A Method to Evaluate In Vivo CD8⁺ T Cell Cytotoxicity in a Murine Model

Abstract

Herein, a method to measure in vivo CD8⁺ T cell cytotoxicity in a murine model is presented. The activation of a strong CD8⁺ T cell response is paramount when designing vaccines to tackle intracellular infections and for cancer therapy. CD8⁺ T cells can directly kill infected and transformed cells and are directly associated with beneficial protection in many disease models. CD8⁺ T cell cytotoxicity can be measured using multiple methods including measuring IFNγ production by ELISPOT or measuring intracellular cytokines or cytotoxic granules by flow cytometry. However, to determine the ability of CD8⁺ T cells to kill their target in the context of its cognate receptor and in their native environment, the in vivo cytotoxic T cell assay (in vivo CTL) is ideal. The in vivo CTL assay provides a snapshot of the whole ability of the host to kill “Target” cells by measuring the loss of injected target cells relative to “Non-target” cells. The assay involves isolating splenocytes from donor mice, forming “Target” and “Non-target” cellular samples and injecting them intravenously into naïve and experimental mice at a chosen time-point in the experiment. Mice are humanely sacrificed 20 h later, and their spleens are excised and processed for flow cytometric analysis. The extent of “Target” cell killing relative to “Non-target” cells is determined by comparing the surviving proportions of these cells among experimental mice relative to naïve mice. The in vivo CTL assay is a rapid, sensitive, and reliable method to measure the potency of CD8⁺ T cells in their host to kill their target.