Endogenous presentation of HLA-B*3501–restricted CD8⁺ T-cell epitope from human cytomegalovirus encoded protein, pUL138, in primary human fibroblasts and TAP-deficient T2.B35 cell line. Cells were infected with recombinant adenovirus encoding the UL138 transgene (AdUL138), and antigen presentation was measured at 16 hours after infection using an intracellular cytokine secretion assay. (A) Flow cytometric dot plots showing IFN-γ secretion by pUL138-specific CD8⁺ T cells after activation by virus-infected cells. (B-D) T-cell response to virus-infected cells treated with chemical inhibitors of the proteasome (B), aminopeptidases (C), or lysosomal proteases (D). Fibroblasts or T2.B35 cells were treated with individual inhibitors starting at 1 hour before the infection and cultured in the presence of these inhibitors until the time of antigen presentation assay at 16 hours after infection. The T-cell response was normalized to that of untreated controls (B,D) or controls treated with the reducing agent dithiothreitol alone (C). The absolute (mean) percentages of T-cell response to reference cells were 23.3%, 20.7%, and 23.3% for fibroblasts, and 11.0%, 7.5%, and 11.9% for T2.B35 cells in panels B, C, and D, respectively. (E) Fibroblasts and T2.B35 cells were infected by AdUL138 at low and high MOI, with or without treatment with chloroquine. The absolute (mean) percentages of T-cell response to reference cells at MOI 5 and MOI 100 were 9.3% and 15.5% for fibroblasts and 7.4% and 11.0% for T2.B35 cells, respectively. (B-E) Data are from 3 independent experiments, each performed in triplicate samples (mean ± SEM). **P < .05 (2-tailed Student t test).