IDE is not involved in endogenous MHC-I presentation of viral antigens and a self antigen.

A, 150,000 CFSE-labeled OT-I T cells were incubated with EG7 cells complemented to 100,000 cells with EL4 cells, with the percentage of EG7 cells in the mixture indicated in the legend. As control, 100,000 EL4 cells were incubated with S8L or irrelevant peptide TSYFESEV (T8V). After 16 h (left) and 48 h (right), T cell activation was assessed by measuring the IL-2 concentration in the supernatants and T cell proliferation by the dilution of CFSE, respectively. B, HeLa-Kb cells were transfected with siRNA. Seventy-two hours later, the cells were infected with a recombinant vaccinia virus encoding ovalbumin or the H-2Kb restricted epitope SIINFEKL, or pulsed with 10−10M SIINFEKL peptide and incubated with SIINFEKL-specific OT-I T cells, using different effector to target ratios. Secretion of IFN-γ by OT-I cells was measured by ELISA. In panel C, formation of S8L/H2-Kb complexes at the cell surface of IDE wt and ko C57BL/6 MEFs was evaluated 6 h after infection with vaccinia viruses expressing OVA or not (CTRL), by staining cells with mAb 25D1.16; numbers indicate the MFI for 25D1.16. Panel D shows an equivalent experiment with siRNA transfected HeLa-Dd cells and CTL recognizing a peptide from HIV gp160. Here the antigens were (from left to right) 10−7M cognate peptide G9I, wt vaccinia virus, vaccinia virus encoding HIV-env, and a plasmid encoding peptide G9I (pM-G9I). Presentation was assessed using a standard kill assay with an effector to target ratio of 2∶1. E, HEK293-Kd cells stably expressing an IGRP-GFP fusion protein were transfected with siRNA and acid stripped 56 h after transfection (IGRP). Prior to addition of CTLs at an E:T ratio of 1∶1, 72 h after transfection, part of the target cells was incubated with epoxomicin (Epo). Secretion of IFN-γ by CTLs was measured by standard ELISA. Cells treated as described and pulsed with 10−6M super-agonist peptide NRP-V7 were positive controls. One out of 3 (A, B, D), 2 (C) or 5 (E) experiments is shown.