[Isolation, amplification and identification of human natural CD4⁺CD25⁺ regulatory T cells in vitro].

OBJECTIVE: To establish a method for in vitro expansion of human natural CD4⁺CD25⁺ T regulatory cell (Treg) cells for clinical study and immunotherapy. METHODS: Human natural CD4⁺CD25⁺ Treg were isolated from peripheral blood monocyte cells (PBMCs) by magnetic activated cell sorting (MACS) and expanded by CD3/CD28 expansion beads, IL-2 and rapamycin. The number and the viability of the freshly isolated and expanded Treg were detemined by trypan blue staining. The phenotype and the purity of the freshly isolated and expanded Treg were analyzed by FACS. Treg suppression activity was assessed by mixed lymphocyte reaction (MLR) assay. RESULTS: Human natural Treg were expanded up to 2 000 folds after 3 weeks in culture, and the activity was more than 97%. The expanded Treg retained Treg phenotype as shown by their freshly isolated counterparts, and the purity of CD4⁺CD25⁺FoxP3⁺ Treg was (94.22 ± 2.12)%. The expanded Treg demonstrated a similar potent suppression of both proliferating auto- and allo- CD4⁺CD25⁻ effector T cells in vitro in a cell number-dependent manner. CONCLUSION: An in vitro expansion of human natural Treg was established to obtain large numbers of human Treg with highly suppressive phenotype and function, thereby providing a solution to the availability of sufficient human natural Treg in clinical study and immunotherapy.