Antigen Microarrays
Introduction
Antigen arrays provide a powerful approach to study autoimmune disease. Autoimmune responses activate B cells to produce autoantibodies that recognize self-molecules termed autoantigens, many of which are proteins or protein complexes. Antigen arrays enable profiling of the specificity of autoantibody responses against panels of peptides and proteins representing known autoantigens as well as candidate autoantigens. In addition to identifying autoantigens and mapping immunodominant epitopes, proteomic analysis of autoantibody responses will further enable diagnosis, prognostication, and tailoring of antigen-specific tolerizing therapy.
Autoantibody specificity reflects the specificity of autoimmune responses
Autoimmune diseases result from aberrant activation of T and B lymphocytes which attack self-molecules (termed autoantigens). Most but not all autoantigens are proteins or protein complexes. Aberrant autoimmune responses destroy cells and tissues containing these self-proteins, thereby causing the clinical syndromes classified as autoimmune diseases. Autoimmune responses are coordinated by autoreactive CD4+ T lymphocytes. These autoreactive T cells reciprocally activate B cells, which then differentiate into memory B cells as well as plasma cells. The sole purpose of plasma cells is to produce high-affinity, high-avidity antibodies directed against the original activating antigen. In the case of autoreactive plasma cells, large amounts of autoantibodies are produced and secreted into the blood where they circulate and can deposit in tissues and organs, generating acute and chronic inflammation.
The specificity of B cell autoantibody responses reflects the overall specificity of the autoimmune response. B cells are professional antigen presenting cells (APCs) that provide and receive help from CD4+ T cells. B cells bind, internalize, process, and present major histocompatibility (MHC) -bound peptides derived from macromolecular antigens that are specifically recognized by their rearranged cell surface immunoglobulin (Ig) receptors. B cells can only provide help to, and receive help from, T cells that recognize these MHC-bound peptide epitopes. The reciprocal nature of T and B cell activation results in the activation of autoreactive T and B cells that recognize epitopes derived from the same macromolecular complex. This system likely evolved to insure that B and T cells could coordinate their attack against invading pathogens without also damaging the host. The specificity of the B cell autoantibody response therefore reflects the overall specificity of the autoreactive T cell response. This provides the rationale for the use of protein array profiling of autoantibody responses to gain insights into the overall immune response.
Autoreactive B and T cells are very rare. For example, on the order of 1 out of 10,000 or fewer lymphocytes from a diseased patient is autoreactive, based on limiting dilution and ELISPOT experiments. The detection of individual autoreactive T lymphocytes requires highly-specialized reagents such as tetramers1, which are tedious to produce, are specific for only a single epitope, and are not amenable for detecting rare populations of autoreactive T cells. Because B cells produce and secrete large quantities of soluble antibodies which are readily detectable in the serum, it is a simpler task to study autoantibodies using a variety of different techniques, including enzyme linked immunosorbent assays (ELISAs), western blot analysis, immunoprecipitation analysis, and flow-based assays.
Antigen arrays are well-suited for the study of autoantibody responses for a number of reasons. Our labs as well as several other laboratories in academia and in industry have taken advantage of the abundant, high-affinity autoantibodies that are present in the serum of patients with rheumatic diseases to develop specific protein array technology that can be applied directly to studying human disease. In this review we will summarize advances in protein array technology that have catalyzed our ability to analyze the "serum autoantibody proteome." There are 5 main areas in which we and others are currently developing or employing protein array technology for the study of autoantibodies: (i.) to improve the diagnosis of autoimmune diseases; (ii.) to study the natural progression of the immune response, both in autoimmunity and following vaccinations and infections; (iii.) to identify "serum autoantibody biosignatures" that might identify subsets of patients with certain clinical features, prognostic outcomes, or who might be expected to respond well or have an adverse event related to a therapeutic intervention; (iv.) to develop "antigen-specific tolerizing therapy" based on the presence or absence of serum autoantibodies; and (v.) to discover unique, novel autoantigens. Each of these uses will be described in more detail below.
Autoantibodies for the diagnosis of autoimmune disease: why develop antigen arrays?
Autoantibodies are a hallmark of many autoimmune diseases. For certain autoimmune diseases, the detection and quantification of autoantibodies provide diagnostic utility, and are routinely used in the clinic for diagnosis (Table 1). Routine assays for detection of autoantibodies are generally performed by enzyme linked immunosorbent assays (ELISA) and fluorescence immunoassays. Individual assays are performed in microtiter plates, with each well representing a single antigen. For many clinical entities, clinicians order a host of individual ELISAs or fluorescence immunoassays to establish the diagnosis and to provide prognostic data to assist with clinical decision-making. These tests are performed one-at-a-time, are laborious, and can be expensive. As will be discussed below, antigen arrays to characterize autoantibodies have tremendous potential to improve the quantity, and perhaps the quality, of serologic information that is made available to the practicing clinician.
While there is little debate that detection of autoantibodies can be of tremendous importance to clinicians, there is great debate within the literature regarding the significance of autoantibodies as mediators of disease2. Table 1 lists select autoimmune diseases for which determination of autoantibody specificities provides important diagnostic utility. Examples of autoimmune diseases in which the pathophysiology is mediated by autoantibodies include Grave's disease (a destructive, inflammatory disease of the thyroid gland), myasthenia gravis (an autoimmune disease in which autoantibodies are produced against the acetylcholine receptor, leading to muscle weakness and its complications), antiphospholipid antibodies (in which antibodies bind to cell surface lipids, causing spontaneous abortions and clot formation in both the venous and arterial circulation), and Wegener's granulomatosus (in which antineutrophil cytoplasmic antibodies can be transferred in animal models, producing inflammatory kidney and lung disease). Detection of specific serum autoantibodies is an important component of the diagnostic criteria for such diseases.
Proteomics Technologies for Detecting Autoantibodies
Early immunoassays capable of multiplex analysis include: enzyme-linked immunosorbent assays (ELISA), fluorescence-based immunoassays, and radioimmunoassays performed in microtiter plates; arrays of peptides synthesized on plastic pins; Western blot analysis; and genetic plaque and colony-based assays. All of these technologies are limited by requirements for relatively large quantities of reagents and clinical samples. Genetic plaque- and colony-based assays are further limited by incomplete addressibility; DNA sequence analysis is required to determine the identity of the antigens at each location on the array.
In the late 1980's Ekins as well as Fodor and colleagues proposed miniaturized and addressable immunoassays, including 'multianylate microspot immunoassays' and photolithography-generated peptide arrays3,4. Another major advance was the development of robotic printing devices by Patrick Brown and colleagues for precise deposition of cDNA to fabricate DNA microarrays5. These devices are inexpensive and widely available, and several groups recently extended their use to generate ordered arrays of proteins6,7. In recent years, major advances have been made towards development and application of miniaturized, addressable arrays of proteins, peptides and other biomolecules. These other proteomics methodologies are reviewed in detail elsewhere.
 Figure. 'Connective Tissue Disease' Array. 'Connective tissue disease' arrays were produced by printing common lupus antigens, including DNA, histone proteins, and additional nuclear proteins, on poly-L lysine-coated microscope slides. The array presented was incubated with serum derived from a patient with SLE, and binding of autoimmune antibodies detected with Cy3-conjugated goat-anti-human IgM/G (the green spots). The yellow spots are used as 'marker features' to orient the arrays. |
Our antigen array technology utilizes a robotic arrayer to attach proteins, protein complexes, peptides, nucleic acids, and other biomolecules in an ordered array on poly-L lysine-coated microscopic slides8. Approximately 1 nl containing 200 pg of antigen is deposited on each array to produce antigen features measuring 100-200 micrometers in diameter. Individual arrays are incubated with serum from patients or controls, followed by fluorescently-labeled secondary antibody. We typically use 1:150 dilutions of human or animal serum to probe arrays, requiring 2 l of serum per array under standard protocols and only 0.15 l serum per array when employing cover slips8. Other biological fluids such as cerebrospinal fluid, synovial fluid, and tissue eluates may also be used (our unpublished observations). Arrays are scanned using a fluorescence-based digital scanning device. Algorithms are available for nearest-neighbor (cluster) and statistical analysis of the data (http://rana.lbl.gov/; http://www-stat.stanford.edu/~tibs/). Detailed protocols are presented in our manuscript9 and on the World Wide Web at http://www.stanford.edu/groups/antigenarrays. Information for construction of robotic arrayers is available at http://cmgm.stanford.edu/pbrown.
Antigen arrays proved 4-8-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) analysis for detection of autoantibodies specific for 5 recombinant autoantigens 9. Moreover, antigen arrays demonstrated linear detection of antibody concentrations over a 3-log range 9.
Specialized proteomes for specific autoimmune diseases
We are developing specialized arrays representing the 'proteomes' of the tissue targets in various autoimmune diseases.
'Connective tissue disease' arrays. Our 'connective tissue disease' arrays contain 200 distinct proteins, peptides, nucleic acids, and protein complexes targeted in a host of autoimmune diseases, including systemic lupus erythematosus (SLE), polymyositis, limited and diffuse scleroderma, primary biliary sclerosis, and Sjögren's disease 8. Specific antigens include Ro, La, histone proteins, Jo-1, hnRNPs, snRNPs, Sm/RNP complex, topoisomerase I, CENP B, thyroglobulin, thyroid peroxidase, RNA polymerase, cardiolipin, pyruvate dehydrogenase, serine-arginine splicing factors, and DNA.
'Synovial proteome' arrays. We developed 'synovial proteome' arrays to study autoimmune arthritis involving synovial joints, including rheumatoid arthritis (RA) and its animal models. Our 'synovial proteome' arrays contain 650 candidate RA autoantigens, including deiminated fibrin, citrulline-modified filaggrin and fibrinogen peptides, vimentin, BiP, glucose-6-phosphate isomerase, hnRNP A2/B1, collagens and overlapping peptides derived from several of these proteins.
'Myelin proteome' arrays. Our 'myelin proteome' arrays contain 500 proteins and peptides derived from the myelin sheath, the target of the autoimmune response in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). These myelin antigens include myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), myelin oligodendrocytic glycoprotein (MOG), golli-MBP, oligodendrocyte-specific protein, cyclic nucleotide phosphodiesterase and overlapping peptides derived from these proteins. We are utilizing our 'myelin proteome' arrays characterize the autoantibody response in EAE, MS patient serum and cerebral spinal fluid, and to guide selection of antigen-specific therapies in relapsing EAE10.
References
- Altman, J.D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94-6. (1996).
- Smolen, J.S. & Steiner, G. Are autoantibodies active players or epiphenomena? Curr Opin Rheumatol 10, 201-6. (1998).
- Ekins, R.P. Multi-analyte immunoassay. J Pharm Biomed Anal 7, 155-68 (1989).
- Fodor, S.P. et al. Light-directed, spatially addressable parallel chemical synthesis. Science 251, 767-73. (1991).
- Schena, M., Shalon, D., Davis, R.W. & Brown, P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467-70. (1995).
- MacBeath, G. & Schreiber, S.L. Printing proteins as microarrays for high-throughput function determination. Science 289, 1760-3. (2000).
- Haab, B.B., Dunham, M.J. & Brown, P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol 2, research0004.1-0004.13 (2001).
- Robinson, W.H. et al. Autoantigen microarrays for multiplex characterization of autoantibody responses. Nat Med 8, 295-301. (2002).
- Robinson, W., Genovese, M. & Moreland, L. TNF alpha antagonism: Bad actor or innocent bystander in demyelinating disease. Arthritis Rheum In Press(2001).
- Robinson, W.H. et al. Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis. Nat Biotechnol 21, 1033-9 (2003).
