Natural HLA class II epitopes can be divided in HLA-DM–sensitive and –resistant antigens. The cervix carcinoma HeLa cell line was retrovirally transduced with the invariant chain (Ii), HLA-DM, or a combination of both. (A) Indicated is the HLA-DR (left) and class II–associated invariant chain peptide (CLIP; right) surface expression as measured by flow cytometry. (B) All HeLa variants were cotransduced with the appropriate HLA class II restriction molecules, and specific release of IFN-γ by the CD4⁺ T-cell clones after exogenous loading with recombinant proteins was measured in ELISA. Antigens in the upper row display a DM-resistant phenotype as defined by retained antigen presentation on expression of HLA-DM, whereas DM-sensitive antigens with impaired presentation in the presence of HLA-DM (DM-sensitive) are depicted in the lower part. Mean ± SD release of IFN-γ in duplicate wells is shown. (C) HLA-DM was retrovirally transduced into EBV-transformed B-cell lines (EBV-LCL) from patient MRJ, and tested for T-cell recognition in IFN-γ ELISA. CD4⁺ T-cell clones specific for PI4K2B, MR1, PTK2B, LY75, and MTHFD1 were isolated from patient MRJ and selected on the basis of recognition of patient-derived EBV-LCL. HLA-DM was also introduced into the HLA-DQB1*0502–positive Raji cell line derived from a male patient with Burkitt lymphoma to measure T-cell recognition of endogenous DBY antigen. Solid squares and open circles indicate T-cell recognition of the endogenous antigens as expressed in the wild-type and HLA-DM transduced EBV-LCL, respectively. Numbers of titrated target cells are depicted on the x-axis. Mean ± SD release of IFN-γ in duplicate wells is shown.