T_reg cells persistently interacted with T_eff cells in damaged tissue, despite an imbalance in T_reg versus T_eff numbers. CD4⁺ T cell behavior was analyzed in the graft tissue that was being destroyed due to failed immune regulation or protected by successful T_reg cell regulation. Protected islet grafts had at least 80% of original islet mass, whereas damaged islet graft had 20% or less. (A) Representative fluorescence micrographs (3D rendering) showing protected (left) and damaged (right) islet grafts (gray; visualized by laser backscatter) in NOD.SCID mice reconstituted with GFP-labeled CD4⁺ BDC2.5 T_reg cells and CFP-labeled CD4⁺ BDC2.5 T_eff cells. (B) T_reg–T_eff interaction time in protected versus damaged grafts (mean ± SEM). (C) Ratios of T_reg–T_eff cell pairs within protected versus damaged grafts. The T_reg–T_eff cell pairs outside of the target graft tissue, in the iris in the same ACE, were analyzed as controls for the target tissue (mean ± SEM). (D) Interaction index (ratio of T_eff cells with or without T_reg interactions during the entire imaging sessions) in protected vs. damaged grafts (mean ± SEM). Imaging experiments in A–D were done on days 18–25 after T cell transfer. (E–G) T_eff and T_reg cell numbers in the target tissue at the initial phase of T cell infiltration (days 10–12, all animals were free of diabetes) and a later stage (days 17–20) when some animals developed diabetes with near complete (>80%) damage of the islets and some animals were protected, even though they received the same kind of T_eff and T_reg cell mixture in the same experiment. T_eff (E) and T_reg (F) cell numbers and T_reg/T_eff ratios (G) in the target tissue were analyzed in the same graft before and after extensive damage occurred (if it occurred). Results represent 3 experiments in a total of 4–6 mice in each group (the protected vs. damaged groups). Each data point represents one islet. Unpaired Student’s t tests were performed in B and D and one-way ANOVAs with Tukey’s post-hoc analyses were performed in C and E–G; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bar, 50 µm.