In this grant proposal we plan to study the role of CD154-CD40 interactions in orchestrating inflammation and fibrosis in scleroderma. Additional, we will utilize new technology to study the TCR repertoire and antigen specificity of T cells cloned from dermal lesions of patients with scleroderma. Scleroderma is an autoimmune disease characterized by endothelial cell and fibroblast activation resulting in obliterative vasculopathy and fibrosis. Antigen activated CD4+ T cells play central roles in orchestrating the inflammation and fibrosis characteristic of the disease. The identity of antigens driving the cellular immune response in tissues of patients are not known. Additionally, the T cell effector antigens driving the cellular immune response in tissues of patients are not known. Additionally, the T cell effector molecules regulating inflammation and tissue injury in scleroderma are not precisely known but it is likely that CD154 mediated signals participate in the process. In this regard, CD154 is an activation induced cell surface molecule predominantly expressed on antigen activated CD4+ T cells. The CD154 counter-receptor is CD40, expressed on a variety of cells, including endothelial cells and fibroblast. CD154-CD40 interactions play key roles in both humoral and cellular immune responses and blocking CD154 mediated signals in vivo inhibits murine models of systemic lupus erythematosis and rheumatoid arthritis. With regard to potential roles in regulating cellular immune responses, we have previously demonstrated that CD40 ligation induces endothelial cell and fibroblast activation. In more recent studies, we have demonstrated that CD154-CD40 interactions induce fibroblasts and endothelial cells to secrete prostaglandins and/or leukocyte chemoattractants. Moreover, CD154 mediated signals appear to regulate leukocyte migration in vivo because anti-CD154 mAB therapy prevents the perivascular accumulation of inflammatory cells in the murine bleomycin induced pulmonary fibrosis model. Evidence for CD154-CD40 interactions playing pathogenic roles in scleroderma is provided by our observation of infiltrating CD154+ lymphocytes in dermal biopsy specimens from patients with scleroderma. Because T cells and T cell effector molecules in particular CD154, are likely key mediators of scleroderma pathogenesis, we plan the following studies: 1) characterize the functional role of CD40 on scleroderma fibroblasts in vitro. In particular, we will determine if CD154-CD40 interactions modulate fibroblast growth, extracellular matrix production or chemokine secretion. 2) Utilize the technology provided in Core Laboratories A and B of this project to clone T cells from dermal lesions and characterize the TCR repertoire and antigen specificity of the cells.. In particular, T cells will be immortalized with herpes virus saimairi (HVS). HVS immortalized cells grow indefinitely in culture and retain there antigen specificity. 3) pilot a study of anti-CD154 mAb in treating early diffuse scleroderma. We plan to study the efficacy of a humanized anti-CD154 mAb, initially developed at Columbia, in modulating the cellular performance and humoral immune responses and clinical outcome in patients.
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