American Society of Hematology

Figure 1

$Figure 1. Evidence for increased surveillance of cells in mitosis by human NK cells. (A) Because no cell surface protein is known to be uniquely expressed during mitosis and flow cytometry using DNA dyes cannot specifically separate cells in mitosis, images of conjugates stained for α-tubulin were used to distinguish cells in mitosis (top row) from target cells in other stages of the cell cycle (bottom row). Micrographs show bright-field images (left column) and corresponding fluorescence images (right column) of NK cells (small cells) bound to target cells. (B) The percentage of various susceptible target cells bound by NK cells was assessed after 10 minutes of coincubation. Data from at least 3 independent experiments are shown, and the total numbers of cells indicated. (C-D) Target cells (721.221) were arrested in mitosis by incubation overnight with colchicine (▪) or treated with DMSO as a control (♦). Alternatively, 721.221 cells were treated with a higher concentration of colchicine for 1 hour to disrupt the cytoskeleton without arresting cells in mitosis (▴). Target cells were incubated with CFSE-labeled NK cells and the percentage of CD107a+ NK cells was determined by flow cytometry. (C) Representative flow cytometric dot plots after 10 minutes of coincubation. (D) Percentages of CD107a+ NK cells after different times of coincubation. (E) 721.221 target cells were arrested in mitosis by overnight incubation with nocodazole (▪) or the DMSO vehicle only, as a control (♦). Target cells were then incubated with NK cells, and the percentage of CD107a+ NK cells was determined by flow cytometry. (F) The fraction of HT1080 cells in mitosis was enriched using the shake-off method. The percentages of NK cells that were CD107a+ after incubation with free-floating HT1080 cells (containing ~40%-50% of cells in mitosis) or adherent HT1080 cells (in other stages of the cell cycle) were compared using flow cytometry. (G) The percent of autologous T cells bound by NK cells was assessed after 10 minutes of coincubation by imaging, as in panel B. (H) Autologous T cells were arrested in mitosis by overnight incubation with colchicine (~40% of cells in mitosis) or treated with DMSO as a control (3% of cells in mitosis). The percent of CD56+ NK cells that were CD107a+, that is, degranulated, was determined by flow cytometry after 1 hour of coincubation with T cells or positive control cells 721.221. (B,G) Data are from 3 independent experiments, and the total numbers of cells are indicated. (C-F,H) Representative data from 3 experiments are shown. *P <.05, **P <.005.$

Evidence for increased surveillance of cells in mitosis by human NK cells. (A) Because no cell surface protein is known to be uniquely expressed during mitosis and flow cytometry using DNA dyes cannot specifically separate cells in mitosis, images of conjugates stained for α-tubulin were used to distinguish cells in mitosis (top row) from target cells in other stages of the cell cycle (bottom row). Micrographs show bright-field images (left column) and corresponding fluorescence images (right column) of NK cells (small cells) bound to target cells. (B) The percentage of various susceptible target cells bound by NK cells was assessed after 10 minutes of coincubation. Data from at least 3 independent experiments are shown, and the total numbers of cells indicated. (C-D) Target cells (721.221) were arrested in mitosis by incubation overnight with colchicine (▪) or treated with DMSO as a control (♦). Alternatively, 721.221 cells were treated with a higher concentration of colchicine for 1 hour to disrupt the cytoskeleton without arresting cells in mitosis (▴). Target cells were incubated with CFSE-labeled NK cells and the percentage of CD107a⁺ NK cells was determined by flow cytometry. (C) Representative flow cytometric dot plots after 10 minutes of coincubation. (D) Percentages of CD107a⁺ NK cells after different times of coincubation. (E) 721.221 target cells were arrested in mitosis by overnight incubation with nocodazole (▪) or the DMSO vehicle only, as a control (♦). Target cells were then incubated with NK cells, and the percentage of CD107a⁺ NK cells was determined by flow cytometry. (F) The fraction of HT1080 cells in mitosis was enriched using the shake-off method. The percentages of NK cells that were CD107a⁺ after incubation with free-floating HT1080 cells (containing ~40%-50% of cells in mitosis) or adherent HT1080 cells (in other stages of the cell cycle) were compared using flow cytometry. (G) The percent of autologous T cells bound by NK cells was assessed after 10 minutes of coincubation by imaging, as in panel B. (H) Autologous T cells were arrested in mitosis by overnight incubation with colchicine (~40% of cells in mitosis) or treated with DMSO as a control (3% of cells in mitosis). The percent of CD56⁺ NK cells that were CD107a⁺, that is, degranulated, was determined by flow cytometry after 1 hour of coincubation with T cells or positive control cells 721.221. (B,G) Data are from 3 independent experiments, and the total numbers of cells are indicated. (C-F,H) Representative data from 3 experiments are shown. *P <.05, **P <.005.

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