Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Summary

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen¹. One measure of CTL quality is their ability to kill specifically². CFSE labeling of target cells can be used to investigate this CTL response quality in vivo^3,4.

Abstract

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells^4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing^7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities⁹. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Protocol

1. Eliciting Target-Specific Effector CTLs by Immunization (Day 0)

1. Before beginning, decide upon a specific effector:target system. In this example of a classical in vivo CTL assay, we will use a purified peptide immunization to elicit specific CD8 + T cells. The same peptide will be used to pulse the syngeneic target cells, creating a specific effector:target system. Alternatively, you may choose to use whole pathogen or protein to obtain your specific effectors and targets.
2. Decontaminate the work surface inside of a biosafety hood to ensure sterility of the injections. Prepare the peptide immunization by first placing 50 μg of....

Discussion

This assay can be modified to investigate the proliferative capacity of cells, including clonal expansion, because CFSE divides relatively equally among progeny^10,11. It is also possible to use CFSE labeling to investigate cell migration^6,12, though there are other cell tracing methods that may be more appropriate depending on your hypothesis, for example tetramers and bioluminescence¹³, which also may be combined with CFSE labeling.

It is important to note th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mm glass beads	Biospec Products	11079110
70 μm cell strainer	BD Biosciences	352350
96-well plate	Costar	3799
Adjulite Incomplete Freund’s Adjuvant	Pacific Immunology	n/a
DMSO	Sigma-Aldrich	D-5879
FBS	GIBCO, by Life Technologies	16000-077
FC 500 Flow Cytometer	Beckman Coulter Inc.	n/a
Kontes glass tissue grinder	Kontes Corp	885300-0015
Mini Beadbeater	Biospec Products	n/a
Needle	BD Biosciences	305175
Parafilm "M"	Pechiney Plastic Packaging	PM992
Paraformaldehyde	Electron Microscopy Sciences	157-8
PBS	GIBCO, by Life Technologies	10010-023
PharmLyse lysing reagent	BD Biosciences	555899
RPMI 1640	GIBCO, by Life Technologies	11875-093
Syringe	BD Biosciences	309602
Vybrant CFSE Kit	Invitrogen	V12883