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

Abstract

Dissection of the role and function of human γδ 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 γδ T cells is crucial to facilitate comparability between published and emerging research. The application of commercially available reagents to classify γδ 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 γδ 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 γδ 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.

Cancer immunotherapy with γδ T cells: many paths ahead of us

“… 71 Further optimization of staining protocols and antibody clone selection, which includes suitable mAbs directed against γδ T cell subsets, will help to improve immunohistological studies in the future. 72 We anticipate that interest in γδ T cell subset analysis within tumors in situ will be invigorated with innovative technologies such as fully automated high-content imaging and quantitative whole-slide imaging analysis. 73 , 74 …”

“…As yet, limited information is available on the TCR repertoire of γδ TILs in human tumors. Based on antibody staining with available mAbs to identify expressed TCR Vγ chains, 72 an increase in non-Vδ2 γδ T cells coexpressing Vγ2/3/4 among ascites and TIL γδ T cells compared to levels in peripheral blood was observed in ovarian cancer patients. 77 In glioblastoma multiforme, unique TCR clonotypes were identified by next-generation sequencing in intratumoral Vγ9Vδ2 γδ T cells compared to peripheral blood, suggesting specific recruitment of selected γδ T cells to the tumor site.…”

Tumor resistance mechanisms and their consequences on γδ T cell activation

“…58 Similar to the results in ovarian cancer measured by flow cytometry, a co-expression of Vγ2, 3, or 4 on Vδ1 TIL is also detected by IHC in frozen sections of colon adenocarcinoma patients. 53 In contrast, an enhanced number of Vδ2 was found in glioblastoma multiforme, 75 whereas no significant difference in the percentage of Vδ1 and Vδ2 T cells was observed in prostate cancer tissue in comparison with PBL. 76 In the majority of the above described tumor entities, γδ TIL are described to be in an activated stage accompanied by an increased number of Vδ1 TIL with a T CM or T EM -phenotype and Vδ2 TIL with an T EM or TEMRA-phenotype.…”

“…52 In addition, analysis of paraffin-embedded sections from patients with Epstein-Barr virus (EBV)-associated Hodgkin's lymphoma demonstrated that most of the γδ T cells from these patients expressed Vγ9, whereas γδ T cells expressing Vγ2, 3, or 4 were nearly absent. 53 γδ T cell infiltration and correlation with patient outcome have been also demonstrated in epithelial and solid tumor cells including melanoma, colorectal, breast, pancreatic, and ovarian cancer. [54][55][56][57][58] Immunohistochemistry (IHC)-based analysis allows analysis of tumor-infiltrating γδ T lymphocytes (γδ TIL) within the tumor as well as in the context of the TME or the surrounding tissue.…”

Accurate determination of γδ T cells in multi‐channel mass and flow cytometry

“…In the context of the proposal by Bagwell et al, we believe that optimization of the proposed antibody panel by substituting the pan-γδ T cell receptor (TCR) antibody deriving from the clone "B1" with a different clone, for example, "11F2," is essential. "B1-antibodies" may not be able to identify the entirety of the γδ T cell population when used in combination with a CD3 antibody, probably due to close proximity of the recognized epitopes causing steric hindrance (BD datasheet "clone B1", Wistuba-Hamprecht, Pawelec, & Derhovanessian, 2014;Beucke et al, 2020). Another advantage of "11F2-antibodies" is that they are known to work well in combination with additional TCR γδ antibodies such as those specific for Vδ1 (clone REA173) orVδ2 (clone 123R3).…”