Flow Cytometric Analysis of Human Antigen-Specific T-Cell Proliferation

Abstract

This chapter reviews the use of a technique based on serial dilution of a stably binding intracellular fluorochrome, carboxy fluorescein diacetate succinimidyl ester (CFSE) that allows monitoring of cell division by flow cytometry, to monitor antigen-specific T-cell responses. CFSE is capable of freely diffusing across the cell membrane where it reacts with free amine groups of cellular proteins. Carboxyl groups are removed from CFSE by enzymatic action of ubiquitously expressed esterases and CFSE emits strongly at 519 nm after excitation by 488 nm argon laser light. Advantages of monitoring cell division by flow cytometry over traditional thymidine incorporation assays are (1) number of cell divisions can be directly quantified using the flow cytometric assay, (2) phenotype of cells undergoing proliferation can be directly ascertained, (3) phenotypic changes and other properties that occur with respect to cell division can be monitored, and (4) flow cytometric analysis of proliferation allows for sorting of viable cells and based on rounds of proliferation. The critical aspects to be taken care of when using CFSE staining are background proliferation and instrument compensation. The CFSE technology adds a huge advantage over assays using incorporation of tritiated thymidine because these proliferation platform calculations cannot be performed with tritiated thymidine experiments. Monitoring cell division in conjunction with production of effector cytokines might enable detection of more vaccine-induced antigen-specific T cells. Flow cytometric technology, capable of monitoring more than six fluorescent parameters, is slowly becoming more widespread. Such technology will allow dissection of cytokine production and cell division within defined of phenotypically distinct lymphocytes.