Identification Of Autoantigens Research Articles

Identification of tribbles homolog 2 as an autoantigen in autoimmune uveitis by phage display

Autoimmune uveitis is a group of ocular inflammatory disorders with unknown causes. As in other autoimmune diseases, identification of autoantigens from uveitis patients would markedly improve our understanding of the disease mechanism. Here, we report that a candidate autoantigen was identified by phage display in an unbiased fashion. A bacteriophage T7 display cDNA library was generated from human eye and characterized. Patient-specific phages were enriched by four rounds of phage display with purified patient IgG. Enriched phages demonstrated a 20-fold increase in binding specificity to the patient IgG compared with control IgG. Two clonal phages with particularly high relative binding specificities were isolated and characterized. The encoded genes, tribbles homolog 2 (TRB2) and an unknown protein, had 170- and 42-fold increases in their binding specificities to the patient IgG, respectively. The patient-specific immunoreactivities were further confirmed by Western blotting. Anti-TRB2 antibody activities were detected in several uveitis patients but not in control subjects, suggesting that TRB2 is a uveitis-associated candidate autoantigen. These results demonstrate that autoantigens can be identified by phage display using uveitis patient serum.

Identification of autoantigens in psoriatic plaques using expression cloning.

To search for autoantigens in psoriatic plaques, we screened cDNA libraries of plaque epidermis with psoriatic serum samples. This approach has been highly successful in identifying tumor antigens, but has not been widely applied to autoimmune disease. We identified 11 autoantigens including three with prominent reactivity and plausible disease relevance. These are keratin 13 (K13), heterogeneous nuclear ribonucleoprotein-A1 (hnRNP-A1), and a previously uncharacterized protein, FLJ00294. Serum antibody screening for these demonstrated reactivity in 40%, 38%, and 27% of psoriasis patients, respectively. Most positive samples reacted with all three, and we found that this was due to cross-reactivity among them. Enzyme-linked immunospot assay (ELISPOT) analysis of psoriatic peripheral blood T cells confirmed that these autoantigens are also recognized by T cells. This demonstrates that this is a feasible method to identify autoantigens in an autoimmune target tissue, and suggests that these antigens warrant further study in psoriasis. Furthermore, but peripheral blood of normal controls reacted to these autoantigens with essentially the same frequencies as patients, suggesting that psoriatics may have not only an immune system which is capable of reacting to certain autoantigens, but also to a skin immunoregulatory alteration which allows this normal reactivity to develop into abnormal inflammation.

Immunological abnormality and regulation in patients with Sjögren's syndrome

The majority of infiltrating cells into labial salivary glands and into lacrimal glands in patients with Sjogren's syndrome (SS) is CD4+TCRab T. Analyses of the T-cell receptor on T cells in both glands support the notion that infiltrating T cells are induced by antigen-driven stimulation. Thus, the identification of autoantigens recognized by T cells in inflamed lesions is important to clarify the pathogenesis of SS. T-cell epitopes on autoantigens in labial salivary glands have been examined using several strategies such as T-cell lines, PCR–single-strand conformational polymorphism, and West-Western methods. Our results showed the following four autoantigens; Ro/SS-A, heat shock protein, α-amylase, and muscarine receptor 3. Especially, the common T-cell epitope on α-amylase in HLA-DR B1*0405-positive SS patients was NPFRPWW-ERYQWPV (amino acids 68–80). The analog peptide of α-amylase might regulate autoimmunity in an antigen-specific fashion. Furthermore, TCRAV24+BV11+ double-negative natural killer T (NKT) cells are thought to be regulatory T cells. In patients with SS, these NKT cells are significantly decreased in peripheral blood, inducing the autoimmune response. In vitro stimulation by α-galactosylceramide, which is one of the antigens for NKT cells, was able to enrich NKT cells more than 10–100 times. These findings suggest that the upregulation of NKT cells by α-galactosylceramide might be a new therapeutic strategy in patients with SS.

Immunoscreening of phage-displayed cDNA-encoded polypeptides identifies B cell targets in autoimmune disease

Characterisation of self-antigens can contribute to an understanding of the aetiology of autoimmune disorders as well as to the development of new therapies and diagnostic methods. The present study was undertaken to investigate the applicability of complementary DNA (cDNA) phage-display technology to the identification of autoantigens recognised by the humoral response in autoimmune disease. Using systemic lupus erythematosus (SLE) as a model system, a pool of patient immunoglobulin G (IgG) was biopanned on a fibroblast cDNA phage-display library constructed in the vector pJuFo. Following three rounds of biopanning, recovered cDNAs were sequenced and then identified using BLAST comparisons with international databases. Both previously reported SLE autoantigens, for example, α-enolase and U1 small nuclear ribonucleoprotein-C (U1snRNP-C), and novel autoantibody targets, including ribosomal protein S20 (RPS20), ribosomal protein S13 (RPS13), ubiquitin-like protein PIC1 (PIC1), and transcription factor-like protein MRG15 (MRG15), were recovered from the biopanning procedure. Radiobinding assays were used subsequently to confirm the reactivity of some putative autoantigens to panels of sera from SLE patients, control patient groups, and healthy individuals. SLE patient sera were positive for reactivity to: U1snRNP-C, 4/15 (27%); α-enolase, 1/15 (7%); RPS20, 3/15 (20%); RPS13, 1/15 (7%); PIC1, 1/15 (7%); and MRG15, 2/15 (13%). Overall, cDNA phage-display technology appears to be applicable to the identification of autoantigens in autoimmune disease.

Identification of candidate endothelial cell autoantigens in systemic lupus erythematosus using a molecular cloning strategy: a role for ribosomal P protein P0 as an endothelial cell autoantigen.

To attempt to characterize the diversity and nature of antigens recognized by anti-endothelial cell antibodies (AECA) in patients with systemic lupus erythematosus (SLE) using a molecular cloning strategy. AECA in sera of 15 SLE patients were measured by ELISA and Western blot analysis was used to examine the diversity of autoantigen targets in two clinically active patients. A human umbilical vein endothelial cell cDNA expression library was immunoscreened with sera from these two patients to identify their autoantigen targets. An anti-ribosomal P peptide antibody ELISA was used to assess the clinical significance of anti-ribosomal P protein antibodies in the sera of one patient. Significantly higher AECA levels were found in five patients with active disease and nephritis than in five patients with clinically inactive disease. Sera from two clinically active patients were found to recognize distinct spectra of autoantigens. The candidate autoantigens that were identified included (1) endothelial cell-specific plasminogen activator inhibitor; (2) the classical lupus antigen, i.e. ribosomal P protein P0; and (3) proteins never before described as putative autoantigens in SLE, including ribosomal protein L6, elongation factor 1alpha, adenyl cyclase-associated protein, DNA replication licensing factor, profilin II and the novel proteins HEAPLA 1 and HEAPLA 2 (human endothelial associated putative lupus autoantigens 1 and 2). In one patient, antibodies against ribosomal P protein P0 were predominant and levels of these antibodies correlated with total AECA levels, anti-DNA antibody titres, overall clinical score and renal disease in a longitudinal study. A panel of candidate endothelial autoantigens in SLE, which includes previously described autoantigens and novel targets, has been identified by a molecular cloning strategy. This novel molecular approach could also be applied to the identification of autoantigens in other autoimmune vascular diseases.

Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.

Type 1 diabetes is an autoimmune disease in which the insulin-producing pancreatic beta cells are destroyed at an early age by an immune process that involves both CD4 and CD8 T lymphocytes. The identification of autoantigens in diabetes is very important for the design of antigen-specific immunotherapy. By screening a pancreatic islet cDNA library, we have identified the autoantigen recognized by highly pathogenic CD8 T cells in the non-obese diabetic mouse, one of the best animal models for human diabetes. This is the first identification, to our knowledge, of a CD8 T-cell epitope in an autoimmune disease. The peptide recognized by the cells is in the same region of the insulin B chain as the epitope recognized by previously isolated pathogenic CD4 T cells. This has very important implications for the potential use of insulin in preventative therapy.

Rp-HPLC fractionation of muscle proteins as an initial step for the identification of autoantigens in inclusion body myositis (IBM)

rp-HPLC fractionation of muscle proteins as an initial step for the identification of autoantigens in inclusion body myositis (IBM)

Development of a procedure for the direct cloning of T-cell epitopes using bacterial expression systems

Although single bacterial recombinant antigens have been used successfully to stimulate individual T-cell clones and elicit recall responses in peripheral lymphocytes, the broader use of molecular cloning systems for the identification of autoantigens recognised by the cellular arm of the immune system has met with only limited success. In a systematic approach to address this problem, a series of bacterial expression vectors were examined for their potential use as cloning vectors to elicit a proliferative response in vitro from a non-obese diabetic (NOD) mouse T-cell clone which recognises the immunodominant ovalbumin epitope (aa 323–339). The use of the vector pRSET, which produces a hexa-histidine tagged fusion protein, was confounded by non-specific responses to bacterial protein contaminants. pGEX, which generates a glutathione- S-transferase hybrid, avoided this problem but suffered from the disadvantage that a universally applicable purification procedure for the hybrid antigen could not be easily developed. A practical screening protocol was developed using the pUEX expression system (β-galactosidase hybrid) and purification based upon electroelution of the hybrid protein from purified inclusion bodies subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This system can be used to screen expression libraries for the detection of T-cell epitopes provided that the T-cell clones give low background responses to irrelevant pUEX recombinant proteins. Low abundance antigens may be obtained using this system in combination with subtractive hybridisation to construct cDNA libraries enriched in the target antigen.

Section Review—Pulmonary-Allergy, Dermatological, Gastrointestinal & Arthritis: Trials and Tribulations in the Development of Antigen-Specific Therapies for Rheumatoid Arthritis

Identification of key autoantigen(s) for any autoimmune disease can potentially provide rational design strategies for antigen-specific therapeutics which can down-regulate, or perhaps cure, the patient's autoaggressive immune response while having little or no effect on attendant ‘normal’ immunity. For rheumatoid arthritis (RA), autoantigen identification(s) lags behind other diseases such as multiple sclerosis, myasthenia gravis or even type I diabetes mellitus where most investigators agree that antigens like myelin basic protein, the acetylcholine receptor and glutamic acid decarboxylase are keys to the patient's loss of self-tolerance. However, recent advances in knowledge and understanding of the multiple and interacting signals involved in driving immune responses have provided strategies for selective inhibition of the autoaggressive response in RA. Clinical trials utilising these strategies are just beginning. Thus, a general but critical review of the two-signal model of immunity and the therapeutic technologies driven by these new basic immunology discoveries seems warranted.

Cloning and identification of autoantigens

Autoantigens are well-conserved, generally intracytoplasmic antigens that are recognized by autoantibodies and/or self-reactive T cells. Until relatively recently, the molecular identities of the autoantigens have remained elusive; the application of recombinant DNA technology has dramatically changed this picture. Here, we focus on two generic methodologies that have been used successfully to clone autoantigens: (1) hybridization screening of cDNA libraries by labeled DNA probes and (2) immunoscreening of expression cDNA libraries. The potential applications and usage of the polymerase chain reaction in the cloning of autoantigens are also discussed. The clones that have been studied are subsequently subcloned onto plasmid vectors and expressed in Escherichia coli. The biochemical identification of autoantigens has been based on nucleotide sequencing data, biochemical activities, and epitope mapping.

Identification of autoantigen recognized by autoimmune ophthalmopathy sera with immunoblotting correlated with orbital computed tomography

We have demonstrated that there is an antibody related to extraocular muscle enlargement in autoimmune ophthalmopathy (Graves' ophthalmopathy, thyroid-associated ophthalmopathy) by an immunoperoxidase technique correlated with orbital computed tomography (CT). This study was designed to identify the autoantigen and to determine whether there are common antigens among the extraocular muscle, the lacrimal gland, and the thyroid. We prepared a 100,000 g sediment fraction of porcine extraocular muscle, lacrimal gland, thyroid, and human thyroid, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with sera from patients with Graves' disease, with or without ophthalmopathy, classified by symptoms and signs combined with orbital CT and normal controls. The results showed there was an approximately 55-kDa protein band which was recognized by the sera in 32.1% ( 9 28 ) of patients with autoimmune ophthalmopathy and in 47.3% ( 9 19 ) of patients with extraocular muscle enlargement demonstrated by orbital CT. It was significantly higher than the positive rates in patients without autoimmune ophthalmopathy and normal controls (15.8 and 11.1%, respectively, P < 0.025). However, there was no common antigen among the extraocular muscles, the lacrimal gland, and the thyroid. To further confirm this eye musclespecific antigen, the approximately 55-kDa protein band was cut and solubilized from the nitrocellulose paper after SDS-PAGE, and electrophoretically transferred and used as an antigen in enzyme-linked immunosorbent assay. The absorbance was significantly higher in patients with autoimmune ophthalmopathy than patients without ophthalmopathy ( P < 0.005), and normal controls ( P < 0.01). Our findings suggest that an approximately 55-kDa protein may be a possible antigen in the eye muscle related to autoimmune ophthalmopathy.

Genetic analysis of susceptibility to type 1 diabetes.

Allelic sequencing combined with comparative analyses of population-specific haplotypes have provided persuasive evidence that the Type 1 diabetes susceptibility determinants associated and linked to the MHC class II region are partly due to DQ allelic polymorphism. A central role for an immune response molecule in this disease is consistent with many of the features of Type 1 diabetes. Identification of autoantigens and cloning of pathogenic T cells will help elucidate the roles of class II alleles in disease. This and mapping of other genes both inside and outside the MHC will eventually lead to a way of preventing or at least decreasing beta cell destruction.

Identification of autoantigens in autoimmune haemolytic anaemia by a non-radioisotope immunoprecipitation method.

In human autoimmune anaemia (AIHA), warm autoantibodies frequently appear to have serological specificity for the Rh complex, but to date, immunochemical techniques have failed to demonstrate that the antibodies react with Rh-associated polypeptides. We describe the immunoprecipitation of red blood cell (RBC) autoantigens, using biotin labelling and a luminescent detection method. In three cases of warm AIHA, a band of 32 kD and a diffuse zone of 38-51 kD or 40-51 kD were specifically precipitated by eluted RBC autoantibody. This pattern corresponds closely with that precipitated by two Rh-specific monoclonal antibodies, BRIC 207 and AB5. Antibody from the three eluates also showed serological specificity for the Rh complex in a haemagglutination assay against a panel of RBC with a range of Rh phenotypes, including rare -D-/-D- and Rh null cells. Eluted autoantibody from another warm AIHA patient immunoprecipitated a peptide of 67 kD that did not correspond in apparent molecular mass either with Rh-associated bands, or with major RBC membrane proteins or sialoglycoproteins (SGP). The haemagglutination assay showed that this eluate contained both Rh-specific and Rh-unrelated antibody. Warm autoantibody eluted from the RBC of a clinically normal, but direct antiglobulin test positive, blood donor was serologically unreactive with the Rh complex, and immunoprecipitated unknown peptides of 26, 29, 35, 48 and 51 kD, together with a band of 90 kD that comigrated with SGP alpha 2 (glycophorin A). In six further warm AIHA cases, no antigens were precipitated by autoantibody-containing RBC eluates. Overall, the results demonstrate that autoantibodies bind to Rh polypeptides in some, but not all, patients with warm AIHA, suggesting that the aetiology of the disease may vary.

Molecular basis of mitochondrial autoreactivity in primary biliary cirrhosis

Primary biliary cirrhosis (PBC), styled as a model autoimmune disease, has a very close association with antibodies to mitochondrial antigens. The cloning of cDNAs for mitochondrial antigens led to their identification as the three enzymes of the 2-oxo-acid dehydrogenase family which includes pyruvate dehydrogenase (PDH). The autoantigen is associated with the E2 subunit of these enzymes. PDH is the 70–74 kDa antigen previously identified by immunoblotting. An immunodominant site (autoepitope) can be mapped to a sequence of PDH-E2 which is the site of attachment of the functionally important lipoic acid prosthetic group. Here, Ian Mackay and Eric Gershwin discuss how autoantigen identification and epitope mapping has now proceeded further in PBC than in other autoimmune diseases, and will allow access to critically important questions on pathogenesis of PBC in particular and autoimmunity in general.

The identification of autoantigens, autoantibodies and their idiotypes.

The identification of autoantigens, autoantibodies and their idiotypes.

Antigens in immune complexes from patients with breast cancer. Identification of autoantigens in immune complexes isolated from breast cancer effusions.

Sera and effusion fluids of patients with breast cancer (BC) contain immune complexes (IC). Antigens present in these complexes were isolated as follows: a pool of effusions from patients with BC was fractionated with ammonium sulfate. The proteins precipitating at 40% saturation were further fractionated by filtration through a Sephadex G-200 column. The material recovered in the first peak (molecules larger than monomeric IgG) was brought to pH 3.0 to dissociate the IC, and the mixture was filtered through a column of Sephacryl S-300 at pH 3.0. Proteins smaller than monomeric IgG were collected, radioiodinated, and used as antigens (125Ag) to search for corresponding antibodies in sera of patients with BC (BCS) and of healthy individuals (NHS). 125Ag was reacted with the sera and the immune complexes obtained were precipitated with an antiserum to human Ig and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Both NHS and BCS contained antibodies against two antigens; one of these appeared as a strong band of 17KD, the other as a doublet of approximately 25KD. It is concluded that some of the proteins in the IC from patients with BC are auto-antigens. No BC-specific antigens were identified.