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Blood, Vol. 113, Issue 14, 3254-3263, April 2, 2009
Notch signaling is required for proliferation but not for differentiation at a well-defined β-selection checkpoint during human T-cell development
Blood Taghon et al.
113: 3254
Supplemental materials for: Taghon et al
Files in this Data Supplement:
- Figure S1. DN-MAML inhibits the induction of T-cell development in human hematopoietic cord blood progenitors (JPG, 85.3 KB)
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Human CD34+ CB precursors, sorted as CD34+CD19−CD56−EGFP+ after infection with MigR1 and DNMAML1 retroviral constructs, were subjected to FTOC. Cultures were analysed after 14 and 30 days, as indicated, and at each time point 3 lobes of each viral construct were pooled. Results shown are derived from 3 independent experiments. (A) Reduction in the frequency of DNMAML1 transduced EGFP+ cells in FTOC compared to the frequency of control MigR1 transduced EGFP+ CB progenitors. Numbers in histograms indicate the percentage of EGFP+ cells. (B) Reduction in the absolute number of DNMAML1 (white bars) transduced EGFP+ cells that are generated in FTOC after 14 and 30 days of culture compared to the MigR1 control (black bars). Numbers indicate the average number of EGFP+ cells (± SD) that were generated per lobe, initiated with 10,000 EGFP+ cells. (C) Flow cytometric analysis of FTOCs after 30 days of culture, gated on EGFP+ cells as shown in histograms on the right in (A). Numbers in dot plots indicate the percentage of cells in the indicated areas.
- Figure S2 Gating strategy for FTOC analysis and sorting strategy of transduced thymocytes for gene expression analysis (JPG, 77.9 KB)
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(A) Flow cytometric analysis for FTOCs as shown for DNMAML1 transduced FTOC cultures from Figure 1 at Day 19 of culture. Dot plot shows propidiumiodide (PI) and anti-mouse CD45 staining versus FSC. Histogram shows EGFP expression for PI− mouseCD45− gated human thymocytes. Histogram at the bottom shows anti-human CD7 staining for both EGFP− and EGFP+ cells as indicated. (B) Dot plots show anti-human CD45 staining versus EGFP expression for MigR1 and DNMAML1 transduced thymocytes pre- and post-sorting, performed for gene expression. Sorting was performed after retroviral transduction and after a 24hour OP9-DL1 coculture as mentioned in Materials and Methods and in the legend of Fig 1. Numbers in dot plots indicate the percentage of cells in the indicated areas.
- Figure S3. CD34+CD1+ thymocytes on OP9-DL1 are of T-lineage origin but delayed in differentiation (JPG, 82.9 KB)
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(A) Flow cytometric analysis of CD4 versus CD8β and CD3 versus TCR-αβ staining after 14 and 21 days of coculture of CD34+CD1+ thymocytes on OP9-DL1. Numbers in dot plots indicate the percentage of cells in the indicated areas. (B) Flow cytometric analysis of CD34+CD1+ thymocytes on OP9-DL1 after 6 days of coculture. Histograms show CD7, CD14, CD19, CD123, and CD56 expression. Dot plots show CD4 versus CD1 and HLA-DR versus CD4 expression. Numbers in dot plots indicate the percentage of cells in the indicated areas. Numbers in histograms indicate the percentage of positive cells for the specific marker.
- Figure S4. Delayed differentiation of CD4+CD28− and CD4+CD28+ thymocytes on OP9-DL1 (JPG, 56.5 KB)
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Flow cytometric analysis of CD4 versus CD8β and CD3 versus TCR-αβ staining after 13 days of coculture of CD4+CD28− and CD4+CD28+ thymocytes on OP9-DL1. Numbers in dot plots indicate the percentage of cells in the indicated areas.
- Figure S5. TCR-β transduction allows differentiation of CD34+CD1− thymocytes in CD3+TCRαβ+ T cells on OP9 cells in the absence of Notch signaling (JPG, 106 KB)
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CD34+CD1− thymocytes, sorted for EGFP after control and TCR-β transduction, were cocultured on OP9-DL1 (A–C) and OP9-control (D–F) stromal cells in the presence 0 or 5 µM GSI. Cultures were analysed after 12 days. Results shown are derived from 2 independent experiments. (A+D) Flow cytometric analysis, gated on EGFP+ cells, shows CD4/CD8β and CD3/TCR-αβ staining of OP9-DL1 (A) and OP9-control (D) cultures. (B+E) Average of the total number of cells (± SD), plotted on a log scale, generated in OP9-DL1 (B) and OP9-control (E) cultures. (C+F) Average number of CD3+TCRαβ+ cells (± SD), plotted on a linear scale, generated in OP9-DL1 (C) and OP9-control (F) cultures.
- Figure S6. TCR-β transduction allows differentiation of CD34+CD1− thymocytes in CD3+TCRαβ+ T cells in FTOC in the absence of Notch signaling (JPG, 95.5 KB)
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Human CD34+CD1− thymocytes, sorted for EGFP after control and TCR-β transduction, were subjected to FTOC in the presence of 0 or 5 µM GSI. Cultures were analysed after 11 days and 3 lobes of each condition were pooled. Results shown are derived from 3 independent experiments. (A) Flow cytometric analysis, gated on EGFP+ cells, shows CD4/CD8β, CD3/TCR-αβ, CD3/TCR-γδ surface profiles and intracellular staining of CD3ε and TCR-β. (B) Average of the total number of cells generated (± SD), plotted on a log scale. (C) Average number of CD3+TCR αβ+ cells generated (± SD), plotted on a log scale.
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