Acute removal of T_reg cells after T_reg–T_eff interaction establishment altered T_eff cell motility but residual T_reg cells maintained contact with T_eff cells. NOD.SCID mice with islet grafts established at the ACE site were injected with CD4⁺ BDC2.5 T_eff cells, together with CD4⁺ BDC2.5 T_reg cells (blue) with or without the diphtheria toxin receptor (green) at a ratio of 1:1. After establishment of protection, all mice were injected with diphtheria toxin (50 ng/g body tissue). (A and B) The effect of acute T_reg cell depletion (with an efficacy of ∼80–90%) on T_reg/T_eff ratio (A) and tissue damage (B) after acute T_reg depletion (mean ± SEM). (C) The effect of T_reg cell removal on T_eff cell motility (physical displacement, flower plot of individual cell tracks), to test if T_eff cells exhibit altered motility after disengagement from T_reg cells. (D) T_eff cell displacement rate (µm/min; mean ± SEM). (E) The average T_reg–T_eff interaction time after acute removal of the majority of the T_reg cells by diphtheria toxin treatment (mean ± SEM). Data represent four mice per group from two experiments. It should be noted that the intravital imaging platform enabled us to perform noninvasive imaging at the same tissue spot in the same animal longitudinally, so the pretreatment measurements (day 0) also serve as internal controls for posttreatment measurements within each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.