Inhibition of allogeneic and xenogeneic reactions in vitro and in vivo. Untouched Treg cells were isolated from human PBMCs as shown in Figure 5B. (A) Inhibition of mixed lymphocyte reaction (MLR). PBMCs (105) were incubated with the same number of radiated PBMCs of a haplotype-mismatched second donor (allogen). The reaction was inhibited by adding 2.5 × 104 autologous CD49d−CD127− Treg cells (allogen and Treg). Proliferation was determined by 3H-thymidine incorporation and is expressed as counts per minute (cpm). The figure shows 6 independent experiments carried out in duplicate (average inhibition 72.5% ± 16.8%). (C) Prevention of acute GVHD. Acute xeno-GVHD was induced by the adoptive transfer of 30 × 106 CD25-depleted human PBMC (CD25− PBMC) into Rag2−/−γc−/− mice. Progression of the disease was recorded by monitoring for clinical signs of GVHD, expressed as percent free of GVHD (top panel), and by determining the average relative weight in reference to the start of the experiment, expressed as percent weight difference (bottom panel). One group received only CD25− PBMCs (●), a second group received CD25− PBMCs together with 0.5 × 106 untouched autologous Treg cells in a cotransfer (○). Groups of 6 mice were used.
