Figure 3. ABabDR4-derived TCRs recognized NY-ESO-1 more efficiently than human-derived TCRs.

(A) Protein lysates from cell lines used for coculture experiments in C and D were assessed for the presence of NY-ESO-1 protein. β-Actin was stained as protein loading control. (B) Melanoma cell lines pretreated with IFN-γ and the LCL BSM were stained for HLA-DR (dark gray) or isotype control (light gray) and were measured by flow cytometry. (C and D) TCR-transduced CD4⁺ T cells were cocultured with the LCL BSM (HLA-DR4⁺) and the melanoma cell lines FM3 (NY-ESO-1^–, HLA-DR4⁺), FM6 (NY-ESO-1⁺, HLA-DR4^–), FM82, and FM56 (NY-ESO-1⁺, HLA-DR4⁺). Cell lines FM3-NY and BSM-NY were transduced to express NY-ESO-1; BSM was transduced with mCherry (BSM-mCh) as a control. NY-ESO-1₁₁₆ (NY116), PMA and ionomycin (P/I), and blocking antibody αHLA-DR or αHLA-ABC were added where indicated. After overnight incubation, IFN-γ or IL-2 was measured in the supernatant. Mean values of intra-assay duplicates with SD are shown. The results are representative of 3 independent experiments performed with PBLs from different donors (B, C, and D).