Panel Preferences: Which Markers are Best for Staining Regulatory T Cells?

T-Cell

Regulatory T cells (Tregs) are an important cell subpopulation because they can modulate immune responses that target self-antigens but are also associated with poor outcomes in cancer patients. Tregs are found in peripheral blood and are defined by both surface and intracellular markers, but there is still some debate as to the optimal staining panel for monitoring Tregs, particularly during immunotherapeutic interventions for cancer therapy and autoimmune diseases.

CD25, FoxP3, and CD127 have been the most commonly used markers for the analysis of Tregs by flow cytometry, but other markers, including CTLA-4, PD-1, and OX40, have also been used in combination with the prototypical markers to characterize both human and murine Tregs. If you are considering adding a Treg staining panel to your next experiment, consider which markers may work best for your application.

FoxP3

Forkhead box protein 3 (FoxP3) is a transcription factor that has been broadly accepted as a marker of Tregs as it is a master regulator of the development and function of this cell subset. Many early Treg studies used CD4, CD25, and FoxP3 staining exclusively to identify this subset, and FoxP3 staining can still be used for many basic in vitro Treg studies. Unfortunately, FoxP3 staining can be highly variable, especially in human samples, and can vary depending on the tissue from which Tregs are isolated. Recent studies have also revealed that other T cell subsets can express Foxp3, thus limiting its utility as a decisive Treg marker. FoxP3 is located within cells and requires cell permeabilization for staining, which may limit downstream applications.

TREG-Data

CD127

More recently, reduced expression of the cell surface marker CD127 has been defined as a surrogate marker of Tregs in combination with other markers, thus allowing Tregs to be identified as CD4⁺CD25⁺CD127^lo/- T cells. Indeed, the early use of FoxP3 in staining panels was critical for the identification of better Treg markers like CD127. CD127 staining can be used on live or fixed cells and does not require cell permeabilization, thus making it a marker appropriate for downstream applications.

As you develop your Treg staining panel, consider your cell source and downstream applications in order to pick the best markers for your experiment.