Regulatory T Cells

Enrichment of Tregs with CD4, CD25, and CD127

Tregs represent a small population of cells; enrichment is often necessary for downstream analysis. Several methods exist for the enrichment of whole or subpopulations of Tregs. While FoxP3 currently is considered the most accepted marker for Tregs, its intracellular localization prohibits its use for the isolation of viable Tregs. Other markers used for enrichment are either negative, positive, or used in combination. One reported method of negative selection is the removal of cells expressing CD127 and CD49d.10 Cells expressing CD4+ and the highest levels of CD25 are used for positive selection. Combination methods can include the use of magnetic beads to remove contaminating populations prior to cell sorting. One of the best characterized methods enriches for CD4+, CD25+, CD127- cells.

Enrichment of Tregs with CD4 and CD25

In humans, initial analysis of Treg populations revealed that only those ex vivo cells that express the highest levels of CD25, which represent approximately 2-3% of total CD4 T cells, demonstrate an in vitro suppressive activity11,12 in contrast to mouse cells in which all CD25 cells are considered Tregs.4,5 Furthermore, cells expressing low to intermediate levels of CD25 were thought not to exhibit any suppressive activity directly ex vivo.

Additionally, the definition of high and low levels of CD25 expression lacks consensus and has limited its use for obtaining viable human Tregs via flow cytometric cell sorting. As a result, many researchers only select cells with the highest expression of CD25, dramatically reducing the yield of isolated Tregs. These results intensified research to identify cells surface markers other than CD4 and CD25 that are exclusive to human Tregs.

Cell surface characterization and isolation of viable human Tregs using CD127

Staining human CD4 T lymphocytes with CD4, CD25, and CD127 enables the enrichment of viable regulatory T cell fractions for further culture and/or use in other in vitro assays. Using this method, viable cells can be quickly isolated through cell sorting. The BD Pharmingen™ brand Human Regulatory T Cell Cocktail, a pre-mixed three-color reagent, simplifies enrichment of viable T cells using a gating strategy that relies on CD4+CD25int/high CD127low cells. This strategy enhances the recovery of CD25int/high sorted Treg cells by 2 to 4 times compared to gating on CD25high cells alone, while eliminating contaminating CD25-/int effector cells.

Magnetic based pre-enrichment of CD4+ T cells to increase yield of human Tregs

Human Tregs represent a small population of total cells. One method to increase the yield of highly enriched human Treg cells combines magnetic pre-enrichment of CD4+ cells with BD IMag™ technology with CD127-based cell sorting.

The BD IMag Human CD4 T Lymphocyte Enrichment Set (Cat. No. 557939) offers a convenient way to pre-enrich CD4+ T cells. Purities of greater than 90% were routinely attained for the enrichment. The procedure captures leucocytes that are not CD4 T lymphocytes, by magnetically removing them from the sample to create an enriched CD4 population. Enriched cells are then stained and shown in the figure on the left.

Cells were stained with anti-human CD4 PerCP (Cat. No. 345770), anti-human CD25 FITC (Cat. No. 555431), and anti-human CD127 PE (Cat. No. 557938). The enriched cells were sorted using a BD FACSAria™ cell sorter. A detailed protocol and recommended gating strategy can be found at