Fig. 6.
Fig. 6. (A) Daudi cells transfected with the PAF-receptor cDNA express membrane PAF receptors. Wild-type Daudi cells were transfected by the plasmid pCI-neo vectors expressing the sense PAF receptor cDNA gene (pCI-neo.PAF-R s), the PAF receptor antisense cDNA gene (pCI-neo. PAF-R as), and by a plasmid expressing no gene (pCI-neo) control vector. At 4 days later, PAF receptor membrane expression was quantified by flow cytometry using an mAb anti-PAF receptor on nonpermeabilized Daudi transfected cells (solid lines). Cells were stained with propidium iodide to facilitate discrimination among live and dead cells. The background signal is shown (dotted lines). Inset numbers indicate the percentage of cells expressing the PAF receptor on untransfected Daudi cells (wild-type), Daudi cells transfected with pCI-neo PAF receptor sense gene (pCI-neo PAF-R s), Daudi cells transfected with the pCI-neo PAF receptor antisense gene (pCI-neo PAF-R as), and Daudi cells transfected with pCI-neo. (B) PAF-receptor cDNA transfection in the MHC class I and FasR-negative NK-resistant Daudi cells restores susceptibility to NK perforin-dependent lysis. Wild-type Daudi cells and PAF-receptor–positive (pCI-neo PAF-R s), PAF-receptor–negative (pCI-neo PAF-R as and pCI-neo plasmids) transfected Daudi cells were used independently as targets in short-term 51Cr-release assays using naive NK cells as effectors. PAF-receptor–positive Daudi cell susceptibility to NK cells was tested with and without a PAF-receptor antagonist, WEB 2086, added at 2 μmol/L. Resting NK cells were mixed with labeled target cells at the E-to-T ratios of 50:1, 25:1, 12:1, and 6:1, and perforin-induced Daudi cell lysis was measured at 4 hours. Naive NK cells were totally unable to lyse PAF-receptor–negative Daudi cells (wild-type and PAF-receptor–negative transfectants) but were efficient in killing PAF-receptor–positive Daudi cells. The susceptibility of PAF-receptor–expressing Daudi cells to NK lysis was totally reversed in the presence of the PAF receptor antagonist (pCI-neo PAF-R s + WEB).

(A) Daudi cells transfected with the PAF-receptor cDNA express membrane PAF receptors. Wild-type Daudi cells were transfected by the plasmid pCI-neo vectors expressing the sense PAF receptor cDNA gene (pCI-neo.PAF-R s), the PAF receptor antisense cDNA gene (pCI-neo. PAF-R as), and by a plasmid expressing no gene (pCI-neo) control vector. At 4 days later, PAF receptor membrane expression was quantified by flow cytometry using an mAb anti-PAF receptor on nonpermeabilized Daudi transfected cells (solid lines). Cells were stained with propidium iodide to facilitate discrimination among live and dead cells. The background signal is shown (dotted lines). Inset numbers indicate the percentage of cells expressing the PAF receptor on untransfected Daudi cells (wild-type), Daudi cells transfected with pCI-neo PAF receptor sense gene (pCI-neo PAF-R s), Daudi cells transfected with the pCI-neo PAF receptor antisense gene (pCI-neo PAF-R as), and Daudi cells transfected with pCI-neo. (B) PAF-receptor cDNA transfection in the MHC class I and FasR-negative NK-resistant Daudi cells restores susceptibility to NK perforin-dependent lysis. Wild-type Daudi cells and PAF-receptor–positive (pCI-neo PAF-R s), PAF-receptor–negative (pCI-neo PAF-R as and pCI-neo plasmids) transfected Daudi cells were used independently as targets in short-term 51Cr-release assays using naive NK cells as effectors. PAF-receptor–positive Daudi cell susceptibility to NK cells was tested with and without a PAF-receptor antagonist, WEB 2086, added at 2 μmol/L. Resting NK cells were mixed with labeled target cells at the E-to-T ratios of 50:1, 25:1, 12:1, and 6:1, and perforin-induced Daudi cell lysis was measured at 4 hours. Naive NK cells were totally unable to lyse PAF-receptor–negative Daudi cells (wild-type and PAF-receptor–negative transfectants) but were efficient in killing PAF-receptor–positive Daudi cells. The susceptibility of PAF-receptor–expressing Daudi cells to NK lysis was totally reversed in the presence of the PAF receptor antagonist (pCI-neo PAF-R s + WEB).

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