Both EBV-specific and bystander (CMV and Flu specific) CD8⁺ T cells were activated during acute EBV infection. PBMCs from each subject were stained with antibodies against CD3, CD4, CD8, CD38, CD45RA, DR, and GrzmB, as well as pMHCI tetramers. Panels A and B show sample flow cytometric analysis of PBMCs from subject 5036 before, during, and after acute infection with EBV. (A) HLA-A2/YVL (EBV) tetramer staining on CD8⁺ T cells. (B) HLA-A2/NLV (CMV) tetramer staining on CD8⁺ T cells. (A and B) Lower dot plots show CD38, HLA-DR (DR), and GrzmB staining of tetramer⁺ cells. (C) Percentage of tetramer⁺CD8⁺ T cells that expressed CD38 (top), DR (middle), or GrzmB (bottom) before (>40 d prior), during (0–30 d), and after (>150 d) acute primary EBV infection. EBV-specific CD8⁺ T cells were identified with HLA-A2/YVL, A3/RVR, or B8/RAK tetramers; Flu-specific CD8⁺ T cells were identified with HLA-A2/GIL tetramers; and CMV-specific CD8⁺ T cells were identified with HLA-A2/NLV, B7/TPR, B8/ELR, or B35/IPS tetramers (n = 6–19 per virus-specific T cell; ANOVA: *, P < 0.05; **, P < 0.001). Horizontal bars indicate the mean.