Direct contact between CD4⁺ T_eff cells, target tissue cells and T_reg cells, with or without close proximity to myeloid cells. Fluorescence confocal micrographs (z-stacks) acquired within intraocular islet grafts in NOD.SCID recipients in vivo. The mice were reconstituted with CFP-labeled CD4⁺ BDC2.5 T_eff cells (blue) and GFP-labeled T_reg cells (green), and later injected directly into ACE with BV605-conjugated anti-CD11b antibodies to label a broad subset of myeloid cells (yellow) and APC-conjugated Annexin V (red) to detect apoptotic cells in situ. The target β cells were visualized by laser backscatter (gray). (A and B) The Annexin V apoptotic signals were detected in areas of CD4⁺BDC2.5T_eff cells engaging target β cells, in the absence (A) or in the presence (B) of CD11b⁺ myeloid cells. (C) T_reg cells colocalized in the protected cluster of β cells, which persisted amid rejected areas as long as T_reg cells existed. (D) The Annexin V signals on either β cells or T cells were measured as overlap between Annexin V stain volume and that of either β or T cells, normalized to total volume (β and T cells; mean ± SEM). (E) Linear regression analyses correlating β cell apoptosis (Annexin V signal) with the number of intra-graft T cells (n = 10 islet grafts in 4 mice). **, P < 0.01. Bars, 50 µm. See Video 1.