Development of IFN-γ–secreting CD8⁺ T cells recognizing alloantigens expressed by the nonengrafting unit after double-unit CBT. (A) After a 5-hour stimulation, CD8⁺CD45RO^+/−CCR7⁻ IFN-γ–staining cells from PBMCs samples collected 14 to 28 days after transplantation are demonstrable against LCLs generated from the nonengrafting unit (i), but not the dominant unit (ii) or a random third party unit (iii). (B) IFN-γ–secreting CD8⁺ T cells are detectable among 9 of 10 patients establishing single donor dominance. (C) Alloreactive IFN-γ–secreting CD8⁺ T cells decline at later times after CBT. ■ represents time point at which the sample was drawn; solid lines, no administration of steroids for GVHD in the interval between sample assays; dotted lines, administration of steroids for GVHD in the interval between sample assays. Because 3 patients died before second PBMCs samples were collected, serial data for only 6 patients are shown.