FIG 6.

BDLF3 induces more rapid internalization and a delayed appearance of both MHC class I and class II molecules at the cell surface. Internalization and appearance assays were performed on MJS cells transiently expressing control-GFP or BDLF3-GFP. The GFP⁺ population was used to gate BDLF3-expressing cells. Internalization and appearance assays were performed on cells pretreated on ice with saturating amounts of anti-MHC class I antibody or anti-MHC class II antibody. Cells were then washed and incubated at 37°C for up to 60 min. (A) For the internalization assay, viable cells harvested at each time point were stained with an APC-conjugated goat anti-mouse IgG antibody and analyzed using flow cytometry at the indicated times; this identified the prelabeled antibody-bound MHC molecules that remained at the surface, while endocytosed labeled MHC molecules were not detected on the viable cells. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. (B) For the appearance assays, newly arrived MHC-I and MHC-II molecules, which were not prelabeled with unconjugated antibodies, were detected by staining with an APC-conjugated anti-MHC class I antibody or anti-MHC class II antibody. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. Results are representative of three independent experiments.