OVEREXPRESSION OF NOTCH LIGAND IN BONE MARROW STROMA CELLS SUPPORTS T‐LYMPHOPOIESIS

Abstract

The generation of functional T cells in vitro with the potential for therapeutic use was facilitated by the development of OP9 cell lines expressing delta like-1 notch ligand (OP9-DL1). The present research was designed 1) to determine whether T cells that mature in the extrathymic milieu can respond to allogeneic stimuli, and 2) whether DL1-notch receptor interactions induces terminal differentiation of the progenitor cells. Hematopoietic stem cells were isolated from B10.BR mice bone marrow and cultured in the presence of IL7 and FLT-3L on a previously established feeder layer of OP9-DL1 cells. FACS analysis of the cultured cells demonstrates the development of T cell progenitors and a small fraction of single positive T-cells. Importantly, a population of CD8+/TCR− cells, which has been shown to facilitate allogenenic stem cell transplantation, is also generated and maintained under these culture conditions. Using this culture system, high levels of PTα, followed by TCRα, and CD28 m-RNA expression were observed, demonstrating T-cell maturation. MLR results show that T cells obtained from the OP9-DL1 culture system proliferated in response to allogeneic spleen cells, but not in response to syngeneic spleen cells. The present culture system has successfully been able to maintain a pool of progenitor and mature T cells for as long as 90 days. We conclude from the present data that both developing and mature T-cells can be produced and maintained with this technique. In addition, a rare population of CD8+/TCR− facilitating cells can be generated in vitro, which can be used for further functional and biochemical characterization.