Figure 3
Figure 3. 1-7F9 interferes with HLA class I-induced inhibitory signaling in KIR2DL-positive resting NK cells. (A) Freshly purified human NK cells were incubated overnight in medium. Then NK cells were incubated for 4 hours at 37°C alone (medium; ▩), with rituximab-coated 221 cells (E:T ratio = 1; □), or rituximab-coated 221-Cw3/Cw4 (E:T ratio = 1; ■) in the presence or not of 1-7F9 (10 μg/mL) and with anti-CD107 and monensin. Cells were then stained with anti-CD3, anti-CD56, and purified 1-7F9 revealed by anti–human IgG4 (HP6025); fixed; permeabilized; and finally stained with anti–IFN-γ. Percentage of IFN-γ positive (top panels) and CD107 positive (bottom panels) was then assessed on KIR2D-negative NK cells (left panels) and KIR2D-positive NK cells (right panels). Results from 1 representative donor are shown. (B) Data represent means ± SD of the percentages of IFN-γ–positive NK cells (left panel) and of CD107-positive NK cells (right panel) collected using method described in panel A, from 6 people. Results in the presence of 221-Cw3/Cw4 are shown. Statistical analysis was performed using first a one-way repeated measures ANOVA test, followed by a Bonferroni test to compare pairs of values (ie, KIR2D− medium with KIR2D− 1-7F9 and KIR2D+ medium with KIR2D+ 1-7F9). **P < .01. Multiple experiments have also been performed with 221 transfected with single HLA-C and provided similar results. (C) Thawed human PBMC from KIR-S–positive (1 and 2) or KIR-S–negative (3 and 4) donors were incubated for 4 hours at 37°C, alone or in the presence of 1-7F9 (10 μg/mL), cognate isotypic control (IgG4, 10 μg/mL), or K562 (E:T ratio = 10) in the presence of anti-CD107 and monensin. After incubation, cells were stained with anti-CD3 and anti-CD56, and then fixed, permeabilized, and stained with anti–IFN-γ. CD107 mobilization and IFN-γ production are then assessed on NK cells (CD3−CD56+ lymphocytes). Results are representative of 1 experiment of 2 done with a KIR-S–positive and a KIR-S–negative donor. Percentage of cells in each quadrant is shown.

1-7F9 interferes with HLA class I-induced inhibitory signaling in KIR2DL-positive resting NK cells. (A) Freshly purified human NK cells were incubated overnight in medium. Then NK cells were incubated for 4 hours at 37°C alone (medium; ▩), with rituximab-coated 221 cells (E:T ratio = 1; □), or rituximab-coated 221-Cw3/Cw4 (E:T ratio = 1; ■) in the presence or not of 1-7F9 (10 μg/mL) and with anti-CD107 and monensin. Cells were then stained with anti-CD3, anti-CD56, and purified 1-7F9 revealed by anti–human IgG4 (HP6025); fixed; permeabilized; and finally stained with anti–IFN-γ. Percentage of IFN-γ positive (top panels) and CD107 positive (bottom panels) was then assessed on KIR2D-negative NK cells (left panels) and KIR2D-positive NK cells (right panels). Results from 1 representative donor are shown. (B) Data represent means ± SD of the percentages of IFN-γ–positive NK cells (left panel) and of CD107-positive NK cells (right panel) collected using method described in panel A, from 6 people. Results in the presence of 221-Cw3/Cw4 are shown. Statistical analysis was performed using first a one-way repeated measures ANOVA test, followed by a Bonferroni test to compare pairs of values (ie, KIR2D− medium with KIR2D− 1-7F9 and KIR2D+ medium with KIR2D+ 1-7F9). **P < .01. Multiple experiments have also been performed with 221 transfected with single HLA-C and provided similar results. (C) Thawed human PBMC from KIR-S–positive (1 and 2) or KIR-S–negative (3 and 4) donors were incubated for 4 hours at 37°C, alone or in the presence of 1-7F9 (10 μg/mL), cognate isotypic control (IgG4, 10 μg/mL), or K562 (E:T ratio = 10) in the presence of anti-CD107 and monensin. After incubation, cells were stained with anti-CD3 and anti-CD56, and then fixed, permeabilized, and stained with anti–IFN-γ. CD107 mobilization and IFN-γ production are then assessed on NK cells (CD3−CD56+ lymphocytes). Results are representative of 1 experiment of 2 done with a KIR-S–positive and a KIR-S–negative donor. Percentage of cells in each quadrant is shown.

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