Effect of autophagy inhibition and enhancement on pUL138 presentation by HCMV-infected cells. (A) T-cell response to HCMV-infected fibroblasts treated with chemical inhibitors. Fibroblasts were pretreated with inhibitors before HCMV infection and cultured in the presence of these inhibitors until the time of antigen presentation assay at 16 hours after infection. (B) T-cell response to HCMV-infected fibroblasts transduced with recombinant lentivirus encoding Atg12 shRNA or a control vector. Antigen presentation assay was performed 14 hours after HCMV infection and 6 to 7 days after lentivirus infection. (A-B) Also shown are T-cell responses to surface-loaded controls. (C) Response of HCMV pp50-specific T cells (VTEHDTLLY/HLA-A1) to HCMV-infected fibroblasts transduced with lentivirus. (D) LC3 immunoblot on primary fibroblast (MRC-5) cell line treated with 50 ng/mL TNF-α for 24 hours in the presence or absence of 40μM chloroquine in the final 12 hours. (E) Effect of autophagy up-regulation on antigen presentation. Fibroblasts were pretreated with 50 ng/mL TNF-α for 24 hours, with or without 3-MA, infected with HCMV, and rested without TNF-α until the time of antigen presentation assay at 14 hours after infection. The presentation of pUL138 was compared with that of a conventionally processed HCMV antigen, pp50. (F) Flow cytometric dot plots showing IFN-γ secretion by pUL138-specific CD8⁺ T cells in response to mock-infected or HCMV (TB40E strain)–infected MoDCs. (G) T-cell response to HCMV-infected MoDCs treated with chemical inhibitors of antigen-processing pathways from 3 hours to 24 hours after infection. Also shown are T-cell responses to surface-loaded controls. (A-C,E,G) Data are from 3 independent experiments performed in at least duplicate samples (mean ± SEM). **P < .05 (2-tailed Student t test). The mean absolute percentages of T-cell response to reference cells were 10.1%, 11.2%, 21.2%, 2.8%, and 4.2% in panels A, B, C, E, and G, respectively.