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 vivo3,4.
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 cells4-6. 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 killing7,8. 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.
1. Eliciting Target-Specific Effector CTLs by Immunization (Day 0)
2. Harvesting Target Cells (Day 8)
3. Treatment and Labeling of Target Cells
4. Adoptive transfer
5. Harvesting Labeled Cells
*Note: Steps 5.1 through 5.11 are the same as steps 2.1 through 2.11
6. Flow Cytometry and Data Analysis
7. Representative Results
A diminishing epitope specific peak on the resulting histogram is an indicator of cell specific killing (Figure 1). Use the following equations (applied in Table 1) to determine the actual value of cell specific lysis:
Ratio = Irrelevant Percentage : Epitope Specific Percentage ( [low] peak:[high] peak),
Percent Specific Lysis = [1-(Non-transferred control ratio/Experimental ratio)] x 100
Figure 1. CFSE profile of recovered BALB/c mouse splenocytes. Numbers indicate the percentage of cells of the high or low CFSE phenotype. A) Non-transferred control. All [high] CFSE peaks represent epitope specific target cells, while [low] CFSE peaks represent targets pulsed with irrelevant peptide. B) CFSE labeled cells recovered from PBS immunized recipient mouse (Control). C) CFSE labeled cells recovered from two different peptide specific recipient mice, note the diminished epitope specific peaks.
Figure 2. Graphical representation of analyzed data. Three data points shown for each group of mice, PBS, Peptide A, and Peptide B.
Sample | CFSE HIGH % Epitope Pulsed | CFSE LOW % Irrelevant | Ratio Low:High | Percent Epitope Specific Killing |
PBS | 42.8 | 57.2 | 1.34 | 16.97 |
PBS | 43.1 | 56.9 | 1.32 | 15.94 |
PBS | 44.3 | 55.7 | 1.26 | 11.74 |
Peptide A | 39.7 | 60.3 | 1.52 | 32.56 |
Peptide A | 38.6 | 61.4 | 1.59 | 35.61 |
Peptide A | 40.6 | 59.4 | 1.46 | 29.99 |
Peptide B | 38.7 | 61.3 | 1.58 | 24.68 |
Peptide B | 38.5 | 61.5 | 1.60 | 25.32 |
Peptide B | 39.3 | 60.7 | 1.54 | 22.76 |
PBS NT Control | 47.4 | 52.6 | 1.11 | |
Pep A NT Control | 49.4 | 50.6 | 1.02 | |
Pep B NT Control | 45.6 | 54.4 | 1.19 |
Table 1. Analysis of representative data. “NT” is non-transferred.
This assay can be modified to investigate the proliferative capacity of cells, including clonal expansion, because CFSE divides relatively equally among progeny10,11. It is also possible to use CFSE labeling to investigate cell migration6,12, 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 that CFSE bleeds over into multiple channels on the flow cytometer, so if you want to co-stain for a surface marker, PeCy7 would be a good choice of fluorophore, while PE might not work as well.
The authors have nothing to disclose.
I would like to thank Bianca Mothé, Carla Oseroff, and Marie-France DelGuercio for introducing me to immunological assays and mouse handling. Work in the lab of G. A. Splitter is funded by NIH grant 1-RO1-AI-073558, GLRCE grant 1-U54-AI-057153, and BARD US-3829-06.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
1 mm glass beads | Biospec Products | 11079110 | ||
70 μm cell strainer | BD Falcon | 352350 | ||
96-well plate | Costar | 3799 | ||
Adjulite Incomplete Freund’s Adjuvant | Pacific Immunology | n/a | ||
DMSO | Sigma | D-5879 | ||
FBS | GIBCO | 16000-077 | ||
FC 500 Flow Cytometer | Beckman Coulter | n/a | ||
Kontes glass tissue grinder | Kontes | 885300-0015 | ||
Mini Beadbeater | Biospec Products | n/a | ||
Needle | B-D | 305175 | ||
Parafilm “M” | Pechiney Plastic Packaging | PM992 | ||
Paraformaldehyde | Electron Microscopy Sciences | 157-8 | ||
PBS | GIBCO | 10010-023 | ||
PharmLyse lysing reagent | BD Biosciences | 555899 | ||
RPMI 1640 | GIBCO | 11875-093 | ||
Syringe | B-D | 309602 | ||
Vybrant CFSE Kit | Invitrogen (Molecular Probes) | V12883 |