Figure 5
Figure 5. Receptor occupancy and functional activity of 1-7F9 in KIR2DL3tg mice. (A) KIR2DL3 is functional in tg mice. Splenocytes from wild-type (□) and from genetically modified mice (▩, KbDbKO cells; ■, KbDbKO Cw3tg cells), labeled with 0.5 and 3 μM CFSE, respectively, were mixed 1:1 (10 million each) and injected (i.v.) into wild-type or KIR2DL3tg mice. Twenty hours later, the mean percentage ± SD (3 to 6 mice per group) of cells with each level of CFSE was determined by flow cytometry. Results were analyzed by ANOVA, followed by Bonferroni posttest (***P < .005). (B) Single injection of 1-7F9 induces rejection of HLA-Cw3–positive splenocytes in a NK-dependent manner. Spleen cells from wild-type and KbDbKO, Cw3tg mice were labeled differentially with CFSE, as in panel A, and were injected into Rag KO, KIR2DL3tg mice. Four hours before the cells, 1-7F9 and NK1.1 mAbs were injected intravenously at the indicated doses. Twenty hours after injection of cells, percentage of cells with each level of CFSE was determined in blood (□) and spleen (▩) by flow cytometry, and the ratio of tg to wild-type donor cells is reported. In some animals, NK cells were depleted by administration of NK1.1 mAb (200 μg per mouse, intravenously). Curve fitting using receptor saturation equation gives an EC50 of 4.6 μg per mouse (95% CI, 1.5-7.7). Experiment shown is representative of 2 different experiments. (C) Maximum effect of 1-7F9 is achieved when KIR2DL3 receptor is saturated. The level of KIR2DL3 receptor occupancy after injection of 1-7F9 was estimated by flow cytometry. Cells from blood and spleen of mice were stained ex vivo with PE-conjugated 1-7F9, and the total MFI of the NK population (a measure of the free receptors) is determined. The level of receptor saturation at a given dose is calculated as the ratio of MFI obtained in mice injected with 1-7F9 relative to MFI obtained in untreated mice (3 mice per group). Receptor occupancy was measured in parallel with the determination of ratio of CFSE-labeled cells in B (24 hours after injection of 1-F9).

Receptor occupancy and functional activity of 1-7F9 in KIR2DL3tg mice. (A) KIR2DL3 is functional in tg mice. Splenocytes from wild-type (□) and from genetically modified mice (▩, KbDbKO cells; ■, KbDbKO Cw3tg cells), labeled with 0.5 and 3 μM CFSE, respectively, were mixed 1:1 (10 million each) and injected (i.v.) into wild-type or KIR2DL3tg mice. Twenty hours later, the mean percentage ± SD (3 to 6 mice per group) of cells with each level of CFSE was determined by flow cytometry. Results were analyzed by ANOVA, followed by Bonferroni posttest (***P < .005). (B) Single injection of 1-7F9 induces rejection of HLA-Cw3–positive splenocytes in a NK-dependent manner. Spleen cells from wild-type and KbDbKO, Cw3tg mice were labeled differentially with CFSE, as in panel A, and were injected into Rag KO, KIR2DL3tg mice. Four hours before the cells, 1-7F9 and NK1.1 mAbs were injected intravenously at the indicated doses. Twenty hours after injection of cells, percentage of cells with each level of CFSE was determined in blood (□) and spleen (▩) by flow cytometry, and the ratio of tg to wild-type donor cells is reported. In some animals, NK cells were depleted by administration of NK1.1 mAb (200 μg per mouse, intravenously). Curve fitting using receptor saturation equation gives an EC50 of 4.6 μg per mouse (95% CI, 1.5-7.7). Experiment shown is representative of 2 different experiments. (C) Maximum effect of 1-7F9 is achieved when KIR2DL3 receptor is saturated. The level of KIR2DL3 receptor occupancy after injection of 1-7F9 was estimated by flow cytometry. Cells from blood and spleen of mice were stained ex vivo with PE-conjugated 1-7F9, and the total MFI of the NK population (a measure of the free receptors) is determined. The level of receptor saturation at a given dose is calculated as the ratio of MFI obtained in mice injected with 1-7F9 relative to MFI obtained in untreated mice (3 mice per group). Receptor occupancy was measured in parallel with the determination of ratio of CFSE-labeled cells in B (24 hours after injection of 1-F9).

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