Early EBV lytic antigen BMLF1-specific CD8⁺T cells require CD27 for expansion and cytotoxicity. (A) CD8⁺ T-cell memory subsets characterized by CD45RA and CD62L expression and depicted as naive, Tcm, Tem, and Temra in groups treated with either anti-CD27 blocking antibody or isotype control antibody in vivo. (B) Weekly representative flow cytometry plots showing the gating strategy and circulating BMLF1- and LMP2-specific CD8⁺ T cells in blood in vivo. (C-D) Longitudinal data examining the expansion of BMLF1- (C) and LMP2-specific (D) CD8⁺ T cells in blood over time after Luc-EBV infection, treated with anti-CD27 blocking antibody vs isotype control antibody. (E-F) Frequency of BMLF1- (E) and LMP2-specific (F) CD8⁺ T cells in multiple organs (blood, spleen, and liver) at termination of the experiment. (G) Representative flow cytometry plot of EBV-specific TCR-transduced CD8⁺ T cells (depicted as CD8 Pent⁺) and the rest of the T cells (depicted as CD8 Pent⁻) using mouse TCRβ-specific antibodies (mTCRb) and either BMLF1 or LMP2 peptide plus HLA-A2 pentamers.²⁴  (H-I) Frequency of Ki67-expressing cells showing the cell proliferation of EBV-specific BMLF1- (H) and LMP2-specific (I) CD8⁺ T cells (depicted as CD8 Pent⁺ cells) vs the rest of CD8⁺ T cells (depicted as CD8 Pent⁻ cells) in blood in different experimental groups. (J-K) In vitro killing assay with BMLF1- and LMP2-specific T-cell clones generated from healthy EBV carriers ex vivo. T cells were pretreated with either anti-CD27 blocking antibody or isotype control antibody and cocultured with PKH-26 prelabeled autologous LCLs for 21 hours at the indicated effector-to-target ratios (J) and at the ratio of 10:1 (K). Data (n = 5-6 per group) pooled from 2 independent mouse experiments were graphed (A,C-I) and displayed with median and interquartile range. Two-way ANOVA analysis and Sidak’s multiple comparisons as a post hoc test (C-D), and the Mann-Whitney U test (A,E-I) were used. Data (J-K) are pooled from 3 experiments and analyzed using a 2-tailed, unpaired t test; dashed line signifies target cell only. *P < .05; **P < .01. ns, not significant. See also related supplemental Figure 5.