Expansion and activation of CD8⁺ T cells and IFN-γ hypercytokinemia in EBV-infected NOG-hCD34 mice. (A-D) Dynamics of human CD8⁺ T cells in PB. PB was routinely collected from mock-infected mice (n = 5) and EBV-infected mice (n = 12) and was analyzed by flow cytometry. Percentages of CD8⁺ cells (A), CD8⁺CD45RA⁺ cells (B), CD8⁺CD45RO⁺ cells (C), and CD8⁺CD45RO⁺CD38⁺HLA-DR⁺ cells (D) in PBMCs are, respectively shown. (E) Expansion of activated CD8⁺ T cells in multiple organs of EBV-infected mice. The cell numbers of CD8⁺CD45RO⁺CD38⁺HLA-DR⁺ cells (activated CD8⁺ T cells) in the spleen, liver, BM, lung, kidney, and ascitic lavage fluid of mock-infected mice (n = 4) and EBV-infected mice (n = 7) are shown. (F) Ki67 expression in CD8⁺ T cells. (Left) Representatives of splenic CD8⁺ T cells of mock-infected and EBV-infected mice. Values on quadrants represent the percentages of CD8⁺ T cells positive for Ki67. (Right) The percentages of Ki67⁺ cells in splenic CD8⁺ T cells of mock-infected mice (n = 4) and EBV-infected mice (n = 7) are shown. (G) mRNA expression in CD8⁺ T cells. Splenic CD8⁺ T cells of mock-infected mice (n = 6) and EBV-infected mice (n = 9) were isolated by cell sorting. The expression levels of IFNG (left) and TNFA (right) were analyzed by real-time RT-PCR and were normalized to that of GAPDH. Results are presented as the fold change compared with the value in mock-infected mice. (H-I) Longitudinal quantification of IFN-γ and TNF-α in plasma. Plasma was routinely collected from mock-infected mice (n = 4) and EBV-infected mice (n = 7), and the concentrations of IFN-γ (H) and TNF-α (I) were quantified by cytokine bead array system. (J) Detection of EBV-specific HLA-A*2402–restricted CD8⁺ cells. Splenic human MNCs isolated from mock-infected mice (n = 4) and EBV-infected mice (n = 7) were stained with an anti-CD8 antibody and either HLA-A*2402 EBV tetramers or HLA-A*2402 HIV-1 tetramers (as a negative control of the assay) and were analyzed by flow cytometry. Representatives (left) and the percentages of CD8⁺ T cells positive for the tetramers in mock-infected and EBV-infected mice (right) are shown. Asterisks in panels A, C, D, and H represent statistic significance (P < .05 by Student t test) versus the value obtained from the mock-infected mice, and daggers represent statistic significance (P < .05 by paired t test) vs the initial value. Asterisks in panel E represent statistic significance (P < .05 by Welch t test) versus the value obtained from the mock-infected mice. Asterisks in panel F, G, and J represent statistic significance (P < .05 by Student t test) versus the value obtained from the mock-infected mice.