Multiplex Screening Assay for Identifying Cytotoxic CD8+ T Cell Epitopes

Front Immunol. 2020 Mar 11:11:400. doi: 10.3389/fimmu.2020.00400. eCollection 2020.


The cytotoxicity of epitope-specific CD8+ T cells is usually measured indirectly through IFNγ production. Existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. Here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to 23 different specificities of CD8+ T cells in a single reaction. This can greatly reduce the amount of starting clinical materials for a systematic screening of CD8+ T cell epitopes. In addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of CD8+ T cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. This technique is shown to be useful to study both human and mouse CD8+ T cells. Besides, our results from human PBMCs and three independent infectious animal models (MERS, influenza and malaria) further reveal that IFNγ expression by epitope-specific CD8+ T cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). Overall, our approach opens up new possibilities for comprehensive analyses of CD8+ T cell cytotoxicity in a practical manner.

Keywords: CD8 T cell; MERS; Plasmodium; cytotoxicity; influenza; multiplex assay.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Epitopes, T-Lymphocyte / immunology*
  • Epitopes, T-Lymphocyte / isolation & purification*
  • Flow Cytometry / methods*
  • Humans
  • Mice
  • Staining and Labeling / methods
  • T-Lymphocytes, Cytotoxic / immunology*


  • Epitopes, T-Lymphocyte