Figure 1.

Purification and in vitro suppressive function of CD4⁺CD25^highCD127^low/−FoxP3⁺ Treg. (A) Fluorescence-activated cell sorter (FACS) gating strategy used to isolate Treg and Tresp in PBMCs of healthy control. PBMCs were stained using CD4-APC, CD25-PE, and CD127-PECy7 antibodies and sorted by flow cytometry. First, a primary gate was set on live lymphocytes according to their forward/sideward scatter properties, and a secondary gate was set on the CD4⁺ population. Within the CD4⁺ T-cell population, Tregs were identified as CD4⁺CD25^highCD127^low/−, whereas Tresp were isolated as CD4⁺CD25⁻ cells. (B) To verify the purity of sorted Tregs, the cells were fixed-permeabilized, stained with anti-Foxp3, and analyzed by flow cytometry. Scatter plots revealed that sorted Treg were > 90% pure. (C) To examine sorted Treg suppressive function in vitro, varying ratios of Tresp : Treg (1:0, 1:0.5, 1:1) were co-cultured (CFSE-labeled Tresp cells) in the presence of irradiated APCs and stimulated with anti-CD3 antibodies. Representative CFSE dilution plots illustrate sorted Treg cells were highly suppressive in a dose-dependent fashion in functional suppressor assays.