Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment

Abstract

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.

Specific Notch receptor–ligand interactions control human TCR-ab/gd development by inducing differential Notch signal strength

“…In the mouse, it is well established that Notch1-DLL4 signaling is essential for inducing T cell differentiation in the immigrating T cell precursors (Wilson et al, 2001;Hozumi et al, 2008;Koch et al, 2008) and we believe that this interaction is also involved in inducing early T cell development in human. From our previous work, it is clear that Notch1 activation is responsible for inducing human T cell development ( Van de Walle et al, 2009, 2011, and although it remains to be clarified whether Delta-like 4 or Jagged2 is responsible for activating this receptor, the notion that a strong Notch signal is critical at this early stage of human T cell development ( Van de Walle et al, 2009) makes Delta-like 4 the most likely ligand .…”

Conversion of the Thymus into a Bipotent Lymphoid Organ by Replacement of Foxn1 with Its Paralog, Foxn4

“…In contrast to the wild-type situation, the thymi of Foxn1 À/À ;Foxn1:Foxn4 mice contained large numbers of proliferating immature B cells ( Figures 6C and 6D), resembling the situation in the bone marrow (Hardy and Hayakawa, 2001) ( Figures S5A-S5C) and indicating the presence of intrathymic B cell poiesis. It is important to note that the hematopoietic phenotype of the thymi of Foxn1 À/À ;Foxn1:Foxn4 mice (that is, the coexistence of T and B cell poiesis) has not been previously observed in situations of impaired T cell development (Akashi et al, 2000;Ceredig, 2002;Hozumi et al, 2008;Koch et al, 2008;Wilson et al, 2001); for instance, the fraction of B cells in the thymus of Foxn1 À/À ;Foxn1:Foxn4 mice is far greater than that observed in T cell-deficient Cd3e À/À mice ( Figure S5D) and thus is not the result of a simple reduction of T cells in the thymus.…”

“…It regulates the expression of key target genes such as those encoding the chemokine CXCL12, which is capable of attracting hematopoietic cells to the thymus (Calderó n and , and the NOTCH1 (Wilson et al, 2001) ligand DLL4, which directs T cell development (Hozumi et al, 2008;Koch et al, 2008;Calderó n and Boehm, 2012) in undifferentiated lymphoid progenitors. The essential role of FOXN1 in supporting thymopoiesis is illustrated by the failure of T cell differentiation in mammals lacking a functional Foxn1 gene (Frank et al, 1999;Nehls et al, 1994).…”

“…With respect to T cell differentiation, the required microenvironment is provided by the thymus (Petrie and Zú ñ iga-Pflü cker, 2007). The thymic stroma, which primarily consists of epithelial cells (Rodewald, 2008), generates chemokine gradients that attract lymphoid progenitors (Calderó n and Boehm, 2011), provides the Notch ligand DLL4 to initiate specification to the T cell lineage (Hozumi et al, 2008;Koch et al, 2008), orchestrates the selection of self-tolerant antigen receptors clonally expressed on differentiating thymocytes via the provision of self-peptide/major histocompatibility complexes (Kyewski and Derbinski, 2004), and provides the relevant cues supporting the exit of mature T cells into the blood circulation (Zachariah and Cyster, 2010). The thymopoietic process has self-organizing properties; in the presence of a normal hematopoietic system, which generates the necessary lymphoid progenitors, differentiation of thymic epithelial progenitor cells leads to the formation of thymopoietic tissue that supports the generation of a functionally competent T cell repertoire (Bleul et al, 2006).…”

Sending the right signal: Notch and stem cells

“…Studies of human thymopoeisis in culture have reported that Notch signaling regulates cell fate decisions in the T cell lineage in a dosedependent manner, suggesting that different levels of Notch signaling might be at play in different cell fate lineages that develop in the thymus [250]. The Notch ligand Dll4 is highly expressed in the thymic epithelial cells and activated Notch1 in the progenitors, driving T cell specification [251]. Loss of the repressor LRF in the hematopoietic progenitors leads to activation of Notch signaling in these cells and to differentiation of these cells into T cells in the bone marrow, as well as suppression of the B cell specification [252].…”