1. Volume 10, Issue 5, Pages 525-535 (May 1999)
Distinct Roles of the Phosphatidylinositol 3-Kinase and STAT5 Pathways in IL-7- Mediated Development of Human Thymocyte Precursors 
Caroline Pallard, Alexander P.A Stegmann, Titia van Kleffens, Fiona Smart, Ashok Venkitaraman, Hergen Spits 
Immunity 
Volume 10, Issue 5, Pages (May 1999)
DOI: /S (00)

2. Figure 1
Anti-IL-7Rα Chain Antibodies Inhibit T but Not NK Cell Development in an FTOC
CD34+ cells were isolated and incubated in FTOC either in the presence of an isotype-matched control antibody (A) or anti-IL-7Rα antibody (B). Cell suspensions were stained with the indicated TriColor (y axis)- and PE-labeled (x axis) antibodies and analyzed on a FACScan.
Immunity  , DOI: ( /S (00) )

3. Figure 2
Quantification of GFP+ Subpopulations Recovered from FTOC Incubated with CD34+ Thymocytes Transduced with C-GFP or with an IL-4/IL-7 Chimeric Receptor
CD34+ cells were isolated, transduced with viruses harboring IRES-GFP (C-GFP) or WTIL-4/IL-7Rα-IRES-GFP (WTIL-4/IL-7Rα), and incubated in an FTOC at 3 × 104 cells/lobe for 24 days in the presence of antibody against the IL-7Rα chain (50 μg/ml once a week) and hIL-4 (30 ng/ml twice a week). (A) Phenotype of the cells. Cell suspensions were stained with the indicated TriColor- and PE-labeled antibodies and analyzed on a FACScan. The input number of GFP+ cells was 20,000 for both the C-GFP and the WT receptor-transduced cells both divided over six thymic lobes. (B) Output numbers of cells of various subsets. The numbers of GFP+ cells (y axis) within each subpopulation (CD4−CD8− [white bars], CD4+ [light gray bars], CD4+CD8+ [dark gray bars], γδ+ [black bars], αβ+ [indicated by a line], and NK or CD56+CD3− [striped bars]) were calculated on the basis of the percentage of each subpopulation. A representative of four similar experiments is shown.
Immunity  , DOI: ( /S (00) )

4. Figure 3
Y449 of the IL-7Rα Chain Mediates the Survival and/or Proliferation of Immature CD34+ Thymocyte Precursors in an FTOC
The number of GFP+ cells given for each subpopulation (CD4−CD8− [white bars], CD4+ [light gray bars], CD4+CD8+ [dark gray bars], γδ+ [black bars], aβ+ [indicated by a line], and NK or CD56+CD3− [striped bars]) recovered after 18 days are indicated. The input numbers were 18,500 for the WT and 37,000 for the Y449F mutant. Because the input number of the mutant was two times higher we divided the output number by two to allow comparison with the WT. Similar results were obtained in two repeat experiments. The right part of the figure shows an expanded scale of the data in the left panel.
Immunity  , DOI: ( /S (00) )

5. Figure 4 IL-7 Induces the Activation of Both PKB and STAT5
Partially purified CD34+ postnatal thymocytes (2 × 106 cells) were incubated with no factor (−) (lane 1) or IL-7 (lane 2) or SCF (lane 3) (50 ng/ml) for 10 min at 37°C, and total Triton X100 cell lysates were analyzed by PAGE and Western blotting using a polyclonal antibody raised against phosphoSTAT5 (top). The blot was stripped and further incubated with a polyclonal antibody against phosphoPKB (bottom).
Immunity  , DOI: ( /S (00) )

6. Figure 5
Residue Y449 of the IL-7Rα Chain Is Crucial for Proliferation and Mediates the Activation of STAT5 and PKB in CTLL-2 Cells
(A) C-GFP, WT, or Y449F CTLL2 cells (5 × 103/point) were incubated with the indicated concentration of either hIL-2 (left) or hIL-4 (right) (ng/ml) for 3 days, and (3H)thymidine was added for the next 8 hr.
(B) C-GFP, WT, or Y449F CTLL2 cells (2 × 106/point) were incubated with no factor (-) (lanes 1, 4, and 7) or hIL-2 (5000 IU/ml, lanes 2, 5, and 8) or hIL-4 (50 ng/ml, lanes 3, 6, and 9) for 10 min at 37°C, and total cellular lysates were analyzed by PAGE and Western blotting using a polyclonal antibody raised against phosphoPKB. Molecular weight markers are indicated on the left side (kDa).
(C) Nuclear extracts from WT CTLL2 cells (5 × 105/point) treated either with no factor (lane 1), hIL-2 (5000 IU/ml, lanes 2–6), or hIL-4 (50 ng/ml, lanes 7–11) for 10 min were analyzed by EMSA in the absence (-) or presence of the indicated antibodies. S1, S3, S5A, and S5B are abbreviations for antibodies against STAT1, STAT3, STAT5A, and STAT5B, respectively. ss indicates the supershifted complexes, and arrows indicate the two STAT5-containing complexes.
(D) C-GFP, WT, or Y449F CTLL2 cells (2 × 106/point) were incubated with no factor (-) (lanes 1, 4, 7) or hIL-2 (5000 IU/ml, lanes 2, 5, and 8) or hIL-4 (50 ng/ml, lanes 3, 6, and 9) for 10 min at 37°C, and total cellular Triton lysates were analyzed by PAGE and Western blotting using a polyclonal antibody raised against phosphoPKB. Molecular weight markers are indicated on the left side (kDa).
Immunity  , DOI: ( /S (00) )

7. Figure 6
PI-3K Activation Is Needed to Maintain Thymocyte Precursor Homeostasis
CD34+ thymocytes were isolated and transduced with IRES-GFP or DNP85-IRES-GFP and incubated in an FTOC for 18 days.
(A) Output numbers of various subsets after 18 days in an FTOC. The number of GFP+ cells (y axis) is given for each subpopulation (CD4−CD8− [white bars], CD4+ [light gray bars], CD4+CD8+ [dark gray bars], TCRγδ+ [black bars], TCRαβ+ [indicated by a line], and NK or CD56+CD3− [striped bars]). The actual output numbers for the DN mutant were multiplied by three to correct for the 3-fold lower input numbers of GFP+ cells compared to the C-GFP control.
(B) Phenotype of cells harvested from FTOC seeded with control-transduced or DNP85-transduced CD34+ thymocytes. The experiment was repeated once with identical results.
Immunity  , DOI: ( /S (00) )

8. Figure 7
A Dominant-Negative STAT5B Mutant Disrupts T Cell Development in an FTOC
Total CD34+ cells (A) or CD34+CD1a− cells (B and C) were purified and transduced either with DNSTAT5B or with control-GFP. Following incubation in an FTOC, single cell suspensions were prepared and stained with the indicated PE- and TRC-labeled antibodies. The phenotypes of the GFP+ cells in the control-transduced samples were similar to those of the untransduced samples and are not shown. Similar results were obtained in two other experiments. (C) Phenotype of the DNSTAT5B-transduced and untransduced CD34+CD1a− cells cultured for 9 days in a combination of IL-7 and SCF. The percentage of GFP+ cells in this sample was 8%.
Immunity  , DOI: ( /S (00) )