In recent years, successful ex-vivo immunotherapy with autologous T cells (adoptive transfer) has been reported for a variety of cancers. However, current efforts to provide therapeutic T cells for such therapy involve isolation of cells from the patient's peripheral blood, expansion and antigen specific "training" ex-vivo followed by return of the trained cells into the patient. These methods are severely constrained by (a) the difficulties and inefficiency of patient cell isolation (b) problems with expansion of primary cells in vitro (c) the morbidity associated with autologous cell therapy and above all (d) the limited availability of donor cells. In addition, the time required to "expand" and "train" patient-isolated cells for adoptive therapy can often prove to be too long for critical diseases. Therefore, technologies leading to efficient generation and expansion of therapeutic T cells from multipotent stem cells in a synthetic, controlled environment could provide a renewable, on-demand and readily available cell source for a variety of disease applications. Despite tremendous advances in the past few years in our understanding of the molecular signals involved in T cell development, the ultimate therapeutic applicability of stem cell-derived T cells require (a) Quantitative understanding of microenvironment- directed hematopoietic progenitor cell (HPC) differentiation into T cells and (b) Development of technologies for high-throughput production of functional, antigen- specific T cells suitable for on-demand transplantation. Our goal here is to engineer artificial T cell development niches (synthetic thymus-like microenvironment) to understand the effects of controlled Notch signaling in T cell development and direct human HPCs into therapeutic T cells in a scalable manner. Specifically, we propose to synthesize notch-ligand functionalized (artificial thymic stromal cells) and HLA tetramer functionalized magnetic microbeads (artificial antigen presenting cells), and evaluate how cord blood-derived human CD34+CD38- stem cells could be directed to functional, therapeutic T cells. PUBLIC HEALTH RELEVANCE: The goal of this two year project is to develop synthetic microbeads that mimic the micro-environmental conditions of the thymus in order to study how cord blood- derived hematopoietic progenitor cells can be directed to the T cell lineage. Specifically we would investigate how efficient notch signaling through these artificial stromal cells could trigger notch specific genes and in the presence of paracrine signals from mouse or human stromal cell signals generate early T cells. We would also investigate if tetramer signaling through magnetic microbeads (artificial antigen presenting cells) can further differentiate these stem cell-derived early T cells into more mature, CD8+ antigen specific T cells.