The re-discovered suppressive T cells have recently come to be called T regulatory cells (Tregs). These essential components of the immune system have attracted many researchers because of their central role in establishing and maintaining peripheral tolerance and immune homeostasis by exerting suppressive activities on cells of the adaptive and innate immune system. There are both natural (or constitutive) and inducible (or adaptive) populations of Tregs. Natural Tregs are CD4+ T cells expressing high level of the alpha-chain of the IL-2 receptor (CD25). Natural CD4+ CD25+ Tregs were originally discovered in mice, and an identical population with similar phenotypic and functional characteristics has also been identified in humans.
Isolation of functional Tregs is essential for their experimental and clinical manipulations. They can be isolated based on co-expression of cell surface markers CD4 and high levels of CD25. Using Miltenyi Biotec’s Human CD4+ CD25+ Regulatory T cell Isolation Kit, peripheral blood mononuclear cells can be fractionated into CD4+ CD25- (T responder cells) and CD4+ CD25+ (T suppressor cells or Tregs) subpopulations. The isolation of these subpopulations is performed in a 2-step procedure. First, all non-CD4+ cells are depleted with a cocktail of biotin-conjugated antibodies and anti-biotin microbeads. Second, CD4+ CD25+ T cells can be positively selected using CD25 microbeads, leaving CD4+ CD25- T cells in the pre-enriched CD4+ T-cell population.
I regularly use Miltenyi Biotec’s CD4+ CD25+ regulatory T cell isolation kit for separation of human T responder cells and Tregs for further phenotypic and functional characterization. Peripheral blood mononuclear cells are first labeled with a cocktail of biotin-conjugated monoclonal anti-human antibodies against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCR-gamma/delta and Glycophorin A. Then anti-biotin microbeads are added to labeled cells in order to remove all subpopulations in the peripheral blood mononuclear cells, leaving CD4+ T cells untouched. Depletion of the magnetically labeled non-CD4+ cells is performed using Miltenyi Biotec’s LD columns with a suitable MACS separator. I always use the auto MACS™ separator with a separation program called “Depl05”. The purified CD4+ T cells are then labeled with microbeads conjugated to monoclonal anti-CD25 antibody, and CD4+ CD25+ Tregs are positively selected using Miltenyi Biotec’s MS columns with a suitable MACS separator or using the auto MACS™ separator. It is recommended to use a double positive selection program such as “Posseld2” for obtaining a highly pure population of CD4+ CD25+ T cells. In cases of using columns instead of autoMACS, then performing 2 consecutive column separations is recommended. Following cell fractionation, it is quite important to examine the purity of CD4+ CD25+ and CD4+ CD25-subpopulations using flow cytometric analysis.
Natural Tregs are identified and isolated based on expression of high levels of CD25; however, activated effector T cells also express CD25. While the biology of Tregs in mice seems to be very straightforward, expression of CD25 on human activated T cells as well as Tregs makes it more difficult to interpret studies of Tregs in humans. Clearly, there is a striking need for a specific cell-surface marker to discriminate activated effector T cells from natural Tregs. In addition, by using this kit, labeled CD4+ CD25+ Tregs are positively selected and it might be necessary to have an optimized protocol for isolating untouched Tregs, but this requires further investigation and identification of more specific cell-surface markers of Tregs.
In summary, this kit is one of the best available tools for isolating functional human T regulatory cells based on the expression of CD4 and CD25 markers.