In vitro assessment of PNH cell sensitivity to mHa-specific CD8⁺ or CD4⁺ T-cell clones. mHa-reactive T-cell clones were tested for differences in their ability to recognize and/or kill GPI-anchored protein–positive (normal) versus GPI-anchored protein–negative (PNH) cells. (A) A CD3⁺/CD8⁺ T-cell clone expressing a single T-cell receptor (TCR Vβ-16, determined by flow cytometry) was expanded with a cytokine profile (B) compatible with MHC class I–restricted recognition of a patient-specific mHa. (C) This clone was cytotoxic to patient but not donor EBV-LCLs and killed GPI-negative (CD19⁺/CD55^–/CD59^–) B cells in a similar fashion as GPI-positive (CD19⁺/CD55⁺/CD59⁺) B cells. (D) A CD4⁺ T-cell clone that expressed a single T-cell receptor (TCR Vβ-17) was isolated by limiting dilution from the same mHa-reactive CTL line and secreted IFN-γ against recipient but not donor EBV-LCLs, confirming patient-specific mHa recognition. (E) A decrease in IFN-γ when patient EBV-LCLs were precultured with an HLA-DR blocking antibody was consistent with T-cell antigen recognition within the context of MHC class II. (F) This CD4⁺ T-cell clone secreted a slightly higher amount of IFN-γ when cocultured with GPI-negative (CD64⁺/CD14^–/CD59^–) compared with GPI-positive (CD64⁺/CD14⁺/CD59⁺) patient monocytes. Error bars represent 1 standard deviation.