As a source of peripheral blood Treg cells, a nonmobilized apheresis unit is obtained from the HSCT donor 18–20 days prior to transplant (left). Non-Treg cell populations are depleted of CD8, CD14, and CD19 with either magnetic beads or flow cytometry techniques. Alternatively, a third-party UCB unit is used that does not require negative selection (right). Treg cells are enriched by CD25-positive selection. These populations can be expanded with CD3 and CD28 mAb-coated microbeads or a cell-based aAPC consisting of K562 cells transduced to express CD86 and an FcR (CD32 or CD64) upon which CD3 mAb is loaded. Exogenous IL-2, rapamycin, and an irradiated CD4⁺CD25 feeder layer were added to the culture for peripheral blood Treg cell expansion, whereas UCB Treg cell expansion required only supplemental IL-2. After quality control studies are completed, Treg cells are infused in the peri-HSCT period either in the context of GVHD prophylactic drugs or T cell mAb or in vitro T cell depletion. The approximate cell yields at each step are listed.