Study on the preparation and function of regulatory T cells from human peripheral blood

Regulatory T cells (Tregs) are an important cell subgroup of CD4⁺ T cells. Treg cells are critically involved in inducing immune tolerance, maintaining immune environment homeostasis, and preventing the occurrence of autoimmune diseases. Under normal conditions, the number of Tregs in the body is very small. This research was designed to establish an effective method to expand human peripheral blood Tregs in vitro and to analyze phenotype, purity, and function of Treg cells post-expansion.

Peripheral blood was obtained from healthy donors. CD4⁺CD25⁺CD127^dim/- Treg cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic-activated cell sorting (MACS), and an optimized culture system was used for amplification. The in vitro amplification ability of Treg cells was evaluated to determine the expression and purity of Treg cell-specific surface markers in different culture cycles. The suppressive function of Treg was determined by in vitro lymphocyte proliferation assay.

Treg cells could be successfully isolated by magnetic activated cell sorting (MACS). After 21 days of in vitro culture, the mean expansion fold was 2,009±452.2 in ≤60 years, and there was a significant difference between the younger group and the older than 60 years group (1,238±330.0). Flow cytometry analysis revealed that the percentages of CD4⁺CD25⁺ cells and FOXP3⁺ cells were (93.25±3.05)% and (94.19±4.21)% on day 14, and (92.86±4.36)% and (91.55±5.62)% on day 21, respectively. In addition, the proportions of CD8⁺ T, CD19⁺ B, CD3^-CD56⁺ natural killer cell (NK), and CD3⁺ CD56⁺ natural killer T cell (NKT) were extremely low. Lymphocyte proliferation assay demonstrated that Tregs could inhibit the proliferation of CD8⁺ T cells more effectively than that of CD4⁺ T cells. Furthermore, the suppressive capacity of Tregs was correlated with Treg-to-PBMCs ratios.

We successfully established a technical protocol for manufacturing a large quantity of Tregs with high efficiency in vitro. The expanded Tregs have a steady FOXP3 expression and exhibited a potent immune suppression, which might have great significance in adoptive Treg therapy for treating graft-versus-host disease and autoimmune diseases.