Acquisition of intact allogeneic human leukocyte antigen molecules by human dendritic cells by Vincenzo Russo, Dan Zhou, Claudia Sartirana, Patrizia Rovere, Antonello Villa, Silvano Rossini, Catia Traversari, and Claudio Bordignon Blood Volume 95(11):3473-3477 June 1, 2000 ©2000 by American Society of Hematology

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. 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. Vincenzo Russo et al. Blood 2000;95:3473-3477 ©2000 by American Society of Hematology

DC acquisition of allogeneic HLA class I molecules DC acquisition of allogeneic HLA class I molecules.DCs from (A) HLA-A2–negative or (B) HLA-DR*0101–negative individuals were cultivated on a monolayer of nonirradiated melanoma cells expressing the HLA-A2 or the HLA-DR*0101 allele, respectively (right panel... DC acquisition of allogeneic HLA class I molecules.DCs from (A) HLA-A2–negative or (B) HLA-DR*0101–negative individuals were cultivated on a monolayer of nonirradiated melanoma cells expressing the HLA-A2 or the HLA-DR*0101 allele, respectively (right panels). As a control, DCs were cocultured with HLA-unrelated melanoma cells (left panels). After 20 hours of cocultivation, the expression of the allogeneic HLA molecules on the cell surface of DC populations was analyzed by flow cytometry using mAbs specific for CD83 and (A) HLA-A2 or (B) DR0101 molecules. Vincenzo Russo et al. Blood 2000;95:3473-3477 ©2000 by American Society of Hematology

DC acquisition of HLA-Bw4 molecules DC acquisition of HLA-Bw4 molecules.DCs were cultured on glass cover slips on a monolayer of nonirradiated melanoma cells. DC acquisition of HLA-Bw4 molecules.DCs were cultured on glass cover slips on a monolayer of nonirradiated melanoma cells. The cocultures were processed for immunofluorescence and confocal microscopy 20 hours later. (A, B) HLA-DR molecules expressed by DCs were visualized using an HLA-DR-FITC mAb and displayed as green staining. (A, B) HLA-Bw4 molecules were visualized by specific primary mAbs followed by a Texas red–conjugated second antibody and displayed as red staining. (A, B) Optically merged confocal images showed the colocalization, displayed as yellow staining, of the HLA-DR marker with HLA-Bw4 molecules on the cell surface of DCs. The transfer of cell membrane molecules starts from the point of contact between DCs and melanoma cells (B, indicated by arrows). Vincenzo Russo et al. Blood 2000;95:3473-3477 ©2000 by American Society of Hematology

DC acquisition of allogeneic HLA class I from activated human lymphocytes.DCs from an HLA-A2–negative individual were cocultivated with PHA-activated human lymphocytes expressing the HLA-A2 allele (right panels). DC acquisition of allogeneic HLA class I from activated human lymphocytes.DCs from an HLA-A2–negative individual were cocultivated with PHA-activated human lymphocytes expressing the HLA-A2 allele (right panels). As a control, DCs were cocultured with HLA-unrelated lymphocytes (left panels). After 20 hours, the expression of the allogeneic HLA molecules on the cell surface of the DC populations was analyzed by flow cytometry using mAbs specific for HLA-A2 and CD83 (upper panels) or CD1a (lower panels). Vincenzo Russo et al. Blood 2000;95:3473-3477 ©2000 by American Society of Hematology

T-cell recognition of foreign HLA class I molecules displayed by DCs T-cell recognition of foreign HLA class I molecules displayed by DCs.DCs from an HLA-A2–negative individual were cultivated on a monolayer of nonirradiated melanoma cells expressing the HLA-A2 allele (•). T-cell recognition of foreign HLA class I molecules displayed by DCs.DCs from an HLA-A2–negative individual were cultivated on a monolayer of nonirradiated melanoma cells expressing the HLA-A2 allele (•). As a control, DCs were either untreated (○) or cocultured with an HLA-unrelated melanoma cell line (▵). After 20 hours, DCs were harvested, purified from the potential melanoma contamination by CD45RO microbead separation, and used as targets in a standard chromium release assay. Only DCs exposed to the HLA-A2 melanoma are recognized by anti–HLA-A2 cytotoxic T-cell effectors. Recognition of the HLA-A2–positive melanoma cells is shown (▪). Vincenzo Russo et al. Blood 2000;95:3473-3477 ©2000 by American Society of Hematology