After clearance of an infection the level of several cytokines essential for the survival of the responding activated lymphocytes falls, triggering lymphocyte death. Our long-term goal is to elucidate the molecular control of apoptosis triggered by cytokine withdrawal in human lymphocytes. A better understanding of the mechanisms regulating this form of apoptosis in lymphocytes may reveal strategies for designing new therapies for autoimmune diseases and hematologic malignancies. In fact, using an approach similar to the proposed in this project we have recently identified a new genetic defect causing a syndrome of lymphoid accumulation, autoimmunity and cancer. Lymphocytes from BIM, PUMA, and BAD knockout mice are resistant to apoptosis induced by cytokine withdrawal. Multiple pathways regulate BIM in different cell types, and the mechanism regulating this protein in human lymphocytes is unknown. BIM is inactivated by binding to dynein light chains in epithelial cells. Transcription factors of the forkhead box o (FOXO) family transcriptionally regulate BIM in several cell lines. The extracellular-signal regulated kinase (ERK)/MAPK pathway can control the baseline levels of BIM, and the c-JUN amino-terminal kinase (JNK) pathway controls the levels of BIM transcriptionally and post-translationally in neurons and other cell types. In addition, autophagy also seems to protect cell lines from cytokine withdrawal-induced cell death. Based on these observations, our specific aims are to: 1) Characterize the mechanism(s) regulating the levels and activation of BIM after cytokine withdrawal. We will investigate the role of the FOXO family, JNK and ERK kinase pathaways on the transcripitional and post- translational regulation of BIM and during IL-2 withdrawal, by the use of siRNA and chemical inhibitors. 2) Determine the relative importance of PUMA and BAD for the initiation of cell death following cytokine withdrawal in human lymphocytes. We will use siRNA to knock down each of these proteins and evaluate the impact on apoptosis, cytochrome C release, and BAX activation induced by cytokine withdrawal. 3) Determine the role of autophagy as an anti-apoptotic mechanism after IL-2 withdrawal in primary lymphocytes. The presence of autophagy after cytokine withdrawal will be measured and drug treatments used to block autophagy. The impact of these manipulations on cell death induced by IL-2 withdrawal will be quantified. Our long-term goal is to elucidate the molecular control of apoptosis triggered by cytokine withdrawal in human lymphocytes. A better understanding of the mechanisms regulating this form of apoptosis in lymphocytes may reveal strategies for designing new therapies for autoimmune diseases and hematologic [unreadable] [unreadable] [unreadable]