STEM CELLS ARE HIGHLY ENRICHED SOURCES OF AUTOANTIGENS TARGETED IN SCLERODERMA – IMPLICATIONS FOR PATHOGENESIS
Cottrell, Tricia Ruth
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Myositis, scleroderma, and other systemic autoimmune diseases are united by many features, including (i) the presence of a self amplifying cycle of tissue destruction and immune system activation with a failure of homeostatic tissue repair; (ii) an increased risk of cancer; and (iii) a strong association of particular clinical phenotypes with disease specific autoantibodies, which are used clinically for diagnosis and prognosis of disease. Interestingly, most autoantigens identified to date are ubiquitously expressed proteins, despite their associations with highly specific patterns of tissue damage. Finally, there is a significant subset of patients with characteristic features of systemic autoimmunity that lack any known autoantibodies. While most autoantigen identification studies to date have used tractable, immortalized cancer cell lines, we proposed that these cell types likely express only a small subset of proteins targeted in systemic autoimmune disease and the use of appropriately perturbed antigen sources that mimic changes in the target tissues are key to the discovery of novel autoantigens. Studies in autoimmune myositis focused on key characteristics of skeletal muscle in this disease, including the presence of a type I interferon signature and increased antigen expression by regenerating muscle cells, to identify two novel autoantigens, IFIT3 and MYL4. These studies demonstrated a systematic triangulation approach to improve the efficiency of autoantigen identification, featuring: (i) antigen sources expressing pathways shown to be active in the diseased tissue in vivo; (ii) confirmation of spot selection by post-pluck immunoblotting prior to LC/MS/MS peptide sequencing; and (iii) the use of data from additional methods to filter the proteomic data, prior to rigorous experimental confirmation of antigen identity. This approach was then applied to the study of scleroderma. Based on the disease model that an antitumor immune response may be misdirected against progenitor cells involved in tissue repair, we sought to prove that differentiating progenitor cells are a target of the immune response in scleroderma. Our studies demonstrate that the expression of known scleroderma autoantigens is altered during stem cell differentiation. Moreover, immunoblot screening of lysates from undifferentiated and differentiating stem cells with scleroderma patient sera revealed that roughly one third of randomly selected patients and 60% of ANA negative patients have autoantibodies targeting progenitor antigens, which are not expressed in HeLa cells and not targeted by autoantibodies in healthy controls. Finally, two of these novel autoantigens were identified as keratin 8 and fetuin A. These studies outline a systematic approach to autoantigen identification that accommodates the complexity of protein dynamics in perturbed cells and limits the investment of time and financial resources necessary for the identification of any particular target. These are the first studies to implicate differentiating progenitor cells as a target of the autoimmune response in scleroderma. Finally, this work demonstrates the importance of screening antigen sources that approximate changes in the target tissue in order to identify novel autoantigens that have potential clinical utility as well as the possibility of providing mechanistic insights into these potentially devastating and poorly understood diseases.