T regulatory cells (Tregs) have been the focus of many research groups due to their central role in establishing and maintaining peripheral tolerance and immune homeostasis by suppressing effector cells of the adaptive and innate immune systems. In the recent few years, different populations of Tregs have been described and characterized. The importance of Tregs is becoming more recognized in different clinical aspects. For example, lower levels of Tregs or defects in their functionality have been described as potential mechanisms for inducing allergic diseases. On the other hand, increased local and systemic frequencies of Tregs have been reported in most cancer types, which suppress antitumour immunity and consequently impair immunotherapy.
Isolation of highly functional Tregs is vital for many research assays and therapeutic interventions. The forkhead transcription factor FOXP3 is the most specific marker for Tregs; however, it cannot be used for Treg isolation because it is an intracellular marker. So far, the alpha-chain of the interleukin (IL)-2 receptor (CD25) is the best-known surface marker expressed on Tregs, which can be isolated based on co-expression of the cell surface markers CD4 and CD25.
I have recently used Dynabeads’ regulatory CD4+CD25+ T cell kit for isolation of the human Tregs. This kit, which employs a tube-based (not column) technology, is designed for magnetic isolation of CD4+CD25+ Tregs from human mononuclear cells (MNCs). The kit contains Antibody Mix Human CD4, Depletion MyOne Dynabeads, Dynabeads CD25, and DETACHaBEAD. The isolation is performed in a three-step procedure. In the first step, the non-CD4+ MNCs are labeled with Antibody Mix and the non-CD4+ MNCs are removed using Depletion MyOne Dynabeads. In the second step, Dynabeads CD25 are used to positively select CD25+ cells, leaving CD4+CD25- T cells in the non-selected fraction. In the third step, Dynabeads CD25 are removed from CD4+CD25+ cells using DETACHaBEAD. It is recommended that the purity of CD4+CD25+ and CD4+CD25- subpopulations is examined using flow cytometric analysis prior to any downstream applications. Invitrogen claims that higher numbers of CD4+CD25+FOXP3+ T cells are obtained using this kit, compared to other column-based methods. I have used the isolated Tregs in suppression assays and they showed typical characteristics of Tregs (anergic and suppressive).
Signaling through the alpha-chain of the high affinity IL-2 receptor (CD25) is essential for the differentiation and survival of Tregs; therefore, CD25 blocking could result in impaired cell function. The distinguished component of this kit is the removal of Dynabeads CD25, producing bead- and antibody-free Tregs for downstream studies.