Abstract
Antigen-specific killing ability of effector CD8+ T cells is critical for protective immunity against infection. Here, we describe in vivo cytotoxic T cell assay to examine effector function of antigen-specific CD8+ T cells. Mice infected with Listeria monocytogenes (L. monocytogenes) expressing chicken ovalbumin as a model antigen mount ovalbumin-specific CD8+ T cell responses. Effector CD8+ T cell function in vivo is determined by mixed transfer of OVA peptide-pulsed target cells with control target cells into the previously immunized mice. Difference in CFSE expression levels clearly marks two distinct populations: Antigen-pulsed target cells-CFSElow vs. unpulsed target cells-CFSEhi. The frequencies between antigen-pulsed target cells and control target cells are used as readouts of antigen-specific killing.
Materials and Reagents
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Splenocytes from a wild type mouse
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PBS (Thermo Fisher Scientific, catalog number: BP399-20 )
Note: 10x solution, diluted to 1x in house in distilled water and sterilized by autoclave.
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RBC lysis buffer (eBioscience, catalog number: 00-4333-57 )
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HBSS without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14175-095 )
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RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-119 )
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Fetal bovine Serum (Atlanta Biologicals, catalog number: S11055H )
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Penicillin/streptomycin (Gemini Bio-Products, catalog number: F52M00E )
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L-Glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
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Trypan blue solution (Life Technologies, Gibco®, catalog number: 15250-061 )
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OVA257-264 synthetic peptide (Sigma-Aldrich, catalog number: S7951 )
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5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma-Aldrich, catalog number: 21888 )
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Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D-8418 )
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Collagenase D (Sigma-Aldrich, catalog number: C-5138 )
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Percoll (Sigma-Aldrich, catalog number: P-1644 )
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Complete RPMI-1640 media (see Recipes)
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100% percoll solution (see Recipes)
Equipment
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Centrifuge (Thermo Fischer Scientific, SorvallTM Legend RT )
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37 °C water bath
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Hemocytometer
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15 ml and 50 ml Falcon tubes
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6 well plates (USA Scientific, CytoOne®, catalog number: CC7682-7506 )
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BD LSRII Flow Cytometer (BD)
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70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
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5 ml polystyrene round-bottom tubes with cell-strainer cap (BD Biosciences, Falcon®, catalog number: 352235 )
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3 ml syringe (BD, catalog number: 14-823-435 )
Procedure
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Target cell preparation under sterile tissue culture conditions
This step is for preparing peptide-pulsed target cells and stain cells with CFSE to distinguish peptide-pulsed target cells from control target cells.
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OVA257-264 peptide-loading for the target cells.
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Splenocytes are RBC lysed followed by washing with PBS twice.
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Resuspend cells in RPMI-1640 complete medium.
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Count the mononuclear cells by trypan blue exclusion using a hemocytometer.
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Resuspend cells at 5 x 106/ml of RPMI-1640 complete medium.
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Divide the cells equally into two separate 50 ml Falcon tubes- one for
peptide-pulsed target cells, and the other for unpulsed target cells.
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Add OVA257-264 peptide at 1 µl/ml from a 200 µM stock to peptide-pulsed target cells.
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Add an equivalent amount of PBS to the unpulsed target cells.
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Incubate the cells in a 37 °C water bath for 1 h.
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Wash cells twice with RPMI-1640 complete medium.
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Centrifuge the cells at 1,500 rpm for 3 min at 4 °C.
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Resuspend the cell pellet in HBSS.
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CFSE cell labeling under sterile tissue culture conditions.
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Count all live cells by trypan blue exclusion using hemocytometer.
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Resuspend the cells in HBSS at 5 x 107/ml.
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Thaw an aliquot of 5 mM stock CFSE solution.
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Make a fresh CFSElow stock solution by diluting 5 mM stock 1:10 in DMSO (a final concentration of 0.5 mM).
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Incubate the unpulsed target splenocytes with the higher concentration
of CFSE (CFSEhigh): Add 1 µl of the 5 mM stock CFSE for each milliliter
of unpulsed target cells (final concentration of 5 µM).
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Incubate the pulsed target splenocytes with the lower concentration of
CFSE (CFSElow): Add 1 µl of the 0.5 mM stock CFSE for each milliliter of
peptide-pulsed cells (final concentration of 0.5 µM).
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Pipette
cells up and down to mix well and incubate in water bath for 10 min at
37 °C. Gently agitate the cells periodically.
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Add 10x the volume of pre-warmed RPMI-1640 complete medium to the CFSE-labeled cells to stop the reaction.
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Pellet cells at 1,500 rpm for 3 min at 4 °C.
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Remove the supernatant and resuspend the pellet in cold RPMI-1640 complete medium.
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Wash the cells two more times with cold RPMI-1640 complete medium.
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Count the mononuclear cells by trypan blue exclusion using a hemacytometer.
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Wash the cells with cold PBS.
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Resuspend each cell populations in PBS at 6.7 x 106/ml.
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Combine an equal volume (~equal numbers) of peptide-pulsed CFSElow cells with unpulsed CFSEhi cells and proceed with flow cytometry
analysis (Figure 1a).
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Intravenous injection of target splenocytes
To investigate OVA257-264-specific CD8+ T cell killing ability, peptide-pulsed and unpulsed target cells were mixed at a 1:1 ratio and transferred to the previously immunized mice.
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Recipient mice were infected with 5,000 colony-forming units (CFU) of Listeria monocytogenes expressing chicken ovalubmin (LM-OVA) 7 days
intravenously before the CFSE-labeled cell injection.
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Inject
intravenously 300 µl of the combined cell populations into the tail vein
of each recipient. Each recipient should receive approximately 1 x 107 peptide-pulsed target cells combined with 1 x 107 unpulsed target cells.
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Wait for 4 h.
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Preparation of splenocytes and lymphocytes in the liver for flow cytometry analysis
This step is analyzing antigen-specific killing ability in the liver and the spleen by flow cytometric analysis of CFSElow and CFSEhi cell populations.
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Prepare splenocytes for flow cytometry analysis.
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Wash once with PBS, and 2-300 µl into a 5 ml round bottom tubes through the cell strainer.
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Isolate lymphocytes from the liver.
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Harvest livers from the recipients and place them on ice.
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Make a fresh collagenase D solution by diluting 20 mg/ml stock 1:20 in PBS (a final concentration of 1 mg/ml).
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Chop liver with a blade on a slide glass and transfer them into a 50 ml falcon tube.
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Add 7 ml of collagenase D (1 mg/ml) and vortex well.
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Incubate in water bath for 30 min at 37 °C. Vortex every 15 min.
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Put tubes on ice and add supernatant on 70 µm filter on a well of a 6
well plate. Grind chunks of the chopped liver with flat portion of 3 ml
syringe. Wash the tube with 5 ml of PBS and repeat grinding.
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Transfer them to a 50 ml Falcon tube and spin down at 2,000 rpm for 5 min at 4 °C.
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Make fresh 44% and 66% percoll solution: Make 44% final concentration
by diluting 100% percoll in PBS, and make 66% final concentration by
diluting 100% percoll in RPMI-1640 medium.
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Resuspend pellet in 7 ml of 44% percoll, and load them on 3 ml of 66% percoll in 15 ml tube.
Note: The gradient separation is sensitive to agitation. Try not to shake the tube.
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Centrifuge at 3,000 rpm for 30 min at 4 °C without brake.
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Transfer the interphase lymphocytes to a new 15 ml tube.
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Pellet cells at 2,000 rpm for 5 min at 4 °C.
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Wash once with PBS, and transfer 2-300 µl into a 5 ml round bottom tubes through the cell strainer.
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Proceed to flow cytometry analysis with spleen and liver samples (Figure 1b).

Figure
1. CFSE expression in antigen-pulsed target cells and unpulsed target
cells. a. Peptide-pulsed CFSElow and unpulsed CFSEhi splenocytes were
mixed at a 1:1 ratio before transferring to the recipients. b. The
mixture of peptide-pulsed and unpulsed splenocytes was transferred into
the mice predisposed with LM-OVA at day 7 post infection. The spleen and
the liver of the recipients were harvested after 4 h to determine
percentages of CFSElow and CFSEhi cells among CFSE+ cells.
Recipes
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Complete RPMI-1640 medium
10% FBS
1% Penicillin/streptomycin with L-Glutamine
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100% percoll solution
90% of percoll
10% of 10x PBS
Notes
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The gradient separation is sensitive to agitation. Try not to shake the tube.
Acknowledgments
The protocol was adapted from a previously described study (Manjunath et al., 2001). This work was supported by the Starr Cancer Consortium (13-A123 to M.O.L. and M.Q.Z.), the Rita Allen Foundation (M.O.L.), the NBRPC (2012CB316503 to M.Q.Z), and the NIH (HG001696 to M.Q.Z.).
References
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Ingulli, E. (2007). Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells. Methods Mol Biol 380: 365-376.
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Kim, M. V., Ouyang, W., Liao, W., Zhang, M. Q. and Li, M. O. (2013). The transcription factor Foxo1 controls central-memory CD8+ T cell responses to infection. Immunity 39(2): 286-297.
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Kim, M. V., Ouyang, W., Liao, W., Zhang, M. Q. and Li, M. O. (2014). Murine
in vivo CD8
+ T Cell Killing Assay.
Bio-protocol 4(13): e1172. DOI:
10.21769/BioProtoc.1172.