Human Autoantigen Research Articles

The critical amino acids of a nephritogenic epitope on human Goodpasture autoantigen for binding to HLA-DRB1*1501

BackgroundAnti-GBM disease is caused by autoimmunity to Goodpasture antigen on α3(IV)NC1 and had strong associations with HLA-DRB1*1501. Previous studies identified α3127-148 (P14: TDIPPCPHGWISLWKGFSFIMF) as a T cell epitope. The present study was aimed to investigate the binding capacity of P14 to HLA-DRB1*1501 and the critical amino acids for this binding. MethodsA line of EBV-transformed human B cells homozygous for HLA-DRB1*1501 was used to detect the binding capacity of peptides to HLA-DRB1*1501 using flow cytometry analysis. P14 was sequentially truncated into 8 peptides with 15 amino acids to identify the core binding motif. A set of alanine substituted peptides of P14-2 was then synthesized to identify its critical residues for binding to HLA-DRB1*1501. The structure of HLA-DR2b-Peptide-TCR complex was constructed by modeling to analyze the interaction of each amino acids of P14-2 with the HLA-DR2b molecule. ResultsP14 could bind to HLA-DRB1*1501 expressed on B cell surface. The N-terminus of P14 was the core binding motif and the truncated peptide P14-2 (DIPPCPHGWISLWKG) 128-142 had the strongest binding capacity. After sequential amino acid substitution, we found the binding capacity of P14-2 was completely lost by the substitution of cysteine (C) 132 and significantly decreased by the substitution of tryptophan (W) 136, lysine (K) 141, or glycine (G) 142, but still at a high level. The modeling showed that (C) 132 had a strong interaction with pocket 4 on the β chain of DR2b. Thus, C132, W 136, K141, and G142 were defined as the critical amino acid residues for the binding capacity of P14 to HLA-DRB1*1501. ConclusionWe identified α3128-142 (DIPPCPHGWISLWKG) as the core binding motif of P14 to HLA-DRB1*1501 molecule. And the critical amino acid residues for this binding were further defined as C132, W 136, K 141, and G 142.

AAgAtlas 1.0: a human autoantigen database.

Autoantibodies refer to antibodies that target self-antigens, which can play pivotal roles in maintaining homeostasis, distinguishing normal from tumor tissue and trigger autoimmune diseases. In the last three decades, tremendous efforts have been devoted to elucidate the generation, evolution and functions of autoantibodies, as well as their target autoantigens. However, reports of these countless previously identified autoantigens are randomly dispersed in the literature. Here, we constructed an AAgAtlas database 1.0 using text-mining and manual curation. We extracted 45 830 autoantigen-related abstracts and 94 313 sentences from PubMed using the keywords of either ‘autoantigen’ or ‘autoantibody’ or their lexical variants, which were further refined to 25 520 abstracts, 43 253 sentences and 3984 candidates by our bio-entity recognizer based on the Protein Ontology. Finally, we identified 1126 genes as human autoantigens and 1071 related human diseases, with which we constructed a human autoantigen database (AAgAtlas database 1.0). The database provides a user-friendly interface to conveniently browse, retrieve and download human autoantigens as well as their associated diseases. The database is freely accessible at http://biokb.ncpsb.org/aagatlas/. We believe this database will be a valuable resource to track and understand human autoantigens as well as to investigate their functions in basic and translational research.

Proteomic profile of circulating immune complexes in chronic Chagas disease

Immune complexes (ICs) are the direct and real-time products of humoral immune responses. The identification of constituent foreign or autoantigens within ICs might bring new insights into the pathology of infectious diseases. We applied immune complexome analysis of plasma to the study of Chagas disease caused by Trypanosoma cruzi. Twenty seropositive plasma samples including cardiac and/or megacolon determinate patients (n = 11) and indeterminate (n = 9) were analysed along with 10 seronegative individuals to characterize the antigens bound to circulating ICs. We identified 39 T. cruzi antigens and 114 human autoantigens specific to patients with Chagas. Among those antigens, two T. cruzi antigens (surface protease GP63, glucose-6-isomerase) and six human autoantigens (CD180 antigen, ceruloplasmin, fibrinogen beta chain, fibrinogen beta chain isoform 2 preprotein, isoform gamma-A of fibrinogen γ-chain, serum paraoxonase) were detected in more than 50% of the patients tested. Human isoform short of complement factor H-related protein 2 and trans-sialidase of T. cruzi were more frequently found in the indeterminate (5/9 for both) compared with in the determinate Chagas (0/11, P = 0·046 for human, 1/11, P = 0·0498 for T. cruzi). The immune complexome could illustrate the difference of immune status between clinical forms of chronic Chagas disease.

Lupus T and B cell cross-reactivity between the human Ro60 autoantigen and Ro60 orthologs from the human microbiota

Abstract Human Ro60 is an RNA binding protein that is commonly targeted in systemic autoimmunity. We identified Ro60 orthologs that are conserved in a subset of skin, oral, and gut commensal species. Since anti-Ro60 antibodies are the earliest autoantibodies detected in lupus patients, we hypothesized that commensal Ro60 orthologs may trigger autoimmunity via autoepitope cross-reactivity in genetically susceptible individuals. While Ro60-producing gut commensals were common among both healthy controls and lupus patients, only antibodies from 4 Ro60-positive lupus patients, but not Ro60-negative subjects, co-immunoprecipitated Ro60 and its bound Y RNA from the Ro60-containing skin commensal P. propionicum. This suggests antibody cross-reactivity between human Ro60 antibodies and commensal Ro60. Next, Ro60-specific CCR6+ and CCR6- CD4 memory T cells clones from lupus patients were generated using a T cell library assay. Ro60 CCR6+ T cell clones proliferated in response to P. propionicum, demonstrating T cell cross-reactivity with commensal Ro60. Finally, germ-free mice monocolonized with B. thetaiotaomicron, a Ro60 ortholog-containing gut commensal, produced fecal anti-human Ro60 IgA antibodies (n=4, p=0.04), linking anti-Ro60 commensal responses in vivo with the generation of human Ro60 autoantibodies. In summary, Ro60 autoimmune T and B cells from human lupus patients cross-reacted with commensal Ro60 in vitro and commensal Ro60 triggered anti-Ro60 antibodies in vivo. Taken together, these data suggest that colonization with autoantigen ortholog-carrying species may sustain chronic autoimmunity in patients. This concept could lead to development of novel therapeutic approaches targeted at the human microbiota.

Abstract B021: Treatment with a novel targeting module, redirecting UniCAR T cells against PSCA, delays subcutaneous tumor growth and prolongs survival of tumor-bearing NSG mice

Abstract Background: The adoptive transfer of T cell engineered with chimeric antigen receptors (CARs) is currently considered a highly promising therapeutic option for treating so far incurable malignant diseases. In order to reduce adverse reactions associated with CAR engineered T cells, a flexible modular universal chimeric antigen receptor (UniCAR) platform is developed, which allows a precise control of CAR T cell reactivity to manage side effects while preserving efficacy. The UniCAR technology splits the signaling and antigen-binding aspects of conventional CARs into two individual components. The UniCAR is structurally similar to second-generation CARs, however the single chain variable fragment (scFv) does not recognize a cell surface antigen but a short peptide motif derived from a human nuclear autoantigen. Antigen-specificity and crosslinking of UniCAR expressing T cells to target cells is provided by a separate targeting module (TM) compromising a scFv, binding to specific cell surface antigens, fused to the aforementioned peptide motif. Thus, T cells engineered to express UniCARs remain inactivated after infusion, since the UniCAR target is not available for binding under physiological conditions. Only in the presence of the TM, antigen specific activation of UniCAR T cells occurs, which allows for a more sophisticated regulation. Methods: We developed a novel radio-immune target module (TM), which consists of an anti-PSCA (prostate stem cell antigen) scFv for redirecting of UniCARs to PSCA expressing tumor cells. Three different tags are fused in a row to the C-Terminus of the scFv to functionalize the molecule for combined immune- and radiotherapy. First, a ten amino acid (aa) long tag mediates the crosslink to T cells genetically modified with the UniCAR. Further downstream, a peptide tag (Rx) for enhanced conjugation of radionuclides and finally, a 6xhistidine (His) tag are located. TM-Rx efficacy was evaluated for target cell killing in-vitro and compared to a TM without Rx-tag. In-vivo efficacy of the TM-Rx was also investigated in a high and low-tumor burden mouse model. Results: Rx-TM and TM equivalently kill PSCA-expressing tumor cells in-vitro in presence of UniCARs in an E:T (Effector:Target) ratio of 1:1 after 24 hours. UniCAR T cell engrafted NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice show no clinical signs of autoimmunity neither in presence nor absence of the TM. One week treatment with TM delays subcutaneous tumor growth and prolongs survival of tumor bearing, UniCAR T cell engrafted NSG mice. Longterm infiltration of tumor tissue by UniCARs could be observed over 16 weeks in the high and low-tumor burden ectopic carcinoma model. Conclusion: The UniCAR technology shows efficacy both in-vitro and in-vivo. Extension of the TM with an additional tag does not impair functionality of the UniCAR platform. Short-treatment prolongs survival of tumor bearing mice but does not lead to cure, therefore combination with other treatment modalities e.g. radiation oncology seems reasonable. Citation Format: Elham Pishali Bejestani, Malte von Bonin, Marc Cartellieri, Armin Ehninger, Mechthild Krause, Michael Baumann, Gerhard Ehninger, Michael Bachmann. Treatment with a novel targeting module, redirecting UniCAR T cells against PSCA, delays subcutaneous tumor growth and prolongs survival of tumor-bearing NSG mice. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B021.

Intrathecal CD4(+) and CD8(+) T-cell responses to endogenously synthesized candidate disease-associated human autoantigens in multiple sclerosis patients.

MS pathology is potentially orchestrated by autoreactive T cells, but the antigens recognized remain unknown. A novel APC/T-cell platform was developed to determine intrathecal CD4(+) and CD8(+) T-cell responses to candidate MS-associated autoantigens (cMSAg) in clinically isolated syndrome (CIS, n = 7) and MS (n = 6) patients. Human cMSAg encoding open reading frames (n = 8) were cloned into an Epstein-Barr virus (EBV)-based vector to express cMSAg at high levels in EBV-transformed B-cells (BLCLs). Human cMSAg cloned were myelin-associated and -oligodendrocyte glycoprotein, myelin basic protein, proteolipid protein, ATP-dependent potassium channel ATP-dependent inwards rectifying potassium channel 4.1, S100 calcium-binding protein B, contactin-2, and neurofascin. Transduced BLCLs were used as autologous APC in functional T-cell assays to determine cMSAg-specific T-cell frequencies in cerebrospinal fluid derived T-cell lines (CSF-TCLs) by intracellular IFN-γ flow cytometry. Whereas all CSF-TCL responded strongly to mitogenic stimulation, no substantial T-cell reactivity to cMSAg was observed. Contrastingly, measles virus fusion protein-specific CD4(+) and CD8(+) T-cell clones, used as control of the APC/T-cell platform, efficiently recognized transduced BLCL expressing their cognate antigen. The inability to detect substantial T-cell reactivity to eight human endogenously synthesized cMSAg in autologous APC do not support their role as prominent intrathecal T-cell target antigens in CIS and MS patients early after onset of disease.

Comment on: Dietary yeast reduce inflammation in central nerve system via microflora.

We recently read with great interest the article on the possible immunomodulatory effects of dietary yeast, specifically Candida kefir, on the central nervous system, notably the experimental autoimmune encephalomyelitis (EAE) mouse model by Takata et al.1 The role of yeast in inflammatory and autoimmune phenomena is nowadays controversial.2 The experiment from Takata et al. raises an important observation that not all yeast lead to the same effect on immunity. For instance, a significant reduction of proinflammatory cytokines such as IL‐17 and IFN‐γ was observed only in mice exposed to Candida kefir. Moreover, changes in microflora were different according to the microorganism used to feed EAE prone mice, with Saccharomyces cerevisiae being unable to reduce the ratio of Bacteroides or to ameliorate toluene‐2,4‐di‐isocyanate‐induced autoimmune dermatitis in mice. On the other hand, commensal microbiota which is not classically considered pathogenic can prompt and flare autoimmunity when fine regulation of immune tolerance does not work properly.2 This was indeed suggested for S. cerevisiae. Anti‐S. cerevisiae autoantibodies (ASCA) are associated with Crohn's disease and can be found even before clinical onset in ~30% of patients.3 Moreover, these antibodies can be found in several other autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and antiphospholipid syndrome (APS), more frequently than in control subjects. Saccharomyces cerevisiae has immune‐modulating properties since cross‐reactive epitopes on β 2‐glycoprotein‐I and S. cerevisiae have been detected in ASCA‐positive APS patients.4 Several other overlaps in molecular sequence between yeast phosphopeptidomannan and conserved protein domains of specific autoantigens in humans have been analyzed and identified through the National Center for Biotechnology Information (NCBI) database.2 Saccharomyces cerevisiae proteins has been used as an adjuvant in vaccines such as hepatitis B vaccination and studies from our group led to the suspicion of an association with postvaccination autoimmune phenomena such as those described in the autoimmune/inflammatory syndrome induced by adjuvants (ASIA).5 Thus, the interesting experiment by Takata et al. may have revealed the importance of different dietary yeast in contribution to inflammation and autoimmunity. From one side, it is possible that some types (such as Saccharomyces boulardii) can be used as probiotics acting in a microbiome competition model leading to amelioration of autoimmune conditions.1 Nonetheless, it is fundamental to address the exact role of those types which, on the contrary, may even worsen an autoimmune/inflammatory condition.

Pep19 drives epitope spreading in periodontitis and periodontitis-associated autoimmune diseases.

Epitope spreading is one of valid mechanisms operating in immunopathological processes of infection-induced autoimmune diseases. We hypothesized that the peptide 19 from Porphyromonas gingivalis heat shock protein (HSP) 60 (Pep19) may be the dominant epitope from which epitope-specific immune response to subdominant epitopes may diversify sequentially into autoimmune responses directed at human neoepitopes in P. gingivalis-induced periodontitis and autoimmune diseases. However, the exact feature and mechanism on how Pep19 may drive epitope spreading into human autoantigens in chronic periodontitis or P. gingivalis-induced experimental periodontitis has not been clarified. The present study was performed with the following specific aims: (i) to delineate retrospectively the features of epitope spreading by human cross-sectional analysis; (ii) to demonstrate prospectively the epitope spreading into new antigenic determinants in an ordered, predictable and sequential manner in experimental periodontitis; and (iii) to clarify the mechanism on how immunization with Pep19 may mobilize helper T cells or elicit B-cell responses to human autoantigens and neoantigen. The study was devised for two independent investigations - a cross-sectional analysis on clinical subjects and a prospective analysis on experimental periodontitis - each being subdivided further into two additional independent observations. Cross-sectional dot immunoblot pattern against a panel of peptides of P.gingivalis HSP60 and human HSP60 was performed among age-dependent healthy subjects and between healthy subjects, patients with chronic periodontitis and patients with autoimmune disease, to identify epitope spreading. A peptide-specific T-cell line was established for phenotype analysis and for proliferation assay to an array of identical peptides. An identical prospective analysis was performed in P.gingivalis-induced experimental periodontitis or in Pep19-immunized mice. Cross-reactivity of anti-Pep19 monoclonal antibody was also investigated. A dominant immune response exclusively to Pep19 prevailed in healthy human subjects (before the age of 40) and mice that persisted in chronic periodontitis and autoimmune diseases without being replaced further by subsequent subdominant epitopes. A sequential epitope spreading provoked by Pep19 to subdominant autoantigen peptide 19 from human HSP60 (Hu19) in most healthy human subjects and mice, and to autoantigen peptide 9 from human HSP60 (Hu9) and neoantigen oxidized low-density lipoprotein (ox-LDL) in P.gingivalis-induced chronic periodontitis and autoimmune diseases could be demonstrated in a reproducible and predictable manner. T-cell proliferative activity to multiple autoantigens Hu19, Hu9 and ox-LDL, and cross-reactivity of anti-Pep19 monoclonal antibody to these epitopes may be proposed as cellular and molecular mechanisms responsible for the phenomenon. Moreover, the predictive value of Pep19 for Hu9 increased remarkably in the disease group when compared with that of the healthy group. Taken together, epitope spreading to Hu19, Hu9 and ox-LDL provoked by Pep19 could be proposed as a solid phenomenon observed in P.gingivalis-induced chronic periodontitis and infection-induced autoimmune diseases in a reproducible and predictable manner. T-cell proliferative activity to these peptides and cross-reactivity of anti-Pep19 antibodies to multiple human autoantigens could be proposed as cellular and molecular mechanisms responsible for this phenomenon.

Gastrointestinal Autoimmunity Associated With Loss of Central Tolerance to Enteric α-Defensins

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is an autoimmune disorder characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency, but patients also develop intestinal disorders. APECED is an autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE, which regulates immune tolerance) that allow self-reactive T cells to enter the periphery. Enteric α-defensins are antimicrobial peptides secreted by Paneth cells. Patients with APECED frequently have gastrointestinal symptoms and seroreactivity against secretory granules of Paneth cells. We investigated whether enteric α-defensins are autoantigens in humans and mice with AIRE deficiency. We analyzed clinical data, along with serum and stool samples and available duodenal biopsies from 50 patients with APECED collected from multiple centers in Europe. Samples were assessed for expression of defensins and other molecules by quantitative reverse transcription polymerase chain reaction and flow cytometry; levels of antibodies and other proteins were measured by immunohistochemical and immunoblot analyses. Histologic analyses were performed on biopsy samples. We used Aire(-/-) mice as a model of APECED, and studied the effects of transferring immune cells from these mice to athymic mice. Enteric defensins were detected in extraintestinal tissues of patients with APECED, especially in medullary thymic epithelial cells. Some patients with APECED lacked Paneth cells and were seropositive for defensin-specific autoantibodies; the presence of autoantibodies correlated with frequent diarrhea. Aire(-/-) mice developed defensin-specific T cells. Adoptive transfer of these T cells to athymic mice resulted in T-cell infiltration of the gut, loss of Paneth cells, microbial dysbiosis, and the induction of T-helper 17 cell-mediated autoimmune responses resembling those observed in patients with APECED. In patients with APECED, loss of AIRE appears to cause an autoimmune response against enteric defensins and loss of Paneth cells. Aire(-/-) mice developed defensin-specific T cells that cause intestinal defects similar to those observed in patients with APECED. These findings provide a mechanism by which loss of AIRE-mediated immune tolerance leads to intestinal disorders in patients with APECED.

Monoclonal antibody against citrullinated peptides obtained from rheumatoid arthritis patients reacts with numerous citrullinated microbial and food proteins.

To investigate the reactivity of monoclonal anti-citrullinated protein antibody (ACPA) obtained from peripheral blood B cells of rheumatoid arthritis (RA) patients with human autoantigens as well as environmental proteins by determining the essential epitope for the ACPA. A human monoclonal ACPA (cyclic citrullinated peptide antibody 1 [CCP-Ab1]) was obtained by screening peripheral blood lymphocytes from 31 patients with RA using a novel monoclonal antibody-secreting cell (ASC) screening system, the immunospot-array assay on a chip. The essential epitope for CCP-Ab1 was determined using epitope mapping. Then, human, microbial, and plant proteins that share the essential epitope identified were searched using BLAST. Finally, representative proteins identified by the search were produced in vitro, and their reactivity with CCP-Ab1 was examined. CCP-Ab1 bound CCP in a citrulline-indispensable manner. In CCP, the 6 amino acid residues required for CCP-Ab1 binding were identified. In the BLAST search, 38 human, 56 viral, 1,383 fungal, 547 bacterial, and 1,072 plant proteins were found to share the essential epitope, and CCP-Ab1 reacted with all of the recombinant citrullinated proteins tested, which included the various environmental factors, such as various plant proteins that are part of the daily diet. Our findings demonstrate, for the first time, that a monoclonal ACPA (CCP-Ab1) derived from RA patients cross-reacts not only with various autoantigens but also with numerous plant and microbial proteins. We propose that countless environmental factors, including microbes and diet, may trigger the generation of ACPAs that then cross-react with various citrullinated human autoantigens through molecular mimicry to induce RA.

Cross‐reactivity of Gut Commensals and Autoantigen in Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is an autoimmune clotting disorder of unknown etiology targeting mainly the autoantigen ß2‐glycoprotein I (β2GPI). Given the vast antigenic potential of the gut microbiota and its general influence on autoimmune disease models, we hypothesized that a homeostatic adaptive immune response to gut commensals can trigger and sustain autoreactive T and B cells via cross‐reactivity with b2GPI. Protein BLAST and Clustal Omega queries identified the common human gut commensal Roseburia intestinalis as containing peptide sequences with high homology to the immunodominant ß2GPI T and B cell epitopes. We present here initial studies examining T and B cell reactivity to gut commensals in human APS patients. Patient PBMCs proliferated significantly more to R. intestinalis protein extracts compared to phylogenetically related Eubacterium rectale, which lacks homologous sequences. We successfully cloned ß2GPI‐specific memory T cells using a T cell library assay to determine next if these clones cross‐react with candidate commensals. Western blot experiments using patient plasma samples showed reactivity with several commensals, notably also with R. intestinalis including one protein at 24 kDa that may be cross‐reactive based on in silico experiments. We next adsorbed plasma from APS and control subjects with recombinant ß2GPI prior to Western blotting observing a decrease in the R. intestinalis 24 kDa band. These data support a working model where lymphocytes specific to commensals cross‐react with human autoantigen and vice versa. This process could sustain APS pathogenesis in vivo.

Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis.

The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

Low‐dose anti‐CD3 antibody induces remission of active autoimmune hepatitis in xenoimmunized mice

Some patients with autoimmune hepatitis (AIH), despite appropriate treatment, progress towards cirrhosis and liver failure, requiring transplantation. New biological agents targeting immune cell subtypes have been developed, with better specificity and longer-lasting effects than conventional wide-spectrum immunosuppressive drugs. The goal of this study was to evaluate the effectiveness of low dose of αCD3 targeting therapy in a model of type 2 AIH. This experimental model is based on xenoimmunization of C57BL/6 mice with DNA coding for human liver autoantigens. Mice with AIH were treated with five daily injections of low dose of αCD3 monoclonal antibody, before disease onset (5.5 months post-xenoimmunization) or during AIH (7 months post-xenoimmunization). Along with serum aminotransferases, autoantibody levels and end-point liver histology, spleen and liver-infiltrating lymphocytes were phenotyped by flow cytometry and immune response measured by lymphoproliferative assays. Before onset of AIH, treatment prevented the development of liver inflammation and tissue injury. During active AIH, low dose of αCD3 antibody therapy resulted in a resorption of liver inflammatory infiltrates, normalization of serum aminotransferas levels, reduced autoantibody titres, increased regulatory T cells and lowered proliferation of autoreactive liver lymphocytes. We report that low dose αCD3 antibody administration is an effective treatment for AIH in an experimental model of type 2 AIH. These data suggest that αCD3 antibody therapy could be tested in clinical trials as a rescue therapy for patients with uncontrolled AIH.

The reactivity of human serum natural autoantibodies with certain autoantigens increases along with aging

IntroductionNatural autoantibodies (NAAs) that recognize autoantigens are mainly generated in the absence of any apparent immunization with foreign antigens (Ags). These antibodies maintain the body homeostasis through functions, such as inhibition of tumor angiogenesis, detection of structural changes in autoantigens, and exertion of anti-inflammatory impacts. The human body is shown to go through immunological and physiological changes during aging. However, data regarding the potential variations in the binding activity of NAAs is still scarce. Therefore, in this study, we were about to explore the trend through which the reactivity of serum NAAs with several autoantigens varies with advancing age. Materials and methodsSerum samples were prepared from healthy individuals of seven age intervals: (0) cord blood, (1) infancy, (2) childhood, (3) adolescence, (4) early adulthood, (5) middle adulthood, and (6) late adulthood (the elderly). The mean immune reactivity (MIR) of the sera with 24 human autoantigens that were obtained from Immunculus research center (Russia) was determined using ELISA and inter-group comparisons were also performed. ResultsIn general, the MIR of serum natural antibodies with the autoantigens was shown to follow an upward trend with advancing age so that the lowest and highest MIRs were detected in the cord blood and late adulthood samples, respectively. Moreover, the results of the inter-group comparisons indicated that the MIRs of the first, second, fourth, and sixth groups were significantly higher than those of their previous groups, i.e. zero, first, third, and fifth groups, respectively. ConclusionThis study showed that the reactivity of human tissue-specific and non-specific NAAs with autoantigens varied along with aging. Regarding the crucial roles NAAs play in maintaining the body homeostasis, the variation in their concentration at different age intervals might account for the immunological and pathological changes that occur in the elderly.

THU0118 Toll-Like Receptor Dependent Autoantigens in Animal Models and Humans in Use to Improve Collagen Induced Arthritis

BackgroundCurrent therapies for autoimmune diseases still mainly suppress inflammation and only indirectly affect antigen-specific immune activities. Therapeutically targeting relevant autoantigens found in humans and animal models of RA has the...

Myasthenia gravis: analysis of serum autoantibody reactivities to 1827 potential human autoantigens by protein macroarrays.

BackgroundMyasthenia gravis is a disorder of neuromuscular transmission associated with autoantibodies against the nicotinic acetylcholine receptor. We have previously developed a customized protein macroarray comprising 1827 potential human autoantigens, which permitted to discriminate sera of patients with different cancers from sera of healthy controls, but has not yet been evaluated in antibody-mediated autoimmune diseases.ObjectiveTo determine whether autoantibody signatures obtained by protein macroarray separate sera of patients with myasthenia gravis from healthy controls.MethodsSera of patients with acetylcholine receptor antibody-positive myasthenia gravis (n = 25) and healthy controls (n = 32) were analyzed by protein macroarrays comprising 1827 peptide clones.ResultsAutoantibody signatures did not separate patients with myasthenia gravis from controls with sufficient sensitivity, specificity, and accuracy. Intensity values of one antigen (poly A binding protein cytoplasmic 1, p = 0.0045) were higher in patients with myasthenia gravis, but the relevance of this and two further antigens, 40S ribosomal protein S13 (20.8% vs. 0%, p = 0.011) and proteasome subunit alpha type 1 (25% vs. 3.1%, p = 0.035), which were detected more frequently by myasthenia gravis than by control sera, currently remains uncertain.ConclusionSeroreactivity profiles of patients with myasthenia gravis detected by a customized protein macroarray did not allow discrimination from healthy controls, compatible with the notion that the autoantibody response in myasthenia gravis is highly focussed against the acetylcholine receptor.

A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease.

Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but identifying disease-relevant autoantigens has been a difficult challenge. In this study we aimed to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, in which infection-induced autoimmunity is thought to play an important role. Using tandem mass spectrometry, naturally presented HLA-DR self peptides from a patient's synovium were identified, synthesized, and reacted with his peripheral blood mononuclear cells (PBMCs). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patient cells or sera. Of 120 HLA-DR-presented self peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMCs to proliferate. T and B cell responses to ECGF occurred systemically in ∼10-30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. Among non-antibiotic-treated historical patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis. T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence of autoimmune T and B cell responses in this illness.

Novel approach to identifying autoantibodies in rheumatoid synovitis with a biotinylated human autoantigen library and the enzyme-labeled antigen method

Synovial tissue in rheumatoid arthritis (RA) shows dense infiltration of plasmacytes. The purpose of the present study is to identify and localize autoantibodies produced in these immunocytes in RA synovitis. We developed a novel screening system for detecting specific autoantigens. Protein antigens recognized by antibodies in the serum and synovial tissue extract from five RA patients were screened with the AlphaScreen method. For screening, a biotinylated human autoantigen library was constructed by the wheat germ cell-free protein synthesis system. The AlphaScreen analysis of 2183 proteins detected a limited number of antigens reactive with the serum and synovial tissue extract. Eighteen biotinylated proteins, containing top five showing high signals in each synovitis tissue extract, were utilized as probes for the enzyme-labeled antigen method, in order to visualize the site of specific antibody production in synovial lesions. Specific antibodies against two proteins, tripartite motif-containing 21 (TRIM21, also known as SSA/Ro52) and F-box only protein 2 (FBXO2), were visualized in the cytoplasm of plasmacytes in two RA synovitis lesions, respectively. Absorption experiments using unlabeled proteins confirmed the specificity of staining. No positive signals against these two proteins were identified in the additionally evaluated RA and osteoarthritis synovial lesions. The present study indicated 1) the usefulness of screening the human autoantigen library with the AlphaScreen assay for detecting autoantibodies in RA synovitis, and 2) the applicability of biotinylated proteins to the enzyme-labeled antigen method for visualizing the site of autoantibody production within the lesion.

Trichinella spiralis shares epitopes with human autoantigens

Like other helminths, Trichinella spiralis has evolved strategies to allow it to survive in the host organism, including the expression of epitopes similar to those present in either expressed or hidden host antigens. To identify T. spiralis-derived antigens that are evolutionarily conserved in the parasite and its host and that could be responsible for its evasion of the host immune response, we examined the reactivity of six different types of autoantibodies to T. spiralis larvae from muscle. T. spiralis antigens that share epitopes with human autoantigens were identified by assessing the cross-reactivity of autoantibody-containing serum samples with T. spiralis antigens in the absence of specific anti-parasite antibodies. Of the 55 autoantibody-containing human serum samples that we analysed by immunohistological screening, 24 (43.6%) recognised T. spiralis muscle larvae structures such as the subcuticular region, the genital primordium or the midgut. Using Western blots, we demonstrated that the same sera reacted with 24 protein components of T. spiralis muscle larvae excretory-secretory L1 antigens. We found that the human autoantibodies predominantly bound antigens belonging to the TSL1 group; more specifically, the autoantibody-containing sera reacted most frequently with the 53-kDa component. Thus, this protein is a good candidate for further studies of the mechanisms of T. spiralis-mediated immunomodulation.

Reversal of normal ovarian function in experimental autoimmune ovarian failure mouse model post-dexamethasone treatment (51.10)

Abstract Glucocorticoids (GCs) are in widespread clinical use for treatment of autoimmune diseases including Premature Ovarian Failure and In Vitro fertilization-Embryo Transfer failures. Immunosuppression by GCs in these patients inhibits potential adverse effects of Anti-Ovarian Antibodies (AOAs) on oocyte quality, gamete interaction, implantation and embryo development. The present study highlights the restoration of normal follicular development and fertility in experimental autoimmune ovarian failure mouse model after dexamethasone treatment, thus establishing the significance of corticosteroids in human autoimmune ovarian failures. However, their mode of action in these cases remains unknown. Female mice were immunized with immunodominant epitopes of three dominant human ovarian autoantigens and monitored for various ovarian parameters. Immunized female mice showed high AOA titers and specific reactivity to different ovarian cell types. Adverse effects on cyclicity, pregnancy, pre-implantation embryos and fertility were noticed. Ovaries from these mice exhibited fewer healthy corpora lutea (CL) and growing follicles and increased atretic follicles. However, the number of healthy follicles, CL, pre-implantation embryos and fertility was restored to normal in dexamethasone treated mice in a dose dependent manner, thereby establishing that the three putative autoimmune ovarian antigens can be used for developing targeted therapies in human autoimmune ovarian pathologies.