Abstract
Enumeration of anti-viral CD8+ T cells to make comparisons between mice, viruses and vaccines is a frequently used approach, but controversy persists as to the most appropriate methods. Use of peptide-MHC tetramers (or variants) and intracellular staining for cytokines, in particular IFNγ, after a short ex vivo stimulation are now common, as are a variety of cytotoxicity assays, but few direct comparisons have been made. It has been argued that use of tetramers leads to the counting of non-functional T cells and that measurement of single cytokines will fail to identify cells with alternative functions. Further, the linear range of these methods has not been tested and this is required to give confidence that relative quantifications can be compared across samples. Here we show for two acute virus infections and CD8+ T cells activated in vitro that DimerX (a tetramer variant) and intracellular staining for IFNγ, alone or in combination with CD107 to detect degranulation, gave comparable results at the peak of the response. Importantly, these methods were highly linear over nearly two orders of magnitude. In contrast, in vitro and in vivo assays for cytotoxicity were not linear, suffering from high background killing, plateaus in maximal killing and substantial underestimation of differences in magnitude of responses.
Highlights
CD8+ T cells play a crucial role in anti-viral immunity [1,2]
Using fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE), target cell populations loaded with different antigenic peptides can be labeled with the dye at different fluorescence intensities and the cytotoxic activity of CD8+ T cells towards multiple target populations can be assessed in the same assay [6,7]
Where cells require activation by stimulation, as in IFNc-ICS it is possible that the dynamics of culture might introduce unexpected threshold effects. We have addressed these issues using vaccinia virus (VACV) and herpes simplex virus type 1 (HSV-1) infections of mice and in vitro activated OT-I CD8+ T cells
Summary
CD8+ T cells play a crucial role in anti-viral immunity [1,2]. Their main functions are the elimination of infected cells by cytotoxicity and production of a range of cytokines after activation through their T cell receptor [3,4]. The earliest method to measure CD8+ T cell effector activity was the chromium (51Cr) release assay which indirectly measures the viability of 51Cr-labeled target cells after incubation with effector T cells [5]. Cytotoxicity assays that use fluorescent dyes and flow cytometry are becoming more common and allow a variant of this assay to be done in vivo Using fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE), target cell populations loaded with different antigenic peptides can be labeled with the dye at different fluorescence intensities and the cytotoxic activity of CD8+ T cells towards multiple target populations can be assessed in the same assay [6,7]. These assays remain limited to detecting the killing of targets, rather than the CD8+ T cells themselves
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