Detection and specific lysis of tetramer-positive, CD8⁺ T cells from PBMCs from multiparous female donors nos. 1, 3, and 4. (A,E,I) Staining of PBMCs with HY^A2, HA-1^A2, and HA-2^A2 tetramers, respectively (y-axis) and CD8 antibody (x-axis) after magnetic bead depletion of CD4⁺, CD14⁺, CD16⁺, and CD19⁺ cells. (B,F,J) Analysis of cells collected after nonstringent sorting of the rare CD8⁺, tetramer-positive cells from region R1 for HY^A2, HA1^A2, and HA-2^A2 tetramer, respectively (y-axis), and CD8 (x-axis) staining. In each donor, tetramer-specific cells could be clearly visualized after reanalysis of the cells sorted from region R1 (B,F,J). (M-N) Phenotype analysis of CD8⁺, HY^A2 tetramer-positive cells from donor no. 1 in R2 for the markers CD45RA (M) and CD27 (N; filled histograms); control staining of tetramer-negative cells is represented by open histograms. Bright tetramer staining of polyclonal cultures expanded after double sorting of CD8⁺ HY^A2 and HA-1^A2 tetamer-staining cells (C,G). (K) Tetramer staining of a CTL clone generated from HA-2^A2 tetramer double-sorted cells. The x-axis represents minor H antigen–specific tetramers; the y-axis represents CD8 staining. (D,H,L) Cytotoxic activity of cultured, double-sorted CTLs. Strong minor H antigen–specific lysis can be observed; x-axis, E/T ratio; y-axis, percent specific lysis. Target cells: ▴, Epstein-Barr virus-/lymphoid cell line (EBV-LCL) positive for the relevant minor H antigen (HY, HA-1, and HA-2 for panels D, H, and L, respectively); ▪, minor H antigen negative EBV-LCL pulsed with the relevant minor H peptide; ▵, EBV-LCL negative for the relevant minor H antigen; ⋄, HLA-mismatched EBV-LCL; ○, K562 cells.