Expansion of EBV-specific T cells in patient 1 after T-cell transfer. (A) EBV DNA levels in peripheral blood cells (copies per 20 000 cells) and in throat lavage fluid (copies per milliliter). (B) Absolute numbers of CD8⁺ and CD4⁺ T-cell subsets in peripheral blood. (C) Frequencies of CD8⁺ T cells specific for EBV epitopes in total peripheral blood lymphocytes (PBLs) were assessed by staining with specific HLA-peptide multimers. Epitopes from immediate-early and early lytic cycle antigens (epitopes YVL and GLC) and from latent antigens (epitopes CLG and RPP) were included in the analysis. (D) Examples of HLA-peptide multimer stainings of PBL samples obtained at 3 different times after adoptive cell therapy as indicated. Cell populations within boxes were counted as multimer-positive. Percentages indicate their proportion in total lymphocytes. (E) Frequencies and specificities of antigen-specific T-cell clones obtained by direct single-cell cloning of PBLs on day 37 after adoptive T-cell transfer. (F) Assessment of interferon-γ–secreting EBV-specific T cells by an ELISPOT assay before (day −4) and after adoptive T-cell transfer (days 3 and 6). PBMCs (200 000 per well) were stimulated, as indicated, with individual EBV peptides (CLG, GLC, YVL), a mix of all 23 peptides, no stimulator (“neg”), or with TPA (12-O-tetradecanoylphorbol-13-acetate) and ionomycin for a positive control.