Colocalization of pUL138-eGFP and LC3 with internalized MHC class I molecules and acid stability of HLA-B35/LPL complexes. (A) T2.B35 pUL138-eGFP stable transfectants were cultured for 4 hours in complete medium containing mouse anti–human MHC class I (clone W6/32) or mouse IgG2aK isotype control, washed, and stained with AlexaFluor-546–conjugated goat anti–mouse IgG (shown in red). Colocalization of pUL138-eGFP with internalized MHC class I is shown in white. (B) Nontransfected T2.B35 cells were cultured in complete medium containing mouse anti–human MHC class I (clone W6/32), in the presence or absence of 80μM chloroquine, for 60 hours, washed, and stained with AlexaFluor-488–conjugated goat anti–mouse IgG (green) and anti-LC3B in AlexaFluor-546 (red). Colocalization of LC3 with internalized MHC class I is shown in white. (A-B) Blue represents nuclei staining with 4,6-diamidino-2-phenylindole (DAPI). Scale bars represent 5 μm. Original magnification ×64 (Olympus IX70 microscope, DeltaVision microscopy system). Image deconvolution and colocalization analysis performed with softWoRx. Right panels: scatter plots and degree of colocalization of anti-MHC class I or isotype control with pUL138 or LC3 by Pearson coefficient of correlation. Results are representative of 3 independent experiments. (C) T2.B35 pUL138-eGFP stable transfectants were treated with citric acid buffer (pH 3.0) for 0 to 30 minutes to dissociate peptide-MHC class I complexes, fixed in paraformaldehyde, and then used as antigen-presenting cells in an intracellular cytokine secretion assay. The acid stability of pUL138 HLA-B35/LPL was assessed in parallel with those of EBV LMP2 HLA-A2–restricted CLG and EBV LMP1 HLA-A2–restricted YLQ epitopes. Data shown were from 3 independent experiments, each performed in duplicate samples (mean ± SEM). The mean absolute percentages of T-cell response at time 0 were 32.0%, 4.5%, and 47.4% for HLA-B35/LPL, HLA-A2/CLG, and HLA-A2/YLQ, respectively.