Tissue and lymphoid defects induced by Birc5 deletion in thymic epithelial cells.

Scripps Florida, Jupiter FL

Investigators

Abstract

? DESCRIPTION (provided by applicant): Like all blood cells, T lymphocytes are lost throughout life, and must be continuously replaced. This process involves inducing multipotent progenitor/stem cells to leave the bloodstream and enter the thymus, followed by a unique series of inductive and supportive events that result in multiple functional T lineages. Since the lymphoid components of the thymus are transient, the durable identity of the thymus is obviously established by its stable stromal components. Stromal cells in the thymus are primarily of epithelial lineages, with a lesser contribution by other non-hematopoietic cells (mostly fibroblasts) and some non-lymphoid hematopoietic cells (dendritic cells, macrophages). Characterization of thymic stromal cells has been problematic, since physical isolation methods (mechanical disruption, enzymatic digestion, cell sorting, etc.) are highly biased towards certain cell types, and further, result in profound changes in the cells of interest. Consequently, thymic stromal biology is very poorly understood. We have devised a computational approach that allows us to deconvolve the stromal transcriptome in situ, using RNA extracted from intact tissue regions (isolated by microdissection), or the corresponding lymphoid components of that tissue (isolated in a pristine state by cell sorting). Using informatic approaches, we have spent the last several years characterizing stromal biology, and attempting to identify critical regulatoy genes and circuits in stromal cells. In this proposal, we show that survivin, the product of the Birc5 gene and a member of the IAP family of caspase inhibitory proteins, is essential for thymic epithelial stromal cells (TEC). Conditional deletion of survivin in TEC (using Foxn1[Cre] and floxed Birc5) results in a very small thymus that lacks cortical/medullary compartmentalization and presents with large cystic structures. Lymphoid development is profoundly impaired under these conditions, with an apparent increase in the DN:DP ratio but dramatically fewer cells at all stages, leading to peripheral T lymphopenia. Genetic complementation of conditional survivin deficiency, using a keratin-14 promoter driven survivin transgene, restored thymic size, organization, and function to normal, providing classical genetic evidence for the essential function of survivin in TEC. In this application, we propose to further characterize the defects induced by survivin deletion in TEC, and to analyze the contextual relationship between those TEC that do remain, and the lymphoid cells that do develop. We also propose to determine whether survivin is important solely for the homeostatic maintenance of differentiated TEC, or whether it also/only plays a role in post-natal TEC stem cells, or during fetal organogenesis. Finally, we will use survivin as a tool to probe how the thymus is organized and differentiated in the embryo, and maintained in the adult.