific and liver-related autoantibodies using immunoserological approaches has been widely used for diagnosis and prognosis. However, unambiguous and accurate detection of the disease requires the identification and characterization of disease-specific autoantigens. In the present study, we have profiled the autoantigen repertoire of patients with AIH versus those with other liver diseases, identifying and validating three novel and highly specific biomarkers for AIH. In phase I, we fabricated a human protein chip of 5011 nonredundant proteins and used it to quickly identify 11 candidate autoantigens with relative small serum collection. In phase II, we fabricated an AIH-specific protein chip and obtained autoimmunogenic profiles of serum samples from 44 AIH patients, 50 healthy controls, and 184 additional patients suffering from hepatitis B, hepatitis C, systemic lupus erythematosus, primary Sjogren's syndrome, rheumatoid arthritis, or primary biliary cirrhosis. With this two-phase approach, we identified three new antigens, RPS20, Alba-like, and dUTPase, as highly AIH-specific biomarkers, with sensitivities of 47.5% (RPS20), 45.5% (Alba-like), and 22.7% (dUTPase). These potential biomarkers were further validated with additional AIH samples in a double-blind design. Finally, we demonstrated that these new biomarkers could be readily applied to ELISA-based assays for use in clinical diagnosis/prognosis.