Allo-HLA reactivity of virus-specific T-cell clones. Forty-one virus-specific T-cell clones, of which 7 are shown in this figure, were stimulated with a panel of EBV-LCLs for 18 hours, and IFNγ production was measured by ELISA. (A) The EBV-EBNA3A/HLA-A3–specific T-cell clone 1 exhibited alloreactivity against all EBV-LCLs expressing HLA-A*3101 and 1 EBV-LCL expressing HLA-A*3001. To confirm allo–HLA-A*3101 reactivity, clone 1 was tested against K562 cells (K562), K562 cells transduced with HLA-A*0201 (K562+A2), K562 cells transfected with HLA-A*3101 (K562+A31), and HLA-A*3101–negative (A31neg LCL) and HLA-A*3101–positive (A31+LCL) EBV-LCLs. To confirm the reactivity against HLA-A*3001, the clone was tested against 3 HLA-A*3001⁺ EBV-LCLs, of which 1 is shown, and 1 HLA-A*3101⁺ EBV-LCLs with or without blocking mAbs directed against HLA class I, HLA-A30/A31, and HLA-A2. (B) The CMV-pp50/HLA-A1–specific T-cell clone 24 exhibited alloreactivity against all HLA-A*1101–expressing EBV-LCLs. To confirm allo–HLA-A*1101 reactivity, clone 24 was tested against K562 cells (K562), K562 cells transduced with HLA-A*0201 (K562+A2), K562 cells transduced with HLA-A*1101 (K562+A11), and HLA-A*1101–positive EBV-LCLs (A11+LCL). (C) The EBV-BRLF1/HLA-A3–specific clone 19 exerted alloreactivity against all HLA-A0201⁺ EBV-LCLs. This T-cell clone did not recognize K562 transduced with HLA-A*0201 (K562+A2). To confirm allo–HLA-A*0201 reactivity, clone 19 was tested against untransduced HLA-A*0201–negative EBV-LCLs (HLA-A2neg LCL) or transduced with HLA-A*0201 (A2 trans), and HLA-A*0201–positive EBV-LCLs (A2+LCL). To investigate whether this clone recognized an EBV-derived peptide in the context of HLA-A*0201, the clone was tested against HLA-A*0201⁺ PHA blasts and HLA-A*0201–positive and –negative EBV-LCLs as controls. (D) The CMV-pp65/HLA-B7–specific T-cell clone 11 exhibited reactivity against all 3 HLA-DRB1*0801⁺ EBV-LCLs. To confirm allo–HLA-DRB1*0801 reactivity, clone 11 was tested against the 3 HLA-DRB1*0801⁺ EBV-LCLs, of which 1 is shown, in the presence of either no blocking mAbs (no block) or anti–HLA class I (class I), anti–HLA class II (class II), anti–HLA-DR (DR), anti–HLA-DR (DQ), and anti–HLA-DR (DP) blocking mAbs. (E) The EBV-EBNA3A/HLA-B8–specific T-cell clone 9 exhibited alloreactivity against all EBV-LCLs expressing either HLA-B*4402 or HLA-B*5501. To confirm HLA-B*4402 and HLA-B*5501 cross-reactivity, clone 9 was tested against K562 cells (K562), or K562 cells transfected with HLA-B*4402 (K562+B4402) or HLA-B*5501 (K562+B5501). As controls, clone 9 was tested against HLA-B*4402– and HLA-B*5501–negative EBV-LCLs (neg LCL), HLA-B*4402⁺ EBV-LCLs (B4402+LCL), HLA-B*5501⁺ EBV-LCLs (B5501+LCL), or HLA-B*0801⁺ K562 loaded with viral peptide (K562+B8+pept). (F) The Flu-HA/HLA-DR4–specific clone 5 recognized all HLA-DRB1*1301⁺ EBV-LCLs. To confirm allo–HLA-DRB1*1301 reactivity, clone 5 was tested against HLA-DRB1*1301⁺ EBV-LCLs (FAQ DR13⁺ and IZA DR13⁺) as well as HLA-DR13–negative EBV-LCLs nontransduced (DR13 neg) or transduced with HLA-DRB1*1301 (DR13 trans). (G) The CMV-pp65/HLA-A2–specific clone HRN 3 recognized EBV-LCLs that did not share 1 particular allo-HLA molecule. To investigate whether this reactivity was based on allo-HLA recognition, the clone was tested against 4 of the recognized EBV-LCLs with and without blocking mAb directed against HLA class I. Experiments were performed in duplicate, mean values are shown ± SD.