Abstract
The suppression of inappropriate immune responses by Treg cells is one of the major ways that the body maintains immune tolerance and homeostasis. Since defects in the suppressive capacity of Treg cells underlie many different immune‐mediated diseases, there is great interest in developing ways to track the number and function of Treg cells as biomarkers of tolerance and in devising ways to enhance their function therapeutically. However, the methods of studying human Treg cells are fraught with technical challenges that can often lead to misinterpretation. The most common way to determine the suppressive capacity of human Treg cells is to measure their ability to suppress the proliferation of responding CD4^+^ T cells. Here, we discuss the technical considerations that must be taken into account when performing suppression of T‐cell proliferation assays with human Treg cells. We also consider how T cells may falsely appear suppressive because of dying cells in the system, improper resting of T‐cell lines prior to the assay, or insufficient proliferation of the responding T cells. We propose that, in the future, classification of a population of cells as “regulatory” should rely on more than a simple test for blockade of CD4^+^ T‐cell proliferation.