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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.

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Comparison of DL1- and DL4-mediated T cell development in vitro. (A) Histograms show flow cytometric analyses of GFP expression of uninfected OP9-cells (dashed line) and DL1 (shaded histogram) and DL4 (continuous line) retrovirally transduced OP9 cells. Ctrl BM HSCs were sorted and cultured side by side on OP9-DL1 and -DL4 cells and anaylzed by flow cytometry for the expression of CD4 and CD8 30 d after culture. (B) Sorted BM HSCs derived from either Ctrl, N1−/−, or N2−/− mice were cultured for the indicated times on either OP9-DL1 or -DL4 cells and subsequently analyzed by flow cytometry for the expression of CD44 and CD25 (electronically gated on lineage-negative cells) and the presence of B220+CD19+ B cells. Data are representative of four independent experiments.
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fig5: Comparison of DL1- and DL4-mediated T cell development in vitro. (A) Histograms show flow cytometric analyses of GFP expression of uninfected OP9-cells (dashed line) and DL1 (shaded histogram) and DL4 (continuous line) retrovirally transduced OP9 cells. Ctrl BM HSCs were sorted and cultured side by side on OP9-DL1 and -DL4 cells and anaylzed by flow cytometry for the expression of CD4 and CD8 30 d after culture. (B) Sorted BM HSCs derived from either Ctrl, N1−/−, or N2−/− mice were cultured for the indicated times on either OP9-DL1 or -DL4 cells and subsequently analyzed by flow cytometry for the expression of CD44 and CD25 (electronically gated on lineage-negative cells) and the presence of B220+CD19+ B cells. Data are representative of four independent experiments.

Mentions: The simplest hypothesis to explain the discrepancy in the ability of N2 to compensate for the loss of N1 during T lineage commitment in vitro but not in vivo is that the N2 gene, although being expressed on HSCs, is not expressed in the earliest thymocyte progenitors. To test this hypothesis, semiquantitative RT-PCR was performed on all DN subsets derived from either BM HSCs that have been cultured on OP9-DL1 cells or from WT thymocytes. As shown in Fig. 4, both N1 and N2 were expressed in all DN subsets (even in DN1, CD117+CD44+CD25− subsets), irrespective of whether they were generated in vitro or were derived from thymocytes in vivo. Furthermore, no noticeable differences were observed in the expression levels of both genes in the different DN subsets (Fig. 4). Thus, the hypothesis that the absence of N2 expression in early thymocyte progenitors accounts for the differential outcome of T lineage commitment in vivo versus in vitro is unlikely. However, an alternative explanation for this discrepancy is that the outcome of Notch signaling might be dependent on the specificity and/or avidity of certain ligand–receptor interactions. Although DL1 is able to interact with N1 and N2 to trigger T lineage commitment in vitro, it is conceivable that other Notch ligands may also trigger T lineage commitment, but only upon selective interaction with one specific Notch receptor. An attractive alternative ligand for N1 is DL4, a close homologue of DL1, which has also been shown to trigger T lineage commitment of WT precursors in vitro when expressed on OP9 cells (30). To test whether DL1 and DL4 can mediate T cell development equally efficiently in vitro, sorted WT BM HSCs were cultured on DL1- or DL4-expressing OP9 cells and analyzed side by side. Although the expression level of DL4 was slightly lower than DL1 on OP9 cells (as judged by GFP expression; Fig. 5 A, left), WT HSCs developed very efficiently into DP thymocytes in both cases within 30 d of culture (Fig. 5 A, right). To investigate whether DL1 and DL4 exhibit different Notch receptor specificities, Ctrl, N2−/−, and N1−/− HSCs were cultured side by side on either DL1- or DL4-expressing OP9 cells. After 11 d of culture, Ctrl HSCs cultured on either DL1 or DL4 progressed to the DN2/DN3 stage, although the progression appeared to be slightly more rapid on OP9-DL4–expressing cells. Whereas all three genotypes developed into DN1, DN2, and DN3 T cell progenitors on DL1-expressing OP9 cells, only Ctrl and N2−/− HSCs, but not N1−/− HSCs, developed into DN1, DN2, and DN3 T cell progenitors when cultured on OP9-DL4 cells (Fig. 5 B). Within the same time frame, N1−/− HSCs could only differentiate into a putative DN1 population (CD44+CD25−) on OP9-DL4 cells. These data indicate that N1−/− HSCs either exhibit a developmental block at the DN1 to DN2 transition or that they may have adopted a B cell fate similar to the phenotype observed after inducible inactivation of N1 in BM progenitors in vivo (Fig. 1 A) (8, 9). Therefore, the different OP9-DL4 cultures were stained with antibodies to the pan–B cell markers B220 and CD19. As shown in Fig. 5 B (right), B220+CD19+ B cells were only observed when N1−/− but not Ctrl or N2−/− BM HSCs were cultured on OP9-DL4 cells. These results demonstrate that DL4 and DL1 are not equivalent in their ability to trigger T lineage commitment, as DL4 can only induce T lineage commitment of BM HSCs that express N1. Moreover, DL4-mediated N2 signaling is not sufficient for T cell commitment nor is it sufficient for the inhibition of B cell development in vitro, suggesting that DL4 signals specifically through the N1 receptor, whereas DL1 can signal through both N1 and N2. To investigate whether DL1 and DL4 exhibit different binding avidities for N1 and/or N2, DL1- and DL4-IgG fusion proteins were generated (Fig. 6 A) and assessed for their ability to bind to Notch receptors expressed on thymocytes. As shown in Fig. 6 B, DL4-IgG fusion proteins bind immature DN thymocytes very efficiently. Binding of DL4-IgG is already observed in the DN1 subset, peaks at the DN2 subset, gradually declines through the DN3, DN4, and immature SP (ISP) subsets, and is virtually absent in more mature DP and CD4 and CD8 SP thymocytes (Fig. 6 C). Surprisingly, DL1-IgG fusion proteins did not stain thymocytes above background levels of the IgG isotype control (unpublished data), with the exception of the DN1 and DN2 subsets, which stained weakly above background. These results demonstrate that the DL4 fusion protein has a considerably higher binding avidity to Notch receptors present on immature thymocytes compared with DL1.


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)

Comparison of DL1- and DL4-mediated T cell development in vitro. (A) Histograms show flow cytometric analyses of GFP expression of uninfected OP9-cells (dashed line) and DL1 (shaded histogram) and DL4 (continuous line) retrovirally transduced OP9 cells. Ctrl BM HSCs were sorted and cultured side by side on OP9-DL1 and -DL4 cells and anaylzed by flow cytometry for the expression of CD4 and CD8 30 d after culture. (B) Sorted BM HSCs derived from either Ctrl, N1−/−, or N2−/− mice were cultured for the indicated times on either OP9-DL1 or -DL4 cells and subsequently analyzed by flow cytometry for the expression of CD44 and CD25 (electronically gated on lineage-negative cells) and the presence of B220+CD19+ B cells. Data are representative of four independent experiments.
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Related In: Results  -  Collection

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fig5: Comparison of DL1- and DL4-mediated T cell development in vitro. (A) Histograms show flow cytometric analyses of GFP expression of uninfected OP9-cells (dashed line) and DL1 (shaded histogram) and DL4 (continuous line) retrovirally transduced OP9 cells. Ctrl BM HSCs were sorted and cultured side by side on OP9-DL1 and -DL4 cells and anaylzed by flow cytometry for the expression of CD4 and CD8 30 d after culture. (B) Sorted BM HSCs derived from either Ctrl, N1−/−, or N2−/− mice were cultured for the indicated times on either OP9-DL1 or -DL4 cells and subsequently analyzed by flow cytometry for the expression of CD44 and CD25 (electronically gated on lineage-negative cells) and the presence of B220+CD19+ B cells. Data are representative of four independent experiments.
Mentions: The simplest hypothesis to explain the discrepancy in the ability of N2 to compensate for the loss of N1 during T lineage commitment in vitro but not in vivo is that the N2 gene, although being expressed on HSCs, is not expressed in the earliest thymocyte progenitors. To test this hypothesis, semiquantitative RT-PCR was performed on all DN subsets derived from either BM HSCs that have been cultured on OP9-DL1 cells or from WT thymocytes. As shown in Fig. 4, both N1 and N2 were expressed in all DN subsets (even in DN1, CD117+CD44+CD25− subsets), irrespective of whether they were generated in vitro or were derived from thymocytes in vivo. Furthermore, no noticeable differences were observed in the expression levels of both genes in the different DN subsets (Fig. 4). Thus, the hypothesis that the absence of N2 expression in early thymocyte progenitors accounts for the differential outcome of T lineage commitment in vivo versus in vitro is unlikely. However, an alternative explanation for this discrepancy is that the outcome of Notch signaling might be dependent on the specificity and/or avidity of certain ligand–receptor interactions. Although DL1 is able to interact with N1 and N2 to trigger T lineage commitment in vitro, it is conceivable that other Notch ligands may also trigger T lineage commitment, but only upon selective interaction with one specific Notch receptor. An attractive alternative ligand for N1 is DL4, a close homologue of DL1, which has also been shown to trigger T lineage commitment of WT precursors in vitro when expressed on OP9 cells (30). To test whether DL1 and DL4 can mediate T cell development equally efficiently in vitro, sorted WT BM HSCs were cultured on DL1- or DL4-expressing OP9 cells and analyzed side by side. Although the expression level of DL4 was slightly lower than DL1 on OP9 cells (as judged by GFP expression; Fig. 5 A, left), WT HSCs developed very efficiently into DP thymocytes in both cases within 30 d of culture (Fig. 5 A, right). To investigate whether DL1 and DL4 exhibit different Notch receptor specificities, Ctrl, N2−/−, and N1−/− HSCs were cultured side by side on either DL1- or DL4-expressing OP9 cells. After 11 d of culture, Ctrl HSCs cultured on either DL1 or DL4 progressed to the DN2/DN3 stage, although the progression appeared to be slightly more rapid on OP9-DL4–expressing cells. Whereas all three genotypes developed into DN1, DN2, and DN3 T cell progenitors on DL1-expressing OP9 cells, only Ctrl and N2−/− HSCs, but not N1−/− HSCs, developed into DN1, DN2, and DN3 T cell progenitors when cultured on OP9-DL4 cells (Fig. 5 B). Within the same time frame, N1−/− HSCs could only differentiate into a putative DN1 population (CD44+CD25−) on OP9-DL4 cells. These data indicate that N1−/− HSCs either exhibit a developmental block at the DN1 to DN2 transition or that they may have adopted a B cell fate similar to the phenotype observed after inducible inactivation of N1 in BM progenitors in vivo (Fig. 1 A) (8, 9). Therefore, the different OP9-DL4 cultures were stained with antibodies to the pan–B cell markers B220 and CD19. As shown in Fig. 5 B (right), B220+CD19+ B cells were only observed when N1−/− but not Ctrl or N2−/− BM HSCs were cultured on OP9-DL4 cells. These results demonstrate that DL4 and DL1 are not equivalent in their ability to trigger T lineage commitment, as DL4 can only induce T lineage commitment of BM HSCs that express N1. Moreover, DL4-mediated N2 signaling is not sufficient for T cell commitment nor is it sufficient for the inhibition of B cell development in vitro, suggesting that DL4 signals specifically through the N1 receptor, whereas DL1 can signal through both N1 and N2. To investigate whether DL1 and DL4 exhibit different binding avidities for N1 and/or N2, DL1- and DL4-IgG fusion proteins were generated (Fig. 6 A) and assessed for their ability to bind to Notch receptors expressed on thymocytes. As shown in Fig. 6 B, DL4-IgG fusion proteins bind immature DN thymocytes very efficiently. Binding of DL4-IgG is already observed in the DN1 subset, peaks at the DN2 subset, gradually declines through the DN3, DN4, and immature SP (ISP) subsets, and is virtually absent in more mature DP and CD4 and CD8 SP thymocytes (Fig. 6 C). Surprisingly, DL1-IgG fusion proteins did not stain thymocytes above background levels of the IgG isotype control (unpublished data), with the exception of the DN1 and DN2 subsets, which stained weakly above background. These results demonstrate that the DL4 fusion protein has a considerably higher binding avidity to Notch receptors present on immature thymocytes compared with DL1.

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