by Caitlin Smith
Holding back the power of our immune systems is no small feat—and that’s just what regulatory T cells (Tregs) do. Their ability to suppress unwanted immune responses makes them one of the hottest topics in immunology today, for the potential treatment of autoimmune diseases and organ transplants, and for cancer immunotherapy, according to Vicki Stronge, product manager for immunology at StemCell Technologies.
But researchers encounter barriers when trying to study Tregs. “High recovery is one of the biggest challenges,” says Hermann Bohnenkamp, product manager for T cells at Miltenyi Biotec. “Sample volumes, especially from patients, are usually limited.” Once Tregs are recovered, purifying them is another challenge. “A major hurdle in unlocking the therapeutic potential of these cells lies in the isolation of highly purified functional Tregs,” says Stronge. “The isolation of Tregs has always been a lengthy, cumbersome and inefficient process due to the lack of specific cell surface markers on Tregs, and the low frequency of these cells in peripheral blood.”
Furthermore, not all Tregs are created equal—you may be interested in only a distinct subset. “The field of Treg research is evolving at a rapid rate, whereby new cell surface markers are discovered that can differentiate Tregs from other types of T cells,” says Stronge. “Exploiting these markers for Treg isolation leads to improved selection of Tregs, resulting in higher purity cells that have a more exclusive phenotype.” Isolating these important immune cells can be time-consuming, and yet still result in low recovery or poor levels of purity. Here are some recent tools that may help to speed Treg isolation and purification.
Immunomagnetic isolation
Separating cells from one another is often accomplished using antibodies attached to magnetic nanoparticles or beads that can be easily immobilized using a magnetic field. Miltenyi Biotec offers their MACS® Technology for magnetic cell separation, including Treg isolation. “New, optimized isolation kits combine optimal purity and unmatched recovery of viable Treg cells that can be immediately used for functional characterization or expansion,” says Bohnenkamp. “Not only CD4+CD25+CD127dim/– Treg cells or CD4+CD25+CD49d– subpopulations can be quickly and easily isolated, but also naive CD45RA+ Treg cells, which are ideally suited for in vitro expansion.”
StemCell Technologies has combined two of their cell separation platforms, RosetteSep® and EasySep®, into Complete Kits for the isolation of human Tregs, in an effort to make the process faster and easier for researchers. “The isolation of Tregs is now a simple two-step process: pre-enrichment of cells by negative selection with RosetteSep®, followed by positive selection of CD25high cells with EasySep®,” says Stronge. “The entire procedure from whole blood or buffy coat to purified Tregs takes only three hours, unlike other magnetic separation methods that take up to five hours to complete. The column-free kits are easy to use and are gentle on cells, providing highly functional Tregs that are immediately ready for use in downstream assays.” StemCell Technologies has just released two new Complete Kits for the isolation of Tregs directly from whole blood or buffy coat: the Complete Kit for Human CD4+CD127lowCD25+ T Cells, and the Complete Kit for Human CD4+CD127lowCD49d-CD25+ T Cells. According to Stronge, the new kits isolate Tregs based on the low expression of CD127 (IL-7 receptor alpha chain), and lack of expression of CD49d, the alpha-chain of the integrin VLA-4 (alpha4beta1).
Being bead-free
Being bead- and antibody-free might be important to your experiments; for example, having magnetic nanoparticles attached to or inside your cells may interfere with further experiments. Life Technologies offers a method of isolating human CD25hiCD4+foxP3+ Tregs without beads or antibodies attached. “This is done in a two-step protocol, where CD4+ cells are isolated by negative isolation followed by a positive isolation of the CD25 high-expressing cells,” explains Karoline Schjetne, staff scientist at Life Technologies. “With this product you can isolate the T effector population along with the T regulatory population from the same starting material, [leaving] you with a highly pure population, which can be used in suppression assays or for expansion. This is the only product available on the market where you get cells without beads or antibodies attached to the cells, in addition to both Teffector and Treg populations.”
For mouse Treg cells, Life Technologies offers isolation based on their FlowComp technology, which (like Miltenyi Biotech and StemCell Technologies) uses beads and antibodies. The end result is that beads are removed, but antibodies remain on the cells. “This makes us more like our competitors; however, only [our technology] offers bead-free cells,” says Schjetne. “Other manufacturers use nanoparticles to isolate cells, which readily enter cells and might interfere with functionality. [But] purity and recovery is about the same.”
Life Technologies’ flow-sorting method can also isolate Tregs into subpopulations based on the CD45RA, CD127, and CD49b surface markers. “You can sort both Treg or Teffector cells; however, all cells will have antibodies linked to the surface of the cells (CD25 receptor, for example, is critical for IL-2 signaling, which is the major proliferation signal to T cells),” says Schjetne. “High purity is essential for efficient expansion. The identification of a truly specific surface marker would speed up the isolation technology as positive isolation gives the highest purities. Excellent purity is of major importance for Treg cells … because you often want to expand the cells, as you get so very few from the starting sample.”
StemCell Technologies offers a tool for researchers using flow cytometry to sort Tregs. “Many researchers require Treg purities of over 99% FoxP3+ cells, and flow cytometric sorting is the most reliable method to achieve this level of purity,” says Stronge. “However, the low frequency of Tregs in human peripheral blood means that flow sorting can take many hours, which can also translate to high equipment costs. StemCell Technologies’ kit for pre-enrichment of Tregs, RosetteSep® Human CD4+ and RosetteSep® Human CD4+CD127low Pre-enrichment Kits can save time and money by eliminating the majority of unwanted cells and allowing for faster sorting of highly purified Tregs.”
StemCell Technologies is also releasing “the first kit for the isolation of untouched Tregs,” says Stronge. “The new Human CD4+CD127lowCD49d- Regulatory T Cell Enrichment Kit is the only kit available for the isolation of untouched Tregs. Isolated Tregs are up to 85% FoxP3 positive without the use of positive selection, ideal for researchers who want a gentle method of isolating Tregs that are bead- and antibody-free.”
Tregs in translational medicine
Efforts of Treg researchers will soon be put to good use in clinical trials for a variety of conditions, such as graft-versus-host disease, autoimmune diseases, and organ rejection. “[Tregs] have huge potential, as they have been shown to be important both in tumor therapy (depletion) and autoimmune diseases (transfusion),” says Schjetne.
Bohnenkamp says that Treg cells for clinical applications can be separated with Miltenyi Biotec’s CliniMACS® System. “Using Treg cells for clinical applications is one of the most exciting developments today,” says Bohnenkamp, “and we at Miltenyi Biotec can match researchers’ needs—from discovery to translational medicine.” Indeed, he notes, the biggest challenge today is to combine isolation and expansion methods for Treg cells that can also be used for clinical applications.