Analysis of Notch1 Function by In Vitro T Cell Differentiation ofPax5Mutant Lymphoid Progenitors

Abstract

Signaling through the Notch1 receptor is essential for T cell development in the thymus. Stromal OP9 cells ectopically expressing the Notch ligand Delta-like1 mimic the thymic environment by inducing hemopoietic stem cells to undergo in vitro T cell development. Notch1 is also expressed on Pax5−/− pro-B cells, which are clonable lymphoid progenitors with a latent myeloid potential. In this study, we demonstrate that Pax5−/− progenitors efficiently differentiate in vitro into CD4+CD8+ αβ and γδ T cells upon coculture with OP9-Delta-like1 cells. In vitro T cell development of Pax5−/− progenitors strictly depends on Notch1 function and progresses through normal developmental stages by expressing T cell markers and rearranging TCRβ, γ, and δ loci in the correct temporal sequence. Notch-stimulated Pax5−/− progenitors efficiently down-regulate the expression of B cell-specific genes, consistent with a role of Notch1 in preventing B lymphopoiesis in the thymus. At the same time, Notch signaling rapidly induces cell surface expression of the c-Kit receptor and transcription of the target genes Deltex1 and pre-Tα concomitant with the activation of TCR Vβ germline transcription and the regulatory genes GATA3 and Tcf1. These data suggest that Notch1 acts upstream of GATA3 and Tcf1 in early T cell development and regulates Vβ-DJβ rearrangements by controlling the chromatin accessibility of Vβ genes at the TCRβ locus.

Making a Notch in the lymphocyte kit

“…within hours of culture of Pax5 -/-pro-B cells on OP9-DL1 stroma. These findings imply a 'close link' between Notch signaling and upregulation of c-Kit in these progenitors, and this marked increase in cKit expression appears to be one of the earliest responses of T cell progenitors to Notch signaling [6,12].…”

“…First, Massa et al took advantage of a pro-B cell line that is blocked in its B cell potential by virtue of Pax5-deficiency (reviewed in [10]). This cell line retains multipotency in vivo and in vitro (reviewed in [11]) and was used here, and in a preceeding paper [12], to study the molecular requirements for Notch signaling for the generation of T or B cell lineages in vitro. Expression of c-Kit is low on Pax5 -/-pro-B cells, a property these cells share with 'normal' pro-B cells and various common lymphocyte progenitor phenotypes (see below).…”

“…Expression of c-Kit is low on Pax5 -/-pro-B cells, a property these cells share with 'normal' pro-B cells and various common lymphocyte progenitor phenotypes (see below). The groups of Rolink [6] and Busslinger [12] both noticed an upregulation of c-Kit expression within 1 to 3 days after culture of Pax5 -/-pro-B cells in the presence of stromal cells (OP9-DL1) expressing one of the Notch ligands, Delta-like-1, but not in the presence of stromal cells (OP-9) lacking Delta-like-1. The OP9-DL1 culture system efficiently supports T cell development from uncommitted stem cells and from committed T cell progenitors [13].…”

“…In adult mice, the growth factor receptor c-Kit is essential for maintenance of T cell progenitors [5]. Massa et al [6], and Höflinger et al [12] have linked these two crucial signals by showing that EPLM from normal mice, or Pax5-deficient mice in which B cell development is blocked beyond the EPLM stage, very rapidly upregulate c-Kit mRNA and protein once in contact with the Notch ligand DL1 in vitro. Subsequent in vitro development into T cells required c-Kit as shown by blocking with the tyrosine kinase inhibitor Gleevec.…”

Critical role for c‐kit (CD117) in T cell lineage commitment and early thymocyte development in vitro

“…Recently, Busslinger and colleagues [12] showed that Pax5 -/-pro-B cells also efficiently differentiated into the T cell lineage when cultured on OP9-DL1. In this study, these authors also demonstrated that as a consequence of Notch signaling, the Pax5 -/-pro-B cells rapidly upregulated the cell surface expression of c-kit.…”