Fig. 1.
Fig. 1. DC acquisition of intact cell surface molecules displayed on the plasma membrane of donor cells. / (A) DC acquisition of the cell surface marker ΔLNGFr is vector-independent. Monocyte-derived DCs were differentiated and cocultured with (b) vector-producing cells19 or with (c) the 3T3-ΔLNGFr cell line expressing the ΔLNGFr on its cell surface, but unable to produce any vector particle. Transduction efficiency was evaluated by immunofluorescence for the expression of the cell surface marker ΔLNGFr encoded by the retroviral vector. ΔLNGFr expression on (a) untreated and (b, c) treated DC populations is shown. (B) DC acquisition of the ΔLNGFr cell surface marker appears to require a cell-to-cell interaction. DCs were cultured on glass cover slips on a monolayer of nonirradiated 3T3-ΔLNGFr. Cocultures were processed for immunofluorescence and confocal microscopy 20 hours later. HLA-DR molecules expressed by DCs were visualized using an HLA-DR-FITC mAb and displayed as green staining. ΔLNGFr surface marker was visualized by the 20.4 specific primary mAb followed by a Texas red–conjugated second antibody and displayed as red staining. Optically merged confocal images showed the colocalization, displayed as yellow staining, of the HLA-DR marker with ΔLNGFr. The dependence of the ΔLNGFr transfer by intercellular contact between cell membranes of DCs and donor cells is suggested by the observation that positive DCs were always in close contact with donor cells, while negative DCs were distant (arrow). (C) DC acquisition of ΔLNGFr cell surface marker is associated with the transfer of plasma membrane lipids. DCs were exposed to 3T3-ΔLNGFr cells previously labeled with PKH26, a stable membrane-soluble red dye that does not exchange spontaneously between membranes for prolonged periods. Cocultures were processed, 20 hours later, for immunofluorescence and confocal microscopy by using an HLA-DR-FITC mAb and a Cy-5-labeled secondary mAb to detect ΔLNGFr expression. The Cy-5–labeled secondary mAb is shown as a light blue color. All DCs that acquired the cell surface marker from ΔLNGFr-expressing cells (arrows) also became positive for the PKH26 red dye. (D) Cell surface distribution of the acquired ΔLNGFr molecules. Cocultures of DCs and 3T3-ΔLNGFr cells were analyzed by double immunolabeling for ΔLNGFr and HLA-DR expression. ΔLNGFr molecules (5-nm gold particles) are interspersed between HLA-DR molecules (15-nm gold particles) on the cell surface of DCs.

DC acquisition of intact cell surface molecules displayed on the plasma membrane of donor cells.

(A) DC acquisition of the cell surface marker ΔLNGFr is vector-independent. Monocyte-derived DCs were differentiated and cocultured with (b) vector-producing cells19 or with (c) the 3T3-ΔLNGFr cell line expressing the ΔLNGFr on its cell surface, but unable to produce any vector particle. Transduction efficiency was evaluated by immunofluorescence for the expression of the cell surface marker ΔLNGFr encoded by the retroviral vector. ΔLNGFr expression on (a) untreated and (b, c) treated DC populations is shown. (B) DC acquisition of the ΔLNGFr cell surface marker appears to require a cell-to-cell interaction. DCs were cultured on glass cover slips on a monolayer of nonirradiated 3T3-ΔLNGFr. Cocultures were processed for immunofluorescence and confocal microscopy 20 hours later. HLA-DR molecules expressed by DCs were visualized using an HLA-DR-FITC mAb and displayed as green staining. ΔLNGFr surface marker was visualized by the 20.4 specific primary mAb followed by a Texas red–conjugated second antibody and displayed as red staining. Optically merged confocal images showed the colocalization, displayed as yellow staining, of the HLA-DR marker with ΔLNGFr. The dependence of the ΔLNGFr transfer by intercellular contact between cell membranes of DCs and donor cells is suggested by the observation that positive DCs were always in close contact with donor cells, while negative DCs were distant (arrow). (C) DC acquisition of ΔLNGFr cell surface marker is associated with the transfer of plasma membrane lipids. DCs were exposed to 3T3-ΔLNGFr cells previously labeled with PKH26, a stable membrane-soluble red dye that does not exchange spontaneously between membranes for prolonged periods. Cocultures were processed, 20 hours later, for immunofluorescence and confocal microscopy by using an HLA-DR-FITC mAb and a Cy-5-labeled secondary mAb to detect ΔLNGFr expression. The Cy-5–labeled secondary mAb is shown as a light blue color. All DCs that acquired the cell surface marker from ΔLNGFr-expressing cells (arrows) also became positive for the PKH26 red dye. (D) Cell surface distribution of the acquired ΔLNGFr molecules. Cocultures of DCs and 3T3-ΔLNGFr cells were analyzed by double immunolabeling for ΔLNGFr and HLA-DR expression. ΔLNGFr molecules (5-nm gold particles) are interspersed between HLA-DR molecules (15-nm gold particles) on the cell surface of DCs.

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