Abstract
Regulatory T cells (Tregs) are renowned for maintaining homeostasis and self-tolerance through their ability to suppress immune responses. For over two decades, Tregs have been the subject of intensive research. The immunosuppressive and migratory potentials of Tregs have been exploited, especially in the areas of cancer, autoimmunity and vaccine development, and many assay protocols have since been developed. However, variations in assay conditions in different studies, as well as covert experimental factors, pose a great challenge to the reproducibility of results. Here, we focus on human Tregs derived from clinical samples and highlighted caveats that should be heeded when conducting Tregs suppression and migration assays. We particularly delineated how factors such as sample processing, choice of reagents and equipment, optimization and other experimental conditions could introduce bias into the assay, and we subsequently proffer recommendations to enhance reliability and reproducibility of results. It is hoped that prioritizing these factors will reduce the tendencies of generating false and misleading results, and thus, help improve our understanding and interpretation of Tregs functional studies.
Highlights
Regulatory T cells (Tregs) are a specific subset of CD4 T cells endowed with the ability to suppress immune responses, maintaining homeostasis and self-tolerance [1]
Tregs migration assay relies on the principle of chemotaxis, the directional movement of cells towards a chemical gradient often established by signaling proteins
To assess Tregs migration through the endothelium, the transwell insert is layered with a monolayer of endothelial cells prior to treatment with Tregs
Summary
Regulatory T cells (Tregs) are a specific subset of CD4 T cells endowed with the ability to suppress immune responses, maintaining homeostasis and self-tolerance [1]. Suppression is determined by evaluating the ability of Tregs to repress cytokine production by the responder cells [6]. Tregs migration assay relies on the principle of chemotaxis, the directional movement of cells towards a chemical gradient often established by signaling proteins (e.g., chemokines). To assess Tregs migration through the endothelium, the transwell insert is layered with a monolayer of endothelial cells prior to treatment with Tregs. This type of migration assay is often termed transmigration or transendothelial migration (TEM) assay. Migrated cells can be enumerated using hemocytometer, flow cytometer or other dye assays
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