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. 2001 Oct 1;194(7):991-1002.
doi: 10.1084/jem.194.7.991.

Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation

A C Jaleco  1 , H Neves, E Hooijberg, P Gameiro, N Clode, M Haury, D Henrique, L Parreira
Affiliations

Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation

A C Jaleco et al. J Exp Med. .

Abstract

Notch signaling is known to differentially affect the development of lymphoid B and T cell lineages, but it remains unclear whether such effects are specifically dependent on distinct Notch ligands. Using a cell coculture assay we observed that the Notch ligand Delta-1 completely inhibits the differentiation of human hematopoietic progenitors into the B cell lineage while promoting the emergence of cells with a phenotype of T cell/natural killer (NK) precursors. In contrast, Jagged-1 did not disturb either B or T cell/NK development. Furthermore, cells cultured in the presence of either Delta-1 or Jagged-1 can acquire a phenotype of NK cells, and Delta-1, but not Jagged-1, permits the emergence of a de novo cell population coexpressing CD4 and CD8. Our results thus indicate that distinct Notch ligands can mediate differential effects of Notch signaling and provide a useful system to further address cell-fate decision processes in lymphopoiesis.

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Figures

Figure 1
Figure 1
Analysis of GFP and protein expression on transduced S17 cells, of B cell differentiation in unmodified and vector-transduced S17 cells, and functional assays for Jagged-1 and Delta-1 proteins. (a) Flow cytometric analysis of GFP expression 3 wk after sorting for GFP+ transduced S17 cells. Quadrants were set as to exclude GFP-negative cells as determined by analysis of nontransduced cells. (b) Western blot analysis detection of human Jagged-1 and Delta-1 proteins in S17 cells transduced with vectors containing either Jagged-1 (S17 J1) or Delta-1 cDNAs (S17 D1) in the same samples as in a. FBMD J1-FBMD1 cells transduced with vector containing human Jagged-1 cDNA. Molecular weight markers are indicated on the right (kD). (c) Flow cytometric analysis of CD10 and CD19 expression in CD34+ cells cocultured with parental S17 cells (S17 wt, top) and S17 cells transduced with vector alone (S17 Vector, bottom) in three different time points. (d) Delta-1 and Jagged-1 expressed by S17 cells inhibits the differentiation of C2 cells. Pictures were taken with the confocal microscope Zeiss LSM-510. C2, proliferating C2 cells; DM, differentiation medium (reference 11); S17 D1, S17 cells transduced with Delta-1; S17 J1, S17 cells transduced with Jagged-1. Original magnification: ×20. (e) Expression levels of HES-1, Deltex, pre-T-α, and E47 in CD34+ cells cocultured with parental or transduced S17 cells for 48 h, as assessed by quantitative RT-PCR (n = 2 for E47 and n = 3 for other transcripts). Expression of each transcript was measured as a ratio with the GAPDH transcript and expressed in arbitrary units (each transcript in CD34+ cells cocultured with unmodified stroma = 1).
Figure 1
Figure 1
Analysis of GFP and protein expression on transduced S17 cells, of B cell differentiation in unmodified and vector-transduced S17 cells, and functional assays for Jagged-1 and Delta-1 proteins. (a) Flow cytometric analysis of GFP expression 3 wk after sorting for GFP+ transduced S17 cells. Quadrants were set as to exclude GFP-negative cells as determined by analysis of nontransduced cells. (b) Western blot analysis detection of human Jagged-1 and Delta-1 proteins in S17 cells transduced with vectors containing either Jagged-1 (S17 J1) or Delta-1 cDNAs (S17 D1) in the same samples as in a. FBMD J1-FBMD1 cells transduced with vector containing human Jagged-1 cDNA. Molecular weight markers are indicated on the right (kD). (c) Flow cytometric analysis of CD10 and CD19 expression in CD34+ cells cocultured with parental S17 cells (S17 wt, top) and S17 cells transduced with vector alone (S17 Vector, bottom) in three different time points. (d) Delta-1 and Jagged-1 expressed by S17 cells inhibits the differentiation of C2 cells. Pictures were taken with the confocal microscope Zeiss LSM-510. C2, proliferating C2 cells; DM, differentiation medium (reference 11); S17 D1, S17 cells transduced with Delta-1; S17 J1, S17 cells transduced with Jagged-1. Original magnification: ×20. (e) Expression levels of HES-1, Deltex, pre-T-α, and E47 in CD34+ cells cocultured with parental or transduced S17 cells for 48 h, as assessed by quantitative RT-PCR (n = 2 for E47 and n = 3 for other transcripts). Expression of each transcript was measured as a ratio with the GAPDH transcript and expressed in arbitrary units (each transcript in CD34+ cells cocultured with unmodified stroma = 1).
Figure 1
Figure 1
Analysis of GFP and protein expression on transduced S17 cells, of B cell differentiation in unmodified and vector-transduced S17 cells, and functional assays for Jagged-1 and Delta-1 proteins. (a) Flow cytometric analysis of GFP expression 3 wk after sorting for GFP+ transduced S17 cells. Quadrants were set as to exclude GFP-negative cells as determined by analysis of nontransduced cells. (b) Western blot analysis detection of human Jagged-1 and Delta-1 proteins in S17 cells transduced with vectors containing either Jagged-1 (S17 J1) or Delta-1 cDNAs (S17 D1) in the same samples as in a. FBMD J1-FBMD1 cells transduced with vector containing human Jagged-1 cDNA. Molecular weight markers are indicated on the right (kD). (c) Flow cytometric analysis of CD10 and CD19 expression in CD34+ cells cocultured with parental S17 cells (S17 wt, top) and S17 cells transduced with vector alone (S17 Vector, bottom) in three different time points. (d) Delta-1 and Jagged-1 expressed by S17 cells inhibits the differentiation of C2 cells. Pictures were taken with the confocal microscope Zeiss LSM-510. C2, proliferating C2 cells; DM, differentiation medium (reference 11); S17 D1, S17 cells transduced with Delta-1; S17 J1, S17 cells transduced with Jagged-1. Original magnification: ×20. (e) Expression levels of HES-1, Deltex, pre-T-α, and E47 in CD34+ cells cocultured with parental or transduced S17 cells for 48 h, as assessed by quantitative RT-PCR (n = 2 for E47 and n = 3 for other transcripts). Expression of each transcript was measured as a ratio with the GAPDH transcript and expressed in arbitrary units (each transcript in CD34+ cells cocultured with unmodified stroma = 1).
Figure 1
Figure 1
Analysis of GFP and protein expression on transduced S17 cells, of B cell differentiation in unmodified and vector-transduced S17 cells, and functional assays for Jagged-1 and Delta-1 proteins. (a) Flow cytometric analysis of GFP expression 3 wk after sorting for GFP+ transduced S17 cells. Quadrants were set as to exclude GFP-negative cells as determined by analysis of nontransduced cells. (b) Western blot analysis detection of human Jagged-1 and Delta-1 proteins in S17 cells transduced with vectors containing either Jagged-1 (S17 J1) or Delta-1 cDNAs (S17 D1) in the same samples as in a. FBMD J1-FBMD1 cells transduced with vector containing human Jagged-1 cDNA. Molecular weight markers are indicated on the right (kD). (c) Flow cytometric analysis of CD10 and CD19 expression in CD34+ cells cocultured with parental S17 cells (S17 wt, top) and S17 cells transduced with vector alone (S17 Vector, bottom) in three different time points. (d) Delta-1 and Jagged-1 expressed by S17 cells inhibits the differentiation of C2 cells. Pictures were taken with the confocal microscope Zeiss LSM-510. C2, proliferating C2 cells; DM, differentiation medium (reference 11); S17 D1, S17 cells transduced with Delta-1; S17 J1, S17 cells transduced with Jagged-1. Original magnification: ×20. (e) Expression levels of HES-1, Deltex, pre-T-α, and E47 in CD34+ cells cocultured with parental or transduced S17 cells for 48 h, as assessed by quantitative RT-PCR (n = 2 for E47 and n = 3 for other transcripts). Expression of each transcript was measured as a ratio with the GAPDH transcript and expressed in arbitrary units (each transcript in CD34+ cells cocultured with unmodified stroma = 1).
Figure 1
Figure 1
Analysis of GFP and protein expression on transduced S17 cells, of B cell differentiation in unmodified and vector-transduced S17 cells, and functional assays for Jagged-1 and Delta-1 proteins. (a) Flow cytometric analysis of GFP expression 3 wk after sorting for GFP+ transduced S17 cells. Quadrants were set as to exclude GFP-negative cells as determined by analysis of nontransduced cells. (b) Western blot analysis detection of human Jagged-1 and Delta-1 proteins in S17 cells transduced with vectors containing either Jagged-1 (S17 J1) or Delta-1 cDNAs (S17 D1) in the same samples as in a. FBMD J1-FBMD1 cells transduced with vector containing human Jagged-1 cDNA. Molecular weight markers are indicated on the right (kD). (c) Flow cytometric analysis of CD10 and CD19 expression in CD34+ cells cocultured with parental S17 cells (S17 wt, top) and S17 cells transduced with vector alone (S17 Vector, bottom) in three different time points. (d) Delta-1 and Jagged-1 expressed by S17 cells inhibits the differentiation of C2 cells. Pictures were taken with the confocal microscope Zeiss LSM-510. C2, proliferating C2 cells; DM, differentiation medium (reference 11); S17 D1, S17 cells transduced with Delta-1; S17 J1, S17 cells transduced with Jagged-1. Original magnification: ×20. (e) Expression levels of HES-1, Deltex, pre-T-α, and E47 in CD34+ cells cocultured with parental or transduced S17 cells for 48 h, as assessed by quantitative RT-PCR (n = 2 for E47 and n = 3 for other transcripts). Expression of each transcript was measured as a ratio with the GAPDH transcript and expressed in arbitrary units (each transcript in CD34+ cells cocultured with unmodified stroma = 1).
Figure 2
Figure 2
Phenotypic analysis of hematopoietic cell populations found after 6 wk of coculture of CD34+ cells with parental and transduced S17 stroma. Supernatant cells were counter-stained with anti-CD10/anti-CD19 for detection of pre-B cells (left) and with anti-CD14/anti-CD33 for detection of myeloid precursors (right). For the sake of simplicity, since results obtained with vector alone–transduced S17 cells were identical to those observed with parental stroma (see Table ), only the latter are depicted.
Figure 3
Figure 3
Expression of surface CD7 and cytoplasmic CD3 on progenitor cells cocultured with S17 cells. CB cells were cultured for 6 wk on parental (left), Jagged-1-transduced (middle), or Delta-1–transduced (right) stroma, and analyzed for expression of surface CD7 and cytoplasmic CD3. Histograms of cells stained with the indicated mAbs are depicted in black and cells stained with isotype-matched mouse IgG are depicted in white. Results are representative of two different experiments.
Figure 4
Figure 4
Differentiation capacity of CB progenitors into the B and NK cell lineages after 3 wk coculture with Delta-1–transduced S17 cells. Supernatant cells harvested after 3 wk culture on Delta-1 stroma were transferred to either parental stroma (wt) to test for their B cell differentiation potential, or to a cytokine cocktail consisting of IL-15, IL-7, and Flt-3L (IL-15) to test for their capacity to differentiate into NK cells. (Bottom) Expression of NK cell markers in CD34+ CB cells cultured for 3 wk in suspension with IL-15, IL-7, and Flt-3L.
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Comment in

  • Coming to grips with Notch.
    von Boehmer H. von Boehmer H. J Exp Med. 2001 Oct 1;194(7):F43-6. doi: 10.1084/jem.194.7.f43. J Exp Med. 2001. PMID: 11581323 Free PMC article. No abstract available.

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