Post-transplant lymphoproliferative disorder (PTLD) is a major complication of solid organ and bone marrow transplantation and is associated with significant morbidity and mortality. The incidence of PTLD is 1-10% depending upon the type of allograft and the immunosuppressive regimen. Epstein-Barr virus (EBV) is the etiologic agent in PTLD, the spectrum of which ranges from benign B cell hyperplasia to malignant lymphoma. The objective of this research is to define the immune alterations that contribute to the autonomous growth of EBV-associated B cell lymphomas. The key determinants to be studied are cytokines and the immunosuppressive drugs cyclosporine (CS) and FK506. To accomplish the objective peripheral blood mononuclear cells and a panel of EBV-infected spontaneous lymphoblastoid cell lines (SLCL) generated directly from allograft recipients with PTLD will be utilized.
Three specific aims are proposed. First, the participation of cytokines as autocrine growth factors for SLCL will be defined. Neutralizing antibodies and soluble receptors will be used to determine the role of cytokines in SLCL viability, proliferation, and cell death as assessed by proliferation assays and cell cycle analysis. Particular focus will be given to the cytokines IL-6, IL-10, and TNF-alpha. In addition, the signal transduction pathways initiated by IL-6 and IL-10 will be determined by Western blotting and electromobility shift assays. These experiments will characterize the activation and phosphorylation of the Janus family of protein tyrosine kinases (Jak) and the signal transducers and activators of transcription (STAT) proteins in EBV- transformed B cells from patients with PTLD. Second, the role of T cells in regulation of the growth of EBV-infected B cells will be examined. The function of T helper cells (cytokine production and proliferation) in response to specific viral peptides, and the contribution of allo-activated T cells to the growth of EBV infected B cells will be determined. Third, cell survival proteins in SLCL will be identified and quantitated by Northern and Western blotting as well as by intracellular staining and flow cytometry. The ability of IL-10, and the immunosuppressive drugs CS and FK506, to directly modulate expression of cell survival proteins and cell viability, and to protect B cell lymphomas from apoptotic stimuli, will be defined. An understanding of the immune mechanisms that contribute to PTLD is important in the development of novel therapies for this potentially fatal complication of transplantation.
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