Isolating and Expanding Human Thymic Regulatory T Cells

Thymic (t)Treg were sort-purified from peripheral blood mononuclear cells by Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) isolated from human apheresis products (Memorial Blood Center, St. Paul, MN) in a two-step procedure whereby CD25⁺ cells (including tTreg) were positively selected using GMP-grade mAb-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) and then sorted for naïve tTreg (CD4⁺25⁺⁺127^-45RA⁺). Purified cells were stimulated with GMP-quality artificial antigen presenting cells (aAPC) and cultured for 14 days in high dose IL-2 (300 U/ml) as previously reported^{46 (link)}. Expanded tTreg were banked (frozen) on day 14. For the experiments described herein, tTreg were thawed and re-stimulated with GMP-compliant anti-CD3/28 beads (Thermo-Fisher Scientific, Waltham, MA) for 7–10 days. tTreg purity and in vitro suppressive function were assessed at the end of the culture. The tTreg are simply indicated as Treg in the text.