Objective Scleroderma patients with autoantibodies to centromere proteins (CENPs) and/or interferon-inducible protein 16 (IFI16) are at increased risk of severe vascular complications. cells in biopsies from scleroderma patients and CUDC-907 normal controls. Flow cytometry analysis revealed IFI16 expression in CPCs, but minimal expression in CECs. Conclusion Expression of scleroderma autoantigens IFI16 and CENPs, which are associated with severe vascular disease, is increased in vascular progenitors and mature endothelial cells. High level, lineage-enriched expression of autoantigens may explain the striking association between clinical phenotypes and the immune targeting of specific autoantigens. Introduction Scleroderma is a systemic disease characterized by pathologic features of autoimmunity, non-inflammatory vasculopathy, and tissue fibrosis (1). Most scleroderma patients produce scleroderma-specific autoantibodies that are associated with distinct clinical phenotypes. For example, anti-topoisomerase-1 antibodies are associated with interstitial lung disease (ILD) and digital ulcers (2-5). In contrast, anti-centromere (CENP) and anti-interferon-inducible protein 16 (IFI16) antibodies are frequently found in scleroderma patients with severe vascular complications, with digital loss significantly more common in patients with anti-CENP autoantibodies (6, 7), and digital gangrene significantly more common in the anti-IFI16 antibody positive group (8). Anti-centromere antibodies were first discovered in patients with the CREST variant of scleroderma (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangectasias) using human epithelial cell line 2 (HEp-2) cell substrates (9). These patients have limited skin disease, a better overall survival, increased cutaneous telangiectasia, and less ILD; however this group CUDC-907 is at a significantly increased risk of ischemic digital loss when compared to scleroderma patients with other serotypes (6). Anti-centromere antibodies target mainly CENP-A, CENP-B, and CENP-C, which are the major components of the active centromere. Studies demonstrate a high concordance in clinical phenotypes of patients with anti-CENP-A and anti-CENP-B antibodies (10). Although many autoantigens in systemic autoimmune diseases are believed to be ubiquitously expressed, the phenotypes associated with specific autoantibody responses are distinct, suggesting that localized tissue autoantigen expression may direct immune-mediated injury toward specific targets. Whether the targets of the immune response associated with scleroderma vascular complications (IFI16 and CENP proteins) are preferentially expressed in cells of the developing and mature blood vessel remains unknown. To answer questions about autoantigen expression across cell lineages in an open-ended way, we established a novel system to examine autoantigen expression in different lineages during differentiation of embryoid bodies (EBs). Human embryonic stem (ES) cells, when differentiating in skin, three millimeter punch biopsies from the dorsal forearm were obtained at the time of the clinic visit. Patients were classified as having diffuse (n=11) or limited (n=9) cutaneous scleroderma based on the extent of skin involvement (19, 20). Control skin CUDC-907 biopsies (n=8) were obtained from non-scleroderma family members or friends of the patient. CUDC-907 Skin biopsies were embedded in paraffin and 6 micron sections were cut for immunohistochemistry studies. To study IFI16 expression in circulating endothelial cells and circulating progenitor cells, PBMCs were prepared from blood obtained from 5 limited and 6 diffuse scleroderma patients, and CUDC-907 5 healthy donors. Immunohistochemistry Skin paraffin sections were rehydrated, then soaked in target retrieval solution (DAKO) for 30 mins at 95C. After blocking, the sections were incubated overnight at 4C with antibodies against anti-IFI16 (mouse monoclonal, 13 g/ml, Sigma) or anti-CD31 (rabbit polyclonal from Invitrogen, 0.2 g/ml). The sections were subsequently incubated with donkey anti-mouse IgG Alexa Fluor 594 and donkey anti-rabbit IgG Alexa Fluor 488 (Abcam, both at 1:200 dilution). After extensive washing, the sections were mounted, viewed and photographed as described above. Flow cytometry PBMCs were isolated from whole blood using Ficoll-plaque plus (GE Healthcare). They were subsequently fixed and permeabilized using the Transcription Factor buffer set per the manufacturer’s instructions (BD Biosciences). Permeabilized PBMCs were stained for 30 minutes at room temperature with monoclonal antibodies against CD3 (BV510, clone UCHT1, BD Biosciences), CD31 (APC, clone WM59, Biolegend), CD45 (PE-Dazzle 594, clone H130, Biolegend) and IFI16 (FITC, Rabbit Polyclonal to EDG2 clone 1G7, Santa Cruz Biotechnology). Data were acquired with a FACSAria I SORP cell sorter (Becton Dickinson) and subsequently analyzed with FCS Express 4 (De Novo Software). Since CD34 was lost during the permeabilization and fixation protocol, circulating hematopoietic progenitor cells were identified by the expression of CD31 and CD45. Results Biochemical levels of CENP-A and IFI16 expression increase over time in the progenitor cell differentiation system, with patterns similar to that of CD31 We utilized a human ES/EB progenitor cell differentiation system to define whether CENP and IFI16.