Immunophenotypic and functional characterization of circulating cytotoxic T lymphocytes and natural killer cells using the BD FACSymphony™ A1 Cell Analyzer

Abstract

Abstract Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells have become attractive therapeutic targets for cancer immunotherapy because of their efficient mechanisms for killing cancerous cells. Both effector CTLs and mature NK cells are endowed with secretory granules containing pre-formed cytolytic proteins that, once released at the immunological synapse, initiate a cascade of reactions leading to the apoptosis of the target cell. CTLs and NK cells may also promote cell killing through death-receptor-mediated and cytokine-induced cell death mechanisms. Here we show a 16-color flow cytometry panel allowing clear identification of CTLs, NK cells and related cell subsets in whole blood. We also describe an optimized intracellular staining protocol for analysis of granules content, including granzyme B, granzyme K and perforin. Optimal resolution of cell populations was achieved by employing a selection of BD Horizon Brilliant™ Reagents and BD Horizon™ Red 718 Reagent, a new small molecule fluorochrome option for the red laser. Cell fixation and permeabilization were carried out directly in whole blood using the BD Phosflow™ Reagents, adding convenience to the workflow and practical analysis of cell frequencies. The data also showed the expression of selected activation and inhibitory surface receptors. These results combined with the analysis of intracellular proteins contribute to characterization of the functional status of cytotoxic cells. Class 1 Laser Product. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD 25443 (v1.0) 0121 BD, the BD Logo, FACSymphony, Horizon, Horizon Brilliant and Phosflow are trademarks of Becton, Dickinson and Company or its affiliates. © 2021 BD. All rights reserved.