The effect of CTLA4 modulation on T_reg–T_eff cell interaction. NOD.SCID mice carrying islet grafts in ACE were injected with CD4⁺ BDC2.5 T_eff and T_reg cell mixture. After T_reg cell protection in the tissue was established (∼30 d after T cell transfer), mice were treated with either PBS control or anti-CTLA4 monoclonal antibody (20 µg/g body weight, n = 3 different mice in each group). Notably, the intravital imaging platform enabled us to noninvasively image the same tissue spot in the same animal longitudinally, as in these experiments and throughout the study. Therefore, the pretreatment measurements also serve as internal controls for posttreatment measurements. (A) T_eff cell numbers and (B) T_reg cells numbers in the target tissues (mean ± SEM). (C) Intra-islet graft T_reg/T_eff cell ratio over time after anti-CTLA4 antibody blockade (n = 10–11 islet grafts per group per time points; mean ± SEM). (D) Interaction index, calculated as the ratio of T_eff cells with or without T_reg cell interaction, after anti-CTLA4 or control treatment (mean ± SEM). Arrows in A–D indicated injection of anti-CTLA4 antibodies. (E and F) The duration of T_reg–T_eff cell interactions after CTLA4 blockade, in actual imaging time (E) and relative to the length of whole imaging session (F; n = 24–40 cell pairs; mean ± SEM). One-way ANOVA across all time points in all groups did not yield statistical significance, likely because of the large variations among individual animals and time points in each group in these longitudinal imaging experiments with live animals; however, there was significant difference among pretreatment controls and posttreatment measurements within the anti-CTLA4 treatment group. *, P < 0.05, one way ANOVA was performed with a Tukey’s multiple comparison’s post-hoc analyses, compared with both pretreatment measurement of the same animal or control-treated animals. **, P < 0.01; ***, P < 0.001.