In vivo imaging of color-coded T cells. (a) FACS sorting of DsRed⁺CD4⁺GFP⁻ red T_eff cells from DsRed–knock-in mice and CD4+GFP+ green nT_reg cells from the original knock-in mice. (b) Graft survival curves of mice treated with CD154-specific mAb plus rapamycin and untreated rejecting controls. The difference in the survival curves is significant, as calculated by either log-rank (Mantel-Cox) (P = 0.0004) or Gehan-Breslow-Wilcoxon (P = 0.0012) tests. (c) Representative image of allograft-infiltrating nT_reg (green), T_eff (red) and iT_reg cells (yellow) acquired by intravital microscopy. Scale bar, 50 μm.

Serial endomicroscopy of infiltrating T cells within islet allografts. Representative endomicroscopy images within islet allografts on days 3, 5, 7, 10, 12 and 14 after transplantation in untreated hosts and hosts treated with CD154-specific mAb plus rapamycin. Each row of images is from the same mouse at the given time points. Infiltrating nT_reg (green), T_eff (red) and iT_reg cells (green + red) accumulate in the allograft over time. Scale bar, 50 μm.

Analysis of infiltrating T cells within islet allografts. (a) Representative intravital microscopy images showing T cell infiltration within islet allografts in untreated hosts and hosts treated with CD154-specific mAb plus rapamycin on week 1 and week 2 after transplantation. Scale bar, 100 μm. (b–d) Summary of cell density of islet allograft–infiltrating nT_reg (b), iT_reg (c) and T_eff (d) cells, as detected by intravital imaging. (e,f) Summary of the ratios of islet allograft–infiltrating nT_reg to T_eff (e) and iT_reg to T_eff (f) cells, as detected by intravital imaging. Error bars represent means ± s.d.

Detection of nT_reg, T_eff and iT_reg cells by in vivo flow cytometry in the peripheral blood. (a) A representative in vivo flow cytometry trace showing the identification of single positive nT_reg (green box), T_eff (red boxes) and double-positive iT_reg (yellow box) cells. The second peak in the DsRed channel occurred about 45 ms before the second peak in the GFP channel. As this time difference was greater than the uncertainty of the measurements, these two peaks were distinguished as separate cells and not a double-positive iT_reg cell. (b) In vivo flow cytometry showing T_eff (red), nT_reg (green) and iT_reg cells (yellow) in the peripheral blood. There is a ten-fold difference in scale between mice treated with CD154-specific mAb plus rapamycin and untreated mice. Each curve represents serial analysis of the same blood vessel of the same animal. (c) Summary of the ratio of circulating T_reg to T_eff cells, as detected by in vivo flow cytometry. Error bars represent means ± s.d.