Abstract:

Objective     To establish a method for isolating and expanding human CD4⁺CD25⁺  regulatory T(Treg) cells in vitro, and to identify the purity, phenotype, activity and function to suppress xenogeneic immune responses of fresh and expanded Treg cells.  Methods      Human Treg cells were isolated by magnetic activated cell sorting (MACS) and expanded by CD3/CD28 expand beads, IL-2 and rapamycin. Activities and the purity of the fresh and expanded cells were detected by trypan blue staining and FACS, respectively. The phenotype and Foxp3 gene expression were analyzed by FACS and real-time PCR, respectively. The suppression of fresh or expanded human Treg cells was detected by the mixed lymphocyte reaction(MLR) assay. Cytokine-producing cells in coculture were examined by ELISPOT. Results      Human CD4⁺CD25⁺ Treg cells were expanded up to 3500-fold the number of fresh Treg cells after 2-3 weeks of culture. The activity of the expanded CD4⁺CD25⁺  Treg cells was >97 %, and the purity of them was >96 %. The expanded Treg cells had the similar phenotype and Foxp3 expression with fresh Treg cells. In the coculture experiment (MLR assay),  expanded Treg cells were more potent at suppressing CD4⁺CD25⁺ - T cell-mediated anti-porcine xenogeneic responses compared with fresh Treg cells, and the difference was significant（71% vs 51%, P<0.05）. The enhanced suppression of expanded Treg cells was associated with increased secretions of IL-4 and IL-10. Conclusions    The method established in our study for isolation and expansion of human CD4⁺CD25⁺ Treg cells is effective to obtain sufficient cells,  which have high purity and activity, and suppressive function uninfluenced.

Key words: Human CD4⁺CD25⁺ regulatory T Cells; Expansion in vitro; Phenotype marker; Immunosuppression; Cytokines