Pitfalls in the characterization of circulating and tissue-resident human γδ T cells

Dissection of the role and function of human gamma delta T cells and their heterogeneous subsets in cancer, inflammation, and auto-immune diseases is a growing and dynamic research field of increasing interest to the scientific community. Therefore, harmonization and standardization of techniques for the characterization of peripheral and tissue-resident gamma delta T cells is crucial to facilitate comparability between published and emerging research. The application of commercially available reagents to classify gamma delta T cells, in particular the combination of multiple Abs, is not always trouble-free, posing major demands on researchers entering this field. Occasionally, even entire gamma delta T cell subsets may remain undetected when certain Abs are combined in flow cytometric analysis with multicolor Ab panels, or might be lost during cell isolation procedures. Here, based on the recent literature and our own experience, we provide an overview of methods commonly employed for the phenotypic and functional characterization of human gamma delta T cells including advanced polychromatic flow cytometry, mass cytometry, immunohistochemistry, and magnetic cell isolation. We highlight potential pitfalls and discuss how to circumvent these obstacles.