Confocal study of OX40 and 4-1BB on activated T cells and transfectant cells. (A) OX40 and 4-1BB colocalize on activated T cells. Mitogen-stimulated total T cells were stained for OX40 (green), 4-1BB (red), or CD27 (red) and analyzed by confocal microscopy. In the top 3 panels, stimulated PBLs were stained with OX40 and 4-1BB. In the right end panel, green and red fluorescence overlapped and cells exhibiting colocalization of OX40 and 4-1BB are indicated by arrows. In Ai-ii, cells positive for OX40 and 4-1BB staining (i) and cells positive for OX40 and CD27 (negative control) staining (ii) were analyzed at higher magnification for colocalization. (B) OX40 and 4-1BB colocalize in gene-transfected HEK293 cells. HEK293 cells were gene-transfected with OX40-GFP and 4-1BB-RFP or CD40L-RFP for 16 hours before confocal microscopy. In the right end panels, green and red fluorescence were overlapped to show colocalization of receptors. (C) OX40 and 4-1BB coendocytose following cross-linking to 4-1BB. HEK293 cells were gene-transfected with OX40-GFP and 4-1BB–RFP for 16 hours. Cells were treated for 30 minutes with anti–4-1BB Ab (1 μg/mL) at 4°C, 37°C, or at 37°C in the presence of sucrose (0.45 M), which prevented clathrin-dependent endocytosis.27 In the right end panels, green and red fluorescences were overlapped to emphasize the coendocytosis of receptors.
