A higher frequency of CD8⁺ T cells specific for minor H antigens is present in the T_N subset than in the T_M subset. (A) Lysis of PHA blasts prepared from the HLA-identical sibling/monocyte donor (“recipient”) and from the from the T-cell donor (“donor”). The symbols represent the percentage lysis of recipient cells by individual wells plated with T_N (○) or T_M (△) that demonstrated both ≥ 10% lysis of recipient PHA blasts and ≥ 5× the lysis of donor PHA blasts. Wells with ≤ 10% lysis of recipient PHA blasts or nonspecific cytotoxicity (lysis of recipient PHA blasts that was not ≥ 5× the lysis of recipient blasts) are not shown. Data are shown for assays from 3 volunteer HLA-identical sibling donors (donors 1, 2, and 3, left to right). (B) Comparison of the minor H antigen–specific T-cell precursor frequency (f) between the T_N and T_M population for HLA-identical siblings. For T_N, f was 1/485 794, 1/1 619 886, and 1/225 188 (broken lines) for donors 1, 2, and 3, respectively; and for T_M, f was 1/2 377 182, 1/5 817 135, and 1/2 803 498 (dotted lines) for donors 1, 2, and 3, respectively. Bold lines represent the frequency estimate; and the nonbold lines, 95% confidence intervals. The P values for the difference in f between T_N and T_M for the 3 pairs were 4.13 × 10⁻⁹, 5.65 × 10⁻⁴, and 2.86 × 10⁻¹⁶, respectively. Each limiting dilution analysis was performed with a minimum of 60 replicates per cell concentration. (C) Repeat analysis of wells with alloreactivity after expansion. Each well plated with T_N (○) or T_M (△) that was positive for specific lysis of recipient cells in the initial assay was expanded and retested for lysis of recipient and donor PHA blasts at an ET ratio of 10:1. The x-axis indicates the level of cytotoxicity detected before expansion; and y-axis, the level of cytotoxicity detected after expansion. The validation assay was performed on expanded cultures from donors 1 and 2.