In vitro generation of EBV-specific CD8⁺ T cells. Peripheral blood mononuclear cells were isolated from donors with AIM (A) and from those who had recovered from AIM at least 1 year previously (B). The percentage of CD8⁺ EBV peptide (RAK)–specific T cells was determined by using HLA class I tetramers. (C) The cell-cycle status of antigen-specific T cells isolated during the acute phase of infection (open histogram) and those cells isolated from donors with chronic infection (filled histogram) were determined by staining cells with antibodies directed against Ki67. PBMCs isolated from donors with chronic infection (D) were stimulated in vitro with RAK peptide-pulsed autologous APCs plus exogenous IL-2 and maintained in culture for 4 weeks (E). (F) Proliferation was assessed by Ki67 staining in freshly isolated CD8⁺ tetramer⁺ cells (filled histogram) and in specifically activated CD8⁺ tetramer⁺ cells that were expanded for 1 month in vitro (open histogram). These results are representative of 6 separate experiments. (A, B, D, E) Numbers refer to percentages of tetramer-positive CD8⁺ cells.