EGFR Inhibition Ameliorates Murine Collagen-induced Arthritis

Abstracts activation. It was demonstrated by the increase in the number of CD80 and CD86 positive cells. Uptake of FITC-labeled Dex was examined by flow cytometry. At FITC-Dex concentration of 80– 100 μg/ml, the number of DCs binding antigen reached “plato.” DC charged by TI-2 antigens were mixed with normal mouse splenocytes and cultivated in the RPMI 1640 medium for 4 days. The numbers of antibody- and immunoglobulin-forming cells were determined by ELISPOT method. The mixtures of splenocytes and naive DC not charged by the antigens were used as control. It was shown that the increase in the numbers of AFC and IFC under the influence of naive DC did not exceed 20%. On the contrary, the addition of DC pulsed by the antigens increased specific immune response more that twice. The data obtained point to the direct interactions of DC with TI-2 antigens. Pulsed DCs present TI-2 antigens to mouse splenocytes and induce specific and polyclonal B-cell activation, i.e., possess immunostimulating activity. doi:10.1016/j.clim.2010.03.179

T.68. Loss of the Soluble CTLA-4 Splice Variant Compromises Immune Regulation Kay Fischer 1, Peilin Zheng 1, Daniel Rainbow 2, Linda Wicker 2, Stephan Kissler 1. 1University of Wuerzburg, Wuerzburg, Germany; 2University of Cambridge, Cambridge, United Kingdom CTLA4 gene variation associates with multiple autoimmune disorders, including type 1 diabetes. The CTLA4 susceptibility allele was found to generate decreased levels of mRNA encoding soluble CTLA-4 (sCTLA-4) relative to the full-length isoform, the functional consequence of which is as yet unknown. To model the disease-associated splicing variation of CTLA4, we generated nonobese diabetic (NOD) mice in which sCTLA-4 mRNA is silenced by RNAi. We found that loss of sCTLA-4 impairs the function of regulatory T cells, accelerates spontaneous diabetes, and makes transgenic mice prone to sudden lethal inflammation following cyclophosphamide challenge. Our results demonstrate that sCTLA-4 participates in immune regulation by potentiating the function of regulatory T cells and provide an explanation for the association of CTLA4 variation with autoimmunity in humans. doi:10.1016/j.clim.2010.03.180

T.69. EGFR Inhibition Ameliorates Murine Collagen-induced Arthritis Christina Swanson, William H. Robinson. Stanford University, Stanford, CA Rheumatoid arthritis (RA) is an autoimmune synovitis affecting approximately 0.5% of the population worldwide. In addition to T cells, B cells, and macrophages, cells such as synovial fibroblasts (RASF) and endothelial cells (EC) promote inflammation in the synovium through the production of cytokines, chemokines and nitric oxide (NO). At the same

S59 time, increased osteoclastogenesis causes bone destruction. Previously, others found that RA serum and synovial fluid contained increased concentrations of epidermal growth factor receptor (EGFR) ligands such as EGF and demonstrated EGFR staining on RASF and EC in the synovium. Thus, we hypothesized that erlotinib (Tarceva), a specific EGFR tyrosine kinase inhibitor, may ameliorate murine collagen-induced arthritis. Erlotinib is currently prescribed to treat non-small cell lung cancer and pancreatic cancer. Here, we demonstrate that erlotinib reduces the severity of established murine collageninduced arthritis (CIA) by acting on RASF, EC, osteoblasts, and osteoclasts. In RASF, EGF synergizes with IL-β and TNF to increase IL-6, IL-8, and stromelysin-1 production. Concurrent treatment of RASF with erlotinib reduces the production of these molecules. Similarly, erlotinib reduces EGF-induced EC production of IL-6, IL-8, and NO. Erlotinib decreases bone resorption directly by inhibiting osteoclast development from bone marrow cells and indirectly by acting on osteoblasts. Osteoblasts produce RANKL and osteoprotegerin (OPG), the balance of which control osteoclast-mediated bone erosion. Here, we show that EGF reduces osteoblast OPG production, which is restored by erlotinib treatment. Together, these results suggest that erlotinib treatment could be beneficial in the treatment of RA. doi:10.1016/j.clim.2010.03.181

T.70. Donor Antigen Specific Regulatory T Cell Function and Outcome in Kidney Transplant Recipients Martin Gasser 1, Igor Tsaur 2, Kai Lopau 1, Christoph Germer 1, Anil Chandraker 3, Ana Maria Waaga Gasser 1. 1University of Wuerzburg, Wuerzburg, Germany; 2University of Frankfurt, Frankfurt, Germany; 3Brigham and Women's Hospital, Boston, MA Chronic allograft dysfunction remains a major impediment to long-term allograft survival. Many different factors have been shown to contribute to deteriorating graft dysfunction, including an ongoing alloimmune response sometimes called chronic rejection. Out of 623 patients transplanted at a single center we selected 107 patients who were mismatched with their donors for one of a number of relevant HLA DR antigens. Patients were categorized into groups according to immunosuppression treatment and then further divided into those with stable or deteriorating graft function (chronic allograft dysfunction or CAD). A total of 37 representative T cell lines were generated from these patients. In patients with CAD, only those with biopsy proven chronic allograft nephropathy were selected. Although cell lines generated from both stable CsA- and Tac-treated patients had much higher percentages of CD4+CD25+ regulatory T cells (Tregs) and Treg-associated gene expression compared to the CAD groups, the Tac-treated stable patients also had a considerably higher Treg population compared with the CsAtreated group. Stable patients were associated with better longterm graft function and T cells from these patients showed increased expression of IL-4 and IL-10 and were also able to regulate the in vitro proliferation and production of IFN-γ by CAD cell lines to mismatched HLA-DR-derived peptides in an IL10-dependent fashion. CAD cell lines produced significantly