Figure 6. DP^84GGPM87-expressing cells constitutively present intracellular peptides generated by the proteasome and TAP-dependent pathway.

(a) K562 aAPCs were transiently transfected with the indicated combinations of genes and cultured in the presence or absence of 0.02 μM bortezomib or 0.02 μM carfilzomib for 48 h. Total cell lysates were immunoblotted with anti-MAGE-A3, anti-HLA-class I or anti-β-actin mAb. (b,c) K562/DP4/Ii cells were transiently transfected with a retrovirus vector encoding IRES-EGFP (control), or a native (b) or endosome-targeted (c) form of MAGE-A3 linked with IRES-EGFP. Cells were then cultured with carfilzomib at the indicated concentrations for 48 h. Transient transfection efficiencies were normalized to EGFP expression measured by flow cytometry. DP4/MAGE-A3_243–258 CD4⁺ T cells were stimulated with the K562/DP4/Ii transfectants and IFN-γ secretion was measured by ELISPOT analysis. Data shown represent means±s.d.'s of triplicates. (d) DP4/MAGE-A3_243–258 CD4⁺ T cells were stimulated with the indicated K562-based aAPCs pulsed with tetanus toxin_947–967 (control) or MAGE-A3_243–258 peptide, and IFN-γ secretion was evaluated by ELISPOT assays. Data shown represent means±s.d.'s of triplicates. (e,f) The indicated K562-based aAPCs were transiently transfected with a retrovirus vector encoding IRES-EGFP (control) or a native (e) or endosome-targeted (f) form of MAGE-A3 linked with IRES-EGFP. Transient transfection efficiencies were normalized to EGFP expression measured by flow cytometry. DP4/MAGE-A3_243–258 CD4⁺ T cells were stimulated with the indicated aAPCs and IFN-γ secretion was measured by ELISPOT analysis. Data shown represent means±s.d.'s of triplicates. Results are representative of three independent experiments. ns, not significant; *P<0.05 by unpaired, two-tailed Welch's t-test.