EBV+ B CELL LYMPHOMAS ASSOCIATED WITH POST-TRANSPLANT LYMPHOPROLIFERATIVE DISEASE DEMONSTRATE UNIQUE DEFECTS IN TRAIL-INDUCED APOPTOSIS (2004)

O86 Aims: Post-transplant lymphoproliferative disease (PTLD) remains a significant complication of immunosuppression in transplant recipients, particularly when the disease manifests as monoclonal Epstein-Barr virus (EBV)-infected B cell lymphoma. EBV-specific cytotoxic T cells prevent the outgrowth of activated, proliferating B lymphoblasts in immunocompetent individuals through induction of apoptosis. TNF-related apoptosis-inducing ligand (TRAIL), a death-inducing molecule of the TNF superfamily, preferentially induces apoptosis in numerous tumor cell types while sparing normal cells. Given the therapeutic potential of TRAIL for tumor elimination, the aim of this study was to examine the sensitivity of EBV+ spontaneous lymphoblastoid B cell lines (SLCL) derived from patients with PTLD to TRAIL-induced apoptosis. Methods: Apoptosis sensitivity in response to treatment with recombinant TRAIL protein was tested in a panel of five SLCL using Annexin V staining and cell cycle analysis. Cell surface expression of death receptors was quantified by flow cytometry. Biochemical characterization of caspase recruitment and activation was carried out using immunoprecipitation and Western blot. Ligand binding and receptor oligomerization assays were conducted using biotinylated TRAIL protein in combination with avidin-PE and examined by flow cytometry and fluorescent microscopy, respectively. Results: Strikingly, all five SLCL tested showed complete resistance to TRAIL-induced apoptosis whereas apoptosis was readily induced by TRAIL in the control cell lines Jurkat and BJAB (54% and 64% respectively). TRAIL resistance could not be attributed to differences in expression of the TRAIL receptors DR4 and DR5, since similar levels of expression were observed on the SLCL as compared to TRAIL-sensitive BJAB cells. SLCL undergo apoptosis in response to etoposide (range = 46–57%) and staurosporine (range = 32–62%), demonstrating intrinsic pathways of apoptosis are intact. We determined that proximal DR4/DR5 signaling events, including the formation of the death-inducing signaling complex (DISC) and subsequent caspase 8 activation, are severely impaired in all SLCL. Consistent with these findings, SLCL demonstrated quantitatively less ligand binding versus BJAB cells, despite expressing similar amounts of DR4 and DR5 on the cell surface. Furthermore, TRAIL receptor oligomerization on the cell surface is defective in SLCL compared to BJAB cells, in which we demonstrated that TRAIL receptors redistribute into large clusters following 60 minutes of TRAIL stimulation. Conclusions: In summation, PTLD-derived EBV+ B cell lymphomas are completely resistant to apoptosis mediated by TRAIL. Our studies describe a novel defect in TRAIL binding and receptor clustering associated with TRAIL resistance in these cells. Because TRAIL is thought to play a critical role for tumor surveillance in vivo, these studies suggest one means by which EBV-infected B cell lymphomas can evade elimination by the immune system. Furthermore, elucidating mechanisms of apoptosis resistance in PTLD-associated B cell lymphomas may aid in the characterization and treatment of other EBV-associated malignancies.