Tetramer staining of human and mouse CD8⁺ T-cell lines. CD8 T-cell lines were incubated with anti-CD8 mAb and peptide–MHC class I–specific tetramers. The percentage of tetramers and CD8 double-positive T cells is shown in the upper right quadrant of each plot. (A) The panel shows that 0.02% of EBV-negative human peripheral blood mononuclear cells (PBMCs) tested positive for LMP₁/HLA-A2 tetramers. (B) The panel shows that 92% of LMP₁ peptide–specific CD8 T cells bound to LMP₁ tetramers. (C) The panel shows that 87% of Flu-specific CD8 T cells bound to Flu_58-66/HLA-A2 tetramers. (D) Less than 1% of the human Flu-specific CD8⁺ T cells tested positive for the LMP₁/HLA-A2 tetramers. (E) The panel shows that 0% normal mouse spleen CD8 T cells bound the LLO_91-99/H-2K^d tetramers. (F) More than 80% of the mouse splenic CD8 T-cell line bound LLO_91-99/H-2K^d tetramers. (G) The panel shows that 90% of the p60_217-225-specific T-cell line tested positive for p60_217-225 tetramer staining. (H) Only mildly decreased binding to their cognate CD8 T cells by dimeric/trimeric compared with trimeric/tetrameric MHC constructs was observed.