Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells

Abstract

The clearance of tumors or virus infected cells is a crucial task of the immune system. Cytotoxic T-cells (CTLs) are able to detect and to kill such altered host cells. Given the recent success of checkpoint inhibitors for tumor therapy, it becomes more and more important to understand the biology of T-cell mediated target cell killing. Tests that allow analyzing the biology of CTLs are either based on flow cytometry or fluorescence microscopy. Thus, they either lack image-based information or have a poor statistical robustness. Therefore, we describe an approach to quantify CTL-mediated cytotoxicity using imaging flow cytometry. Using activated primary human cytotoxic T-cells as CTLs and P815 as target cells, we show that both the evaluation of target cell death and the biology of CTLs can be evaluated in parallel. This enables to gain information about CTL-mediated cytotoxicity in samples from patients important for translational medicine. J. Cell. Biochem. 118: 2528-2533, 2017. © 2017 Wiley Periodicals, Inc.

Guidelines for the use of flow cytometry and cell sorting in immunological studies*

“…This molecule is present on the membrane of cytotoxic granules and becomes detectable on the cell surface of degranulating CTLs. CD107a levels correlate mostly , but not always , with the lytic potential of CTLs. Antibodies to CD107a need to be added at 37°C to capture this rapidly recycling protein.…”

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…However, they can also be differentially regulated . Precise imaging of the accumulation of CD107a, granzyme, and other molecules in the cytotoxic immune synapse between T cells and target cells can be performed by imaging FCM (see also the section on Imaging FCM). mAbs directed against CD107a can be combined with FATAL assays, MHC/peptide multimers, or cytokine‐specific Abs to determine multiple effector functions of individual antigen‐specific CTLs by FCM .…”

“…IFC can also be used to characterize cytotoxic immune synapses for multiparametric analysis of molecular mechanism involved in the cytotoxicity of human CD8 T‐cells .…”

Sulforaphane Inhibits Inflammatory Responses of Primary Human T-Cells by Increasing ROS and Depleting Glutathione

“…Although T-cell/APC conjugate formation can be measured using flow cytometry, this technique is limited for evaluating spatial informations which are important for analysing the formation of the immune synapse. We, therefore, took advantage of MIFC, which combines fluorescence microscopy and flow cytometry and is a suitable means for the analysis of immune synapse formation ( 26 ). By defining regions of interest, MIFC allows the spatial quantification of fluorescence signals within T-cells, and thus of the accumulation of receptors at the T-cell/APC interface.…”