Cotransfer of Tregs leads to amelioration of GVHD pathology. (A) Differential persistence of Tregs in blood of animals on the indicated days after transfer. (B) Comparison of Treg (A2⁺) numbers in circulation on day 7 versus day of death due to disease (open symbols) or conclusion of the experiment (closed symbols). (C,D,F) Disease severity was assessed at time of death (open symbols) or at the conclusion of the experiment (closed symbols). (C) Decreased percentage of human CD4⁺ cells was observed in blood from Treg-treated animals that survived to day 80. (D) Moribund animals were found to have decreased hematocrit readings, except for the one peri-transplant, non–GVHD-related death observed in the 4.1BBL group. Representative example (E) and disease severity scores (F) for H&E staining of liver, lung, and ileum from animals with or without PBMNCs and Tregs. (G) Representative examples of anti-hCD4 peroxidase staining of liver and lung from animals with or without PBMNCs and Tregs expanded with KT32/OX40L. Pathology data from same experiment as Figure 5, and the pathology is representative of 3 experiments analyzed. Images were acquired at room temperature with an Olympus BX51 microscope, Olympus U-plan Apo and 10× objective lens (aperture = 0.40; Olympus, Hamburg, Germany) with an RTT Spot camera and Spot Advanced software (Diagnostic Instruments, Sterling Heights, MI).