OMIP‐020: Phenotypic characterization of human γδ T‐cells by multicolor flow cytometry
2014-04-22

This panel was composed and optimized to investigate the differentiation stages of human γδ T-cells in cryopreserved peripheral blood mononuclear cells (PBMC) from healthy individuals (Tables 1 and 2). As the majority of pan-γδ T-cell antibodies available commercially proved to be inappropriate for detecting all γδ T-cell populations in combination with other markers, this panel provides an essential tool for the analysis of different subsets of human γδ T-cells by flow cytometry. The panel works very well with cryopreserved PBMC. Other tissues have not been tested. Based on our long-standing experience of studying human αβ T-cells [e.g., 1-3], and an extensive survey of the published literature, we aimed to set up an antibody panel for the analysis of the differentiation status of circulating human γδ T-cells. During the initial phase of panel development, we became aware that the majority of commercially available anti-γδ TCR antibodies do not detect all γδ T-cell subsets when used in combination with other markers in a multicolor flow cytometry panel. The following pan-γδ T-cell antibodies were tested: an APC-conjugated antibody from clone B1, a PE-conjugated antibody from clone B1.1, and unconjugated, PE-Cy7- and FITC-conjugated antibodies from clone 11F2. Of these, the only pan-γδ antibody we found able to stain all Vδ1+ T-cells (as identified by antibodies from clones TS8.2 & R9.12) and all Vδ2+ T-cells (as identified by antibodies from clones B6 & Immu 389), in a multicolor panel, was the unconjugated form of the clone 11F2 antibody. Having found a suitable marker for identification of all γδ T-cells, other markers were added to the panel. The panel as finally developed includes a live/dead markerEMA, CD3Alexa 700, CD4PerCP, CD8APC-H7, and the aforementioned pan-γδ TCRindirect PO, as a basis to identify γδ T-cells. This combination allows the analysis of all γδ T-cells and to distinguish the CD4−CD8− cells from those expressing CD8 4, 5 (gating strategy provided in Supporting Information). Furthermore, Vδ1 TCRFITC and Vδ2 TCRPerCP markers were used to discriminate the two main γδ T-cell populations in peripheral blood, also allowing the analysis of the remaining small pool of Vδ1−Vδ2− circulating γδ T-cells. EMA can be replaced by red dead stain without altering the performance of the panel (Supporting Information Fig. 8). It also works well after fixation and permeabilization (Supporting Information Fig. 5). CD27APC and CD28PE in combination with CD45RAPacific Blue was informative for the differentiation phenotype of the γδ T-cell subsets as previously reported in several studies 5-14. Finally, we have included CD16BV711 in this panel, as it reportedly distinguishes between γδ T-cell subsets with different activation pathways and functional properties 15-17 (Fig. 1). In the process of setting up the panel, fluorescence minus one (FMO)-controls were measured for each marker to identify the best antibody and fluorophor combinations in the context of the whole panel. This panel has been used successfully for analyzing more than 250 healthy individuals (and cancer patients). Data using a slimmed-down version of this panel (without CD16) were recently published 17. Thus far, there are no other OMIPs published for γδ T-cells. The authors thank Karin Hähnel and David Goldeck for technical support and Lilly Öttinger for the titration of some antibodies. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.