Chapter 32
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity
in a Murine Model
Felicity C. Stark, Renu Dudani, Gerard Agbayani,
and Michael J. McCluskie
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 “Nontarget” cells. The assay involves isolating splenocytes from donor mice, forming “Target” and “Non-target”
cellular samples and injecting them intravenously into naı ¨ve 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 naı ¨ve 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.
Key words In vivo CTL, Cytotoxic T lymphocyte, CTL, Vaccines, Cytotoxicity, Antigen-specific
CD8
+ T cell killing, Killing assay, Cellular immunity, T cell killing
1 Introduction
When evaluating a candidate vaccine in preclinical studies, it is key
to design assays that can detect the activation of both humoral and
cell-mediated immunity. Historically, the induction of a potent
antibody response was sufficient to advance a new vaccine formulation toward a clinical trial. However, as vaccinologists tackle
tougher, more complex pathogens (influenza, HIV, HCV, cancer,
etc.), new vaccine formulations are frequently also required to show
evidence of the activation of cell mediated immunity [1–4]. In this
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_32, © Springer Science+Business Media, LLC, part of Springer Nature 2021
549
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity
in a Murine Model
Felicity C. Stark, Renu Dudani, Gerard Agbayani,
and Michael J. McCluskie
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 “Nontarget” cells. The assay involves isolating splenocytes from donor mice, forming “Target” and “Non-target”
cellular samples and injecting them intravenously into naı ¨ve 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 naı ¨ve 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.
Key words In vivo CTL, Cytotoxic T lymphocyte, CTL, Vaccines, Cytotoxicity, Antigen-specific
CD8
+ T cell killing, Killing assay, Cellular immunity, T cell killing
1 Introduction
When evaluating a candidate vaccine in preclinical studies, it is key
to design assays that can detect the activation of both humoral and
cell-mediated immunity. Historically, the induction of a potent
antibody response was sufficient to advance a new vaccine formulation toward a clinical trial. However, as vaccinologists tackle
tougher, more complex pathogens (influenza, HIV, HCV, cancer,
etc.), new vaccine formulations are frequently also required to show
evidence of the activation of cell mediated immunity [1–4]. In this
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_32, © Springer Science+Business Media, LLC, part of Springer Nature 2021
549