Blocking of CD27 diminishes the immune control of EBV infection. (A) Workflow of CD27 blocking experiments. One day before infection with 10⁵ RGU luciferase encoding B95-8 EBV (Luc-EBV), huNSG-A2 mice (HLA-A2 transgenic NSG mice reconstituted with HLA-A2⁺ human hematopoietic progenitor cells) were adoptively transferred with 200,000 autologous T cells expressing either BMLF1- or LMP2-specific TCRs that had been transduced ex vivo. The anti-CD27 blocking antibody is the Fc domain re-engineered version of the anti-CD27 depletion antibody of Figure 2 and can no longer engage antibody-directed cellular cytotoxicity. At week 2 after EBV infection, animals were injected (IP) with 6.5 μg/g of either anti-CD27 blocking antibody or isotype control antibody consecutively every 4 days until termination of the experiment. (B) Representative flow cytometry plots of BMLF1- and LMP2-specific TCR-transduced CD8⁺ T cells (left) using mouse TCRβ-specific antibodies (mTCRb) and BMLF1 and LMP2 peptide plus HLA-A2 pentamers.²⁴  Frequency of CD27 expression on transduced and nontransduced CD8⁺ T cells (right). (C) Spleen weight of animals treated with either anti-CD27 blocking antibody (α-CD27 blo.) or isotype control antibody (IsoCtrl. blo.) upon different transfer conditions. (D-E) EBV viral loads quantified by quantitative PCR over time during EBV infection (D) at the termination of the experiment in peripheral blood (E, left), spleen (E, middle), and liver (E, right). Mice treated with either anti-CD27 blocking antibody or isotype control antibody in different transfer conditions were compared. (F-G) Representative immunohistochemistry images of EBNA2 in the respective groups (original magnification ×200) (F), and the quantification of EBNA2⁺ cells/mm² in splenic sections (G). (H-I) Representative IVIS image analysis at week 1 and week 5 after Luc-EBV infection (H) and quantification of defined region of interest of IVIS images (I). (J) Spleen weight of animals infected with either wild-type EBV or BZLF1 knockout EBV (BZkoEBV), treated with anti-CD27 blocking antibody or isotype control antibody without adoptive transfer. (K-L) EBV viral loads of animals over time during EBV infection (K) at the termination of the experiment in peripheral blood (L, left) and spleen (L, right) in the respective groups. Data (n = 14-16 per group) pooled from 2 independent mouse experiments were graphed (C-E,G,I-L) and displayed with median and interquartile range. Graphs (F-I) (n = 7-8 per group) are representative from 1 out of 2 independent experiments. Graphs (K-L) (n = 4-6 per group) are from 1 experiment. One-way ANOVA analysis (Kruskal-Wallis test) followed by Tukey’s post hoc test (J,L), 2-way ANOVA analysis and Sidak’s multiple comparisons as a post hoc test (D,I,K), and the Mann-Whitney U test (C,E,G) were used to assess P values. *P < .05, **P < .01. See also related supplemental Figure 3.