Limits...
Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation.

Besseyrias V, Fiorini E, Strobl LJ, Zimber-Strobl U, Dumortier A, Koch U, Arcangeli ML, Ezine S, Macdonald HR, Radtke F - J. Exp. Med. (2007)

Bottom Line: However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression.Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak.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.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT
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 beta 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.

Show MeSH

Related in: MedlinePlus

N2 cannot compensate for the loss of N1 function during T cell maturation in vitro. (A) KLS cells from Ctrl and induced N1−/− mice were sorted and cultured on OP-DL1 cells for 20 d. A representative flow cytometric analysis of CD4 versus CD8 of WT thymocytes, and Ctrl and N1−/− KLS cells cultured on OP9-DL1 are shown. (B) Indicated cells were electronically gated on lineage-negative DN thymocytes and analyzed for the expression of CD44 and CD25. Representative histograms for intracellular TCRβ (iTCRβ) expression on DN3 and DN4 thymocytes derived from Ctrl and N1−/− KLS cells 20 d after culture on OP9-DL1 are shown. The numbers above the bars indicate the percentage ± SD of iTCRβ+ cells (n = 4 for WT thymocytes and in vitro culture experiments).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2118717&req=5

fig2: N2 cannot compensate for the loss of N1 function during T cell maturation in vitro. (A) KLS cells from Ctrl and induced N1−/− mice were sorted and cultured on OP-DL1 cells for 20 d. A representative flow cytometric analysis of CD4 versus CD8 of WT thymocytes, and Ctrl and N1−/− KLS cells cultured on OP9-DL1 are shown. (B) Indicated cells were electronically gated on lineage-negative DN thymocytes and analyzed for the expression of CD44 and CD25. Representative histograms for intracellular TCRβ (iTCRβ) expression on DN3 and DN4 thymocytes derived from Ctrl and N1−/− KLS cells 20 d after culture on OP9-DL1 are shown. The numbers above the bars indicate the percentage ± SD of iTCRβ+ cells (n = 4 for WT thymocytes and in vitro culture experiments).

Mentions: Notch signaling is not only essential for T lineage commitment but is also continuously required for the successful differentiation of all DN thymocyte subsets into CD4+CD8+ DP cells (14). Because N2 can instruct N1−/− HSCs to adopt a T cell fate on OP9-DL1–expressing stromal cells, it is conceivable that N2 signaling would be sufficient to allow the subsequent DN to DP transition. To this end, Ctrl and N1−/− HSCs were cultured for 28 d on OP9-DL1 stromal cells and subsequently analyzed for the development of DP T cells. Although Ctrl HSCs differentiated very efficiently into DP T cells, >90% of the N1−/− cells appeared to be blocked in the DN compartment (Fig. 2 A).


Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation.

Besseyrias V, Fiorini E, Strobl LJ, Zimber-Strobl U, Dumortier A, Koch U, Arcangeli ML, Ezine S, Macdonald HR, Radtke F - J. Exp. Med. (2007)

N2 cannot compensate for the loss of N1 function during T cell maturation in vitro. (A) KLS cells from Ctrl and induced N1−/− mice were sorted and cultured on OP-DL1 cells for 20 d. A representative flow cytometric analysis of CD4 versus CD8 of WT thymocytes, and Ctrl and N1−/− KLS cells cultured on OP9-DL1 are shown. (B) Indicated cells were electronically gated on lineage-negative DN thymocytes and analyzed for the expression of CD44 and CD25. Representative histograms for intracellular TCRβ (iTCRβ) expression on DN3 and DN4 thymocytes derived from Ctrl and N1−/− KLS cells 20 d after culture on OP9-DL1 are shown. The numbers above the bars indicate the percentage ± SD of iTCRβ+ cells (n = 4 for WT thymocytes and in vitro culture experiments).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2118717&req=5

fig2: N2 cannot compensate for the loss of N1 function during T cell maturation in vitro. (A) KLS cells from Ctrl and induced N1−/− mice were sorted and cultured on OP-DL1 cells for 20 d. A representative flow cytometric analysis of CD4 versus CD8 of WT thymocytes, and Ctrl and N1−/− KLS cells cultured on OP9-DL1 are shown. (B) Indicated cells were electronically gated on lineage-negative DN thymocytes and analyzed for the expression of CD44 and CD25. Representative histograms for intracellular TCRβ (iTCRβ) expression on DN3 and DN4 thymocytes derived from Ctrl and N1−/− KLS cells 20 d after culture on OP9-DL1 are shown. The numbers above the bars indicate the percentage ± SD of iTCRβ+ cells (n = 4 for WT thymocytes and in vitro culture experiments).
Mentions: Notch signaling is not only essential for T lineage commitment but is also continuously required for the successful differentiation of all DN thymocyte subsets into CD4+CD8+ DP cells (14). Because N2 can instruct N1−/− HSCs to adopt a T cell fate on OP9-DL1–expressing stromal cells, it is conceivable that N2 signaling would be sufficient to allow the subsequent DN to DP transition. To this end, Ctrl and N1−/− HSCs were cultured for 28 d on OP9-DL1 stromal cells and subsequently analyzed for the development of DP T cells. Although Ctrl HSCs differentiated very efficiently into DP T cells, >90% of the N1−/− cells appeared to be blocked in the DN compartment (Fig. 2 A).

Bottom Line: However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression.Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak.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.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT
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 beta 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.

Show MeSH
Related in: MedlinePlus