Figure 2
Figure 2. Functional analysis of NK cells generated on coculture with OP9-DL1 or OP9 stromal cells. NK cells were generated on OP9-DL1 (□) or OP9-control stromal cells (▩) in the presence of Flt-3L, SCF, IL-7, and IL15 for 20 days and either (A) incubated with K562 target cells and analyzed for CD107 expression, (B) stimulated with IL-12 and IL-18 and analyzed for IFN-γ expression, or (C) without preincubation stained for granzyme B. (A) Percentages of CD107a+ NK cells after incubation with K562 target cells for 2 hours with different E/T ratios as indicated in the x-axis. Data are shown as the means (± SD) from 2 independent experiments. The left bar diagram shows the comparative analysis of NK-cell activity of NK cells generated on OP9-control (□) or OP9-DL1 stromal cells (▩). The right bar diagram shows the comparative analysis of NK-cell activity of gated cyCD3+ (■) and cyCD3− (□) NK cells generated on OP9-DL1. (B) Frequencies of IFN-γ+ NK cells on cytokine-activated NK cells. After 3 weeks of coculture, the cells were stimulated with rhuIL-12 and rhuIL-18. Data are shown as the means (± SD) from 2 independent experiments. The left bar diagram shows the comparative analysis of the frequency of IFN-γ+ NK cells generated on OP9-DL1 (□) or OP9-control stromal cells (▩). Right bar diagram shows the comparative analysis of the frequency of IFN-γ+ NK cells of gated cyCD3+ (■) and cyCD3− (□) NK cells generated on OP9-DL1. (C) Granzyme B expression as detected by intracellular flow cytometry of cells obtained on OP-control or OP9-DL1 stromal cells. The left panel of histograms shows the fluorescence of the isotype control of the cells cultured on OP9-DL1, the fluorescence of the granzyme mAb staining of NK cells obtained on OP-9DL1 or on OP9-control stromal cells as labelled. The right panel of histograms shows the fluorescence of the isotype control of the cells cultured on OP9-DL1 and the fluorescence of granzyme B on the gated cyCD3+ or cyCD3− NK cells cultured on OP9-DL1. The same picture, but with lower intensity for granzyme B, is observed for the OP9-control–generated NK cells (data not shown).

Functional analysis of NK cells generated on coculture with OP9-DL1 or OP9 stromal cells. NK cells were generated on OP9-DL1 (□) or OP9-control stromal cells (▩) in the presence of Flt-3L, SCF, IL-7, and IL15 for 20 days and either (A) incubated with K562 target cells and analyzed for CD107 expression, (B) stimulated with IL-12 and IL-18 and analyzed for IFN-γ expression, or (C) without preincubation stained for granzyme B. (A) Percentages of CD107a+ NK cells after incubation with K562 target cells for 2 hours with different E/T ratios as indicated in the x-axis. Data are shown as the means (± SD) from 2 independent experiments. The left bar diagram shows the comparative analysis of NK-cell activity of NK cells generated on OP9-control (□) or OP9-DL1 stromal cells (▩). The right bar diagram shows the comparative analysis of NK-cell activity of gated cyCD3+ (■) and cyCD3− (□) NK cells generated on OP9-DL1. (B) Frequencies of IFN-γ+ NK cells on cytokine-activated NK cells. After 3 weeks of coculture, the cells were stimulated with rhuIL-12 and rhuIL-18. Data are shown as the means (± SD) from 2 independent experiments. The left bar diagram shows the comparative analysis of the frequency of IFN-γ+ NK cells generated on OP9-DL1 (□) or OP9-control stromal cells (▩). Right bar diagram shows the comparative analysis of the frequency of IFN-γ+ NK cells of gated cyCD3+ (■) and cyCD3− (□) NK cells generated on OP9-DL1. (C) Granzyme B expression as detected by intracellular flow cytometry of cells obtained on OP-control or OP9-DL1 stromal cells. The left panel of histograms shows the fluorescence of the isotype control of the cells cultured on OP9-DL1, the fluorescence of the granzyme mAb staining of NK cells obtained on OP-9DL1 or on OP9-control stromal cells as labelled. The right panel of histograms shows the fluorescence of the isotype control of the cells cultured on OP9-DL1 and the fluorescence of granzyme B on the gated cyCD3+ or cyCD3− NK cells cultured on OP9-DL1. The same picture, but with lower intensity for granzyme B, is observed for the OP9-control–generated NK cells (data not shown).

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