The crucial role of Notch signaling in cell fate decisions in hematopoietic lineage and T lymphocyte development has been well established in mice. Overexpression of the intracellular domain of Notch mediates signal transduction of the protein. By retroviral transduction of this constitutively active truncated intracellular domain in human CD34+ umbilical cord blood progenitor cells, we were able to show that, in coculture with the stromal MS-5 cell line, depending on the cytokines added, the differentiation toward CD19+ B lymphocytes was blocked, the differentiation toward CD14+ monocytes was inhibited, and the differentiation toward CD56+ NK cells was favored. The number of CD7+cyCD3+ cells, a phenotype similar to T/NK progenitor cells, was also markedly increased. In fetal thymus organ culture, transduced CD34+ progenitor cells from umbilical cord blood cells or from thymus consistently generated more TCR-γδ T cells, whereas the other T cell subpopulations were largely unaffected. Interestingly, when injected in vivo in SCID-nonobese diabetic mice, the transduced cells generated ectopically human CD4+CD8+ TCR-αβ cells in the bone marrow, cells that are normally only present in the thymus, and lacked B cell differentiation potential. Our results show unequivocally that, in human, Notch signaling inhibits the monocyte and B cell fate, promotes the T cell fate, and alters the normal T cell differentiation pathway compatible with a pretumoral state.

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IntroductionStudies with Notch-1-deficient and transgenic mice have shown that signaling through the Notch-1 transmembrane receptor is necessary and sufficient for T-cell commitment. [1][2][3] In humans, we showed that retroviral overexpression of the intracellular domain of Notch-1 (ICN) in cord-blood CD34 ϩ cells results in inhibition of B-cell development both in vitro and in vivo. Instead, cells are forced to differentiate along the T/NK pathway, resulting in ectopic development of T cells in the bone marrow (BM) of mice injected with ICN-transduced human hematopoietic stem cells (HSCs). 4 A technique to direct HSCs toward the T-cell lineage could be of great therapeutic value to develop strategies to enhance T-cell development from transplanted donor HSCs after myeloablative therapy. Manipulation of HSCs with ICN is not an option however, since constitutive Notch-1 expression eventually leads to the development of T-cell neoplasms, 5 and mutations in the NOTCH1 gene have been involved in most cases of human T-cell acute lymphoblastic leukemia (T-ALL). 6 Understanding the cellular events leading to T-cell commitment after ICN overexpression might lead to the identification of an effector molecule that does not cause tumor development when overexpressed in HSCs.An important target gene of Notch-1 signaling is the basic helix-loop-helix transcription factor HES1, 7 which is expressed in thymocytes and thymic stroma and is essential for normal T-cell development. 8,9 HES-1 is up-regulated in hematopoietic precursors after ICN overexpression 10 and after activation of endogenous Notch-1 signaling by binding with the Delta-like-1 ligand. 11,12 Here, we investigated whether HES-1 is the mediator of the effects on human hematopoietic differentiation seen previously with ICN overexpression. Study design Production of HES-1 retrovirusHuman HES-1 cDNA was cloned in the retroviral vector pLZRS-IRES-EGFP (LIE) 13 upstream of the internal ribosomal entry site (IRES). ICN-LZRS was prepared by subcloning ICN from the ICN-MSCV construct used before. 4 Infectious retrovirus was produced by transfection of Phoenix-A cells. 14 Expression of HES-1 protein was verified by Western blotting on nuclear extracts of HES-1-transduced HEK-T cells using a polyclonal rabbit anti-HES-1 antibody.Sorting of CD34 ؉ Lin ؊ cells, retroviral transduction, and differentiation assays CD34 ϩ cells were isolated from cord-blood mononuclear cells by positive selection with EasySep magnetic beads (StemCell Technologies, Vancouver, BC, Canada), and CD34 ϩ Lin Ϫ cells were sorted after staining with anti-human CD34-APC, CD3-FITC, CD14-FITC, CD19-FITC, or CD19-PE monoclonal antibodies (Becton and Dickinson Immunocytometry Systems, San Jose, CA) and CD56-FITC (Serotec, Oxford, United Kingdom). The purity was always more than 98%. Retroviral transduction and coculture on MS-5 stromal cells were performed as described. 4 Labeling of cells with the indicated antibodies and flow cytometric analysis were performed as described. 4 Calculation of absolute cell ...