Variable allo-HLA recognition by T-cell clones with the same specificity but different TCR Vβ usage. To investigate whether virus-specific T cells sharing the same antigen specificity but expressing different TCRs exert the same allo-HLA reactivity, 3 VZV-IE62/HLA-A2–specific T-cell clones expressing different TCRs were stimulated for 18 hours with a panel of EBV-LCLs and IFNγ production was measured by ELISA. (A) VZV clone 5 showed alloreactivity against all HLA-B*5501⁺ EBV-LCLs. To confirm allo–HLA-B*5501 reactivity, the clone was tested against K562 cells (K562), K562 cells transduced with HLA-A*0201 (K562+A2), K562 cells transduced with HLA-B*5501 (K562+B55), and HLA-B55⁺ EBV-LCLs (B55+LCL). (B) VZV clone 6 was alloreactive against the HLA-B*5701⁺ EBV-LCLs. Clone 6 did not show reactivity against K562 cells transduced with HLA-B*5701 (K562+B57). To confirm allo–HLA-B*5701 reactivity, clone 6 was tested against HLA-B*5701–negative EBV-LCLs (HLA-B57neg LCL) or transduced with HLA-B*5701 (B57 trans), HLA-B*5701⁺ EBV-LCLs (B57+LCL), and HLA-B*5701⁺ PHA blasts (B57+PHA). (C) VZV clone 7 exhibited cross-reactivity against all HLA-A*0205⁺ and HLA-A*0207⁺ EBV-LCLs. Allo–HLA-A*0205 reactivity was confirmed by testing the clone against K562 cells (K562), K562 transduced with HLA-HLA-A*0205 (K562+HLA-A0205), HLA-A*0205–negative EBV-LCLs (HLA-A0205neg LCL), or these EBV-LCLs transduced with HLA-A*0205 (A0205 trans), HLA-A*0205⁺ EBV-LCLs (A0205+LCL), and HLA-A*0205⁺ PHA blasts (A0205+PHA). The results demonstrate that virus-specific T cells with the same antigen specificity, but with different TCR usage, exert alloreactivity against different HLA molecules. Experiments were performed in duplicate, mean values are shown ± SD.