Figure 3
Figure 3. Effect of autophagy inhibition and enhancement on pUL138 TAP-independent antigen presentation. (A) LC3 immunoblot on T2.B35 pUL138-eGFP stable transfectants treated with 10mM 3-MA for 14 hours in the presence or absence of 80μM chloroquine. (B-D) T2.B35 pUL138-eGFP stable transfectants were treated with 3-MA for 72 hours and assessed for the presentation of pUL138 CD8+ T-cell epitope by intracellular cytokine secretion assay (B), and the expression of pUL138-eGFP protein by flow cytometry (C-D). (C) Solid line indicates pUL38-eGFP expression in untreated cells; and dotted line, the expression of this protein in 3-MA–treated cells. Gray-shaded area represents nontransfected T2.B35 cells. (E-G) T2.B35 cells stably expressing pUL138-eGFP protein were transduced with recombinant lentivirus encoding Atg12 shRNA or a control vector, rested for 2 days, selected in puromycin for 3 days, and then used as antigen-presenting cells in a standard intracellular cytokine secretion assay. (E) Presentation of pUL138. (F) Immunoblot for Atg12. (G) The cells were concurrently assessed for the presentation of EBV-encoded LMP2 (HLA-A2–restricted CLG epitope) and LMP1 (HLA-A2–restricted YLQ epitope) using CLG- and YLQ-specific T cells in intracellular cytokine secretion assay. (H-I) T2.B35 pUL138-eGFP stable transfectants were treated with heat shock at 39°C for 12 to 14 hours to up-regulate autophagy, and the presentation of pUL138 by heat shock treated cells was compared with control treated cells in the presence or absence of autophagy inhibition with Atg12 knockdown (H) or 3-MA (I). (J) Effect of heat shock treatment on the amount of pUL38-eGFP as measured by flow cytometry. The mean absolute percentages of T-cell response to reference cells were 8.5%, 20.5%, 10.4%, and 11.3% in panels B, E, H, and I, respectively, and 8.9% for HLA-A2/CLG and 90.7% for HLA-A2/YLQ in panel G. Data shown are from 3 independent experiments (B,D,G-J) or 4 independent experiments (E) performed in at least duplicate samples (mean ± SEM). **P < .05 (2-tailed Student t test). (C,F) Representative examples from 3 experiments.

Effect of autophagy inhibition and enhancement on pUL138 TAP-independent antigen presentation. (A) LC3 immunoblot on T2.B35 pUL138-eGFP stable transfectants treated with 10mM 3-MA for 14 hours in the presence or absence of 80μM chloroquine. (B-D) T2.B35 pUL138-eGFP stable transfectants were treated with 3-MA for 72 hours and assessed for the presentation of pUL138 CD8+ T-cell epitope by intracellular cytokine secretion assay (B), and the expression of pUL138-eGFP protein by flow cytometry (C-D). (C) Solid line indicates pUL38-eGFP expression in untreated cells; and dotted line, the expression of this protein in 3-MA–treated cells. Gray-shaded area represents nontransfected T2.B35 cells. (E-G) T2.B35 cells stably expressing pUL138-eGFP protein were transduced with recombinant lentivirus encoding Atg12 shRNA or a control vector, rested for 2 days, selected in puromycin for 3 days, and then used as antigen-presenting cells in a standard intracellular cytokine secretion assay. (E) Presentation of pUL138. (F) Immunoblot for Atg12. (G) The cells were concurrently assessed for the presentation of EBV-encoded LMP2 (HLA-A2–restricted CLG epitope) and LMP1 (HLA-A2–restricted YLQ epitope) using CLG- and YLQ-specific T cells in intracellular cytokine secretion assay. (H-I) T2.B35 pUL138-eGFP stable transfectants were treated with heat shock at 39°C for 12 to 14 hours to up-regulate autophagy, and the presentation of pUL138 by heat shock treated cells was compared with control treated cells in the presence or absence of autophagy inhibition with Atg12 knockdown (H) or 3-MA (I). (J) Effect of heat shock treatment on the amount of pUL38-eGFP as measured by flow cytometry. The mean absolute percentages of T-cell response to reference cells were 8.5%, 20.5%, 10.4%, and 11.3% in panels B, E, H, and I, respectively, and 8.9% for HLA-A2/CLG and 90.7% for HLA-A2/YLQ in panel G. Data shown are from 3 independent experiments (B,D,G-J) or 4 independent experiments (E) performed in at least duplicate samples (mean ± SEM). **P < .05 (2-tailed Student t test). (C,F) Representative examples from 3 experiments.

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