IL-7 is a cytokine produced by non-lymphoid cells in the thymus and lymphoid organs. Signals from the IL-7 receptor are essential for normal thymocyte development and survival of T cells after leaving the thymus. Clinically, defects in genes for the components of IL-7 receptor are a common cause of human severe combined immunodeficiency disease (SCID). IL-7 is a promising therapeutic in treatment of AIDS, cancer and in immunization, whereas blocking the IL-7 pathway may be desirable in autoimmune diseases and lymphoid cancers. IL-7 protects lymphocytes from apoptosis and our lab has been investigating the intracellular mechanisms. We have shown that IL-7 withdrawal activates p38MAPK, which in turn activates NHE1 inducing intracellular alkalinization which activates the death protein Bax. We showed that Bax is a critical death mediator because deletion of Bax rescues T cell development in mice deficient in the IL-7 receptor. This rescue is incomplete and we are looking for additional death mediators among the Bcl-2 family and by analyzing mitochondrial protein changes by proteomics methods.Withdrawal of IL-7 induces G1 arrest. We have evidence that this arrest occurs through p38MAPK phosphorylating Cdc25a which is required for S phase progression. We also have evidence that P38MAPK leads to phosphorylation and increased stability of p27 which inhibits cdk2. This pathway to p27 stability appears to be a new mechanism of cell cycle regulation that does not involve the previously identified regulators Scp2 and Cks1. This new pathway may be critical in regulating lymphocyte, and lymphoma, proliferation. We find that there are two critical regions of the IL-7 receptor alpha chain: 1)Box 1 binds Jak1 and initiates two pathways. One leads through an unknown kinase to serine phosphorylation of Bad and protects from cell death. The second leads to tyrosine phosphorylation of Y449 of the receptor itself.2)Phospho-Y449 activates Stat5, the pI3K pathway, and an unknown pathway that induces the bcl-2 gene and prevents Bax from translocating to mitochondriaOur previous studies of the IL-7 receptor were conducted in cell lines. We have now developed a retroviral infection of stem cells to examine the function of IL-7 receptor muteins in the development and survival of T cells in mice. We conclude that the same two regions, Box1 and Y449 also are required for thymocyte development, homeostasis, and TCRg rearrangement. We also observe that IL-7 receptor when ectopically expressed in hematopoietic stem cells induces myeloid proliferation. To identify the missing components in the IL-7 signaling pathway, we have developed proteomics methods consisting of 2D gels followed by mass spectroscopy - we have identified over 30 proteins and are evaluating their relevance. We will also attempt phage display to identify the proteins that bind to the Y449 residue of the receptor.We previously focused on the effects of IL-7 on thymic development and are beginning to examine intracellular pathways by which IL-7 regulates homeostasis of peripheral T cells. IL-7 is critical for lymphocyte survival, but little is known about the regulation of its production. We will examine the types of cells that produce it, whether it is recognized in solution or is displayed on surfaces, and if the latter, characterize the binding mechanism.
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