Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia.

Transduced mouse immature thymocytes can be differentiated into T cells in vitro using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

Mutations that collaborate with IL-7Ra signaling pathways to drive ALL.

The IL-7 pathway is required for normal T cell development and survival. In recent years the pathway has been shown to be a major driver of acute lymphoblastic leukemia (ALL), the most common cancer in children. Gain-of-function mutations in the alpha chain of the IL-7 receptor found in ALL patients clearly demonstrated that this pathway was a driver. However mutant IL-7R alone was insufficient to transform primary T cell progenitors, indicating that cooperating mutations were required. Here we review evidence for additional oncogenic mutations in the IL-7 pathway. We discuss several oncogenes, loss of tumor suppressor genes and epigenetic effects that can cooperate with mutant IL-7 receptor. These include NRas, HOXA, TLX3, Notch 1, Arf, PHF6, WT1, PRC, PTPN2 and CK2. As new therapeutics targeting the IL-7 pathway are developed, combination with agents directed to cooperating pathways offer hope for novel therapies for ALL.