The thymus is continuously seeded by progenitors derived from hematopoietic stem cells, which reside in the BM. These progenitors migrate via the blood stream into the thymus, where they adopt a T cell fate, proliferate, and diff erentiate into mature functional T cells. This differentiation process is characterized by multiple developmental stages. The earliest thymic progenitors lack surface expression of CD4 and CD8 and are therefore referred to as doublenegative (DN) thymocytes. They subsequently up-regulate both CD4 and CD8 coreceptors (double positive [DP]) before undergoing positive and negative selection, and maturing to CD4 and CD8 single-positive (SP) thymocytes that emigrate to the periphery. Immature DN thymocytes can be subdivided into four subpopulations according to the surface expression of CD117, CD44, and CD25. The most immature thymocyte progenitors (DN1) express CD117 and CD44 and are negative for CD25, followed by the DN2 population, which upregulates CD25, and the DN3 cells, which downregulate CD117 and CD44 before generating DN4 thymocytes lacking expression of all three markers ( 1, 2 ).Over the last decade, many reports highlighted the importance of the evolutionarily conserved Notch cascade for the lymphoid system ( 3 ). Mammals possess 4 Notch receptors (N1 -4), which are activated by two classes of Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor -mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1 -and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifi cally inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the fi rst steps of intrathymic T cell development.

Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2–DL1–mediated signaling does not allow further T cell maturation beyond the CD25+ stage due to a lack of T cell receptor β expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1–DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch–Delta interactions in which N1–DL4 exhibits the greatest capacity to induce and support T cell development.

Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.