Fig. 8.
Fig. 8. SDF-1α synergizes with IL-7 to stimulate the proliferation of human thymocyte precursors. / (A) Determination of CD34+ thymic cell proliferation after culture for 48 hours in serum-free medium alone or supplemented with rhSDF-1α, IL-7, SCF, or flt3-ligand. Cells were pulsed for 12 hours with BrdU. A specific kit was used to measure BrdU incorporation into newly synthesized DNA. Full details are given in “Materials and methods.” Results are the means of 4 independent experiments, each with 3 cultures per point. Asterisks refer to the statistical significance differences between control and the different treatments. *P ≤ .05; **P ≤ .01. (B) Determination of CD34+ thymic cell proliferation after culture for 48 hours in serum-free medium alone or supplemented with rhSDF-1α (1), IL-7 (2), SCF (3), flt3-ligand (4), or a combination of rhSDF-1α plus IL-7 (5), SCF (6), or flt3-ligand (7). Results are expressed as the percentage of values from untreated cultures (number of cells × 100/number of cells in control cultures). The dashed line represents the control values. The index of synergy between SDF-1α and IL-7 was 1.97, with SCF 0.50 and with flt3-ligand 0.54. This index was calculated as BrdU incorporation in the presence of IL-7, SCF or flt3-ligand with rhSDF-1α divided by the BrdU incorporation by rhSDF-1α alone plus IL-7, SCF, or flt3-ligand alone. (C) Expression of CD127 and CXCR4 was analyzed in CD34+ thymic cell precursors after culture for 48 hours in serum-free medium alone or supplemented with rhSDF-1α or IL-7. The percentages of positive cells and their MFI are indicated in each histogram. Results are representative of 4 independent experiments. (D) SCID fetal thymic lobes were seeded with CD34+ human thymic precursors and organ-cultured for 10 days in the presence of rhSDF-1α (1), rhIL-7 (2), neutralizing anti–SDF-1α antibodies (3), neutralizing anti–IL-7 antibodies (4), rhIL-7 plus anti–SDF-1α antibodies (5), or rhSDF-1α plus anti–IL-7 antibodies (6). The numbers of human cells were calculated by multiplying the total number of cells per lobe by the percentage of human CD45-FITC+ cells determined by flow cytometry analysis. Results are expressed as the percentage of values from the control FTOC (number of cells × 100/number of cells in control culture). The dashed line represents the control values.

SDF-1α synergizes with IL-7 to stimulate the proliferation of human thymocyte precursors.

(A) Determination of CD34+ thymic cell proliferation after culture for 48 hours in serum-free medium alone or supplemented with rhSDF-1α, IL-7, SCF, or flt3-ligand. Cells were pulsed for 12 hours with BrdU. A specific kit was used to measure BrdU incorporation into newly synthesized DNA. Full details are given in “Materials and methods.” Results are the means of 4 independent experiments, each with 3 cultures per point. Asterisks refer to the statistical significance differences between control and the different treatments. *P ≤ .05; **P ≤ .01. (B) Determination of CD34+ thymic cell proliferation after culture for 48 hours in serum-free medium alone or supplemented with rhSDF-1α (1), IL-7 (2), SCF (3), flt3-ligand (4), or a combination of rhSDF-1α plus IL-7 (5), SCF (6), or flt3-ligand (7). Results are expressed as the percentage of values from untreated cultures (number of cells × 100/number of cells in control cultures). The dashed line represents the control values. The index of synergy between SDF-1α and IL-7 was 1.97, with SCF 0.50 and with flt3-ligand 0.54. This index was calculated as BrdU incorporation in the presence of IL-7, SCF or flt3-ligand with rhSDF-1α divided by the BrdU incorporation by rhSDF-1α alone plus IL-7, SCF, or flt3-ligand alone. (C) Expression of CD127 and CXCR4 was analyzed in CD34+ thymic cell precursors after culture for 48 hours in serum-free medium alone or supplemented with rhSDF-1α or IL-7. The percentages of positive cells and their MFI are indicated in each histogram. Results are representative of 4 independent experiments. (D) SCID fetal thymic lobes were seeded with CD34+ human thymic precursors and organ-cultured for 10 days in the presence of rhSDF-1α (1), rhIL-7 (2), neutralizing anti–SDF-1α antibodies (3), neutralizing anti–IL-7 antibodies (4), rhIL-7 plus anti–SDF-1α antibodies (5), or rhSDF-1α plus anti–IL-7 antibodies (6). The numbers of human cells were calculated by multiplying the total number of cells per lobe by the percentage of human CD45-FITC+ cells determined by flow cytometry analysis. Results are expressed as the percentage of values from the control FTOC (number of cells × 100/number of cells in control culture). The dashed line represents the control values.

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