Establishing a method for the expansion of human regulatory T cells with a high-efficiency in vitro

Abstract

The objective of this study is to establish a method to isolate and culture regulatory T cells with a high-efficiency in vitro. The CD4+, CD25+, and CD127low T cells were isolated through fluorescence activated cells sorting and cultured in vitro with anti-CD3-anti-CD28-coated microbeads and IL-2 for two weeks. The number of cells was determined to evaluate the expansion ability, the phenotype of the expanded cells was identified with flow cytometry, and the suppressive function was examined by mixed lymphocyte reaction. The CD4+CD25+CD127low T cells were efficiently amplified in vitro using magnetic beads (Fe3O4). A mean expansion of (755.5 ± 213.5) fold was obtained after the cells were cultured for two weeks. In addition, the expressions of CD4, CD19, CD8, and CD25 in the expanded cells were estimated at (98.3 ± 1.04)%, (0.039 ± 0.021)%, (0.443 ± 0.239)%, and (97.6 ± 1.35)%, respectively. FOXP3+ cells accounted for (87.7 ± 5.5)% of the expanded cells, while Helios+ cells accounted for (73.3 ± 2.9)%. The cultured cells demonstrated the highest suppressive capacity with (84.39 ± 1.98)% suppression of the activated peripheral blood mononuclear cells at a responder: regulatory T cells (Treg) ratio of 1:1. This research suggests a method to obtain a large quantity of Tregs with a high-efficiency in vitro. The expanded cells remain in their original phenotype and demonstrate the ability of immune suppression, which plays enormous significance in the cell therapy for treating autoimmune diseases and transplant rejections.