An Assay to Evaluate Immunodominance in Cytotoxic T Lymphocyte Responses

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Inoculation of C57BL/6 Mice with T Ag-expressing Tumor Cells

1. Grow the SV40-transformed fibrosarcoma cell line C57SV (or a similar T Ag⁺ adherent cell line) in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L D-glucose and L-glutamine (1x) and supplemented with 1 mM sodium pyruvate and 10% heat-inactivated fetal bovine serum (FBS) in tissue culture-treated flasks at 37 °C in humidified atmosphere containing 10% carbon dioxide (CO₂).
2. Once the cells become fully confluent or slightly overconfluent, gently remove and discard the medium and rinse the monolayer with pre-warmed sterile phosphate-buffered saline (PBS).       
   NOTE: Maximal T Ag expression is achieved when T Ag⁺ cells reach 100% confluency.
3. Inside a biological safety cabinet, add pre-warmed trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) (0.25%) to cover the monolayer at room temperature until the cells are dislodged in patches. Tap the sides of the culture flask(s) several times to release the remaining adherent cells.
   NOTE: If necessary and to expedite the trypsinization process, transfer the flask(s) into a 37 °C incubator. Dislodged cells will quickly adopt a rounded shape under a light microscope. This step should last approximately 5 min.
4. Add 5 mL of DMEM medium and dissociate clumps to prepare a single-cell suspension by pipetting the content of each flask up and down.
5. Transfer the cell suspension through a cell strainer with 70-µm pores into a tube.
6. Spin down the tube at 400 x g for 5 min at 4 °C.
7. Discard the supernatant. Resuspend pelleted cells in 10 mL of sterile, cold PBS.
8. Repeat steps 1.6 and 1.7 twice.
9. Count cells using a hemocytometer. Prepare a uniform suspension containing 4 x 10⁷ cells/mL sterile PBS.
10. Inject 500 µL of the above suspension intraperitoneally (i.p.) into each adult (6-12-week-old) male or female C57BL/6 mouse.

2. Treatment Regimens

1. Treatment Regimen to Examine the Contribution of nTreg Cells to T_CD8 Immunodominance
   1. Four days before in vivo priming of C57BL/6 mice with C57SV cells (step 1.10), inject each animal once i.p. with 0.5 mg of a low-endotoxin, azide-free anti-CD25 monoclonal antibody (mAb) (clone PC-61.5.3), which depletes nTreg cells, or with a rat IgG1 isotype control (e.g., clone KLH/G1-2-2, clone HRPN, or clone TNP6A7).
2. Treatment Regimen to Test the In Vivo Significance of Programed cell death proteins (PD-1-PD-L1(2)) Interactions in Shaping T_CD8 Immunodominance 
   NOTE: The engagement of PD-1 by PD-L1 often, but not always, mediates the co-inhibition and/or exhaustion of Ag-specific T_CD8. Therefore, treatment with anti-PD-1 can be performed in parallel with the administration of anti-PD-L1 and anti-PD-L2 mAbs to reveal the exact intercellular interaction involved in a biological phenomenon.

3. Preparation of Target Splenocytes

1. Euthanize sex-matched naïve C57BL/6 mice (6-12 weeks of age) that will serve as splenocyte donors by cervical dislocation.
2. Position each mouse with its abdomen facing up inside a biological safety cabinet. Spray the skin with 70% (v/v) ethanol, EtOH. Using sterile forceps and scissors, lift the skin and make a small ventral midline incision. Then, cut the skin in a cross-like fashion to expose the peritoneum.
3. Using forceps, pull up the peritoneum in a tent-like fashion without snatching any of the internal organs. Cut the peritoneum open to expose the peritoneal cavity and gently remove the spleen.
4. Place the spleen(s) inside a 15 mL Dounce tissue grinder containing 5 mL of sterile PBS. Apply manual pressure using the grinder's glass plunger until the splenic tissue dissipates into a red homogenous cell suspension.
   NOTE: Depending on the number of recipient animals per experimental group, several donor mouse spleens may be needed for target cell preparation. Up to 3 spleens can be homogenized together inside a 15 mL grinder.
5. Transfer the homogenate into a 15 mL tube. Spin down the tube at 400 x g for 5 min at 4 °C.
6. Discard the supernatant. Resuspend pelleted cells in 4 mL of ammonium-chloride-potassium (ACK) lysing buffer for 4 min to eliminate erythrocytes.
   NOTE: This is a time-sensitive step. Overexposing splenocytes to ACK lysing buffer will increase their fragility and render them susceptible to non-specific cell death.
7. To each tube, add 8 mL of Roswell Park Memorial Institute 1640 (RPMI 1640) medium containing 10% heat-inactivated FBS, L-alanyl-L-glutamine, 0.1 mM minimum essential media (MEM) nonessential amino acids, 1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 1x penicillin/streptomycin, which will hereafter be referred to as complete RPMI medium (Table of Materials).
8. Transfer the content through 70 µm pores of a cell strainer into a new 15 mL tube.
9. Spin down the tube at 400 x g for 5 min at 4 °C.
10. Discard the supernatant. Resuspend pelleted cells in 12 mL of complete RPMI.
11. Split the splenocyte suspension into 3 equal portions (4 mL each) in 3 separate tubes.

4. Coating Target Splenocytes with Irrelevant and Cognate Peptides

1. Label the tubes according to the peptides that will be used to pulse target splenocytes. Control splenocytes will be pulsed with an irrelevant peptide, and each population of cognate target splenocytes will be pulsed with a synthetic peptide corresponding to the T Ag-derived immunodominant epitope (site IV) or a subdominant T Ag epitope (site I or site II/III) (Table 1).       
   NOTE: The choice of irrelevant peptides depends on the experimental set-up and the mouse strain used in each investigation. The authors often use gB_498-505 (an H-2K^b-restricted immunodominant peptide epitope of herpes simplex virus [HSV]-1) and/or GP_33-41 (an H-2D^b-restricted immunodominant peptide epitope of lymphocytic choriomeningitis virus ([LCMV]) in C57BL/6 mice (Table 1). These peptides are optimal choices because: (i) they are derived from pathogens not previously encountered in the mouse model described here; (ii) similar to T Ag-derived peptides, gB_498-505 and GP_33-41 are restricted by and binds to H-2^b molecules. In ‘three-peak' in vivo killing assays, each of the two peaks that correspond to cognate target cells may represent splenocytes pulsed with an immunodominant or subdominant peptide. The choice of each peptide set varies according to the objectives of each experiment. See Figure 1 and Figure 2 as examples of such variation. For the remainder of this protocol, T Ag-derived sites I and IV will represent subdominant and immunodominant peptides, respectively.
2. Pulse the content of each labeled tube with 1 µM of the respective peptide for 1 h at 37 °C and 5% CO₂.
3. Use a separate cell strainer (with 70-μm pores) for each tube to remove clumps and debris if necessary.
4. Spin down the tube at 400 x g for 5 min at 4 °C. Discard the supernatant.
5. Resuspend pelleted cells in 12 mL of sterile cold PBS and repeat step 4.4 once more.
   NOTE: It is important to remove as much FBS as possible because FBS can bind carboxyfluorescein succinimidyl Ester (CFSE) in the next step.

5. Labeling target splenocytes with CFSE

1. Resuspend peptide-pulsed splenocytes in 4 mL of sterile PBS.
2. Add CFSE at 0.025 μM, 0.25 μM, and 2 μM into the tubes containing irrelevant peptide-, site I-, and site IV-pulsed splenocytes, respectively.         
   NOTE: To achieve uniform CFSE labeling, hold each tube at a 45° angle before adding CFSE to the side slightly above the cell suspension followed immediately by gentle vortexing. This will ensure the appearance of smooth histograms at the end. Batch-to-batch and age-dependent variations in CFSE intensities are not uncommon. Therefore, one may need to experiment with differential CFSE doses before deciding on the optimal concentrations to be used.     
   CAUTION: CFSE is toxic at concentrations that are higher than 5 μM.
3. Place the tubes inside a 37 °C incubator for 15 min and invert them once every 5 min.
4. Add 3 mL of heat-inactivated FBS to each tube to stop the CFSE reaction. Top up the content with sterile PBS.
5. Spin down the tube at 400 x g for 5 min at 4 °C. Discard the supernatant.
6. Resuspend pelleted cells in 12 mL of sterile PBS and repeat step 5.5.

6. Examination of Adequate/Equal CFSE Labeling of Target Splenocyte Populations

1. Resuspend pelleted cells in 3 mL of PBS.
2. Vortex the tubes gently. Transfer 10 μL, each, of CFSE^low, CFSE^{intermediate (int)}, and CFSE^high cell suspensions into a 5 mL round-bottom polystyrene fluorescence-activated cell sorting (FACS) tube containing 200 μL OF PBS.
3. Interrogate cells using a flow cytometer equipped with a 488 nm laser. Draw a lymphocyte gate based on forward scatter (FSC) and side scatter (SSC) properties of the cells before acquiring 5000 events falling within the lymphocyte gate in the FL-1 channel.
4. Within the ‘parent' CFSE⁺ population, draw additional histogram gates to identify CFSE^low, CFSE^int, and CFSE^high subpopulations.
5. Confirm equal or near-equal event numbers within the three gates. If necessary, adjust cell numbers in the ‘source' tubes (step 6.1) before mixing and injecting target splenocytes into naïve and primed mice in section 7.

7. Injection of CFSE-labeled Target Cells into Naïve and T-Ag-primed Recipients

1. Gently vortex the source tubes. Transfer the three CFSE-labeled cell suspensions in equal ratios into a new tube.
2. Top up the content with sterile PBS.
3. Spin down the tube at 400 x g for 5 min at 4 °C. Resuspend pelleted cells with sterile PBS.
4. Count cells in trypan blue by a hemocytometer to ensure cellular viability of at least 95%.
5. Adjust the volume in order to inject 1 x 10⁷ mixed target cells/200 μL PBS intravenously (i.v.), via tail vein, into each recipient C57BL/6 mouse.
   NOTE: Store the cells on ice in between injections. Gently mix target cells prior to each injection. Record the exact time of injection for each mouse, which will determine when the animal will need to be euthanized. It is important to keep the duration of in vivo cytotoxicity consistent among all animals in the same experiment.

8. Data Acquisition

1. Two or four hours after the injection of CFSE-labeled target cells, euthanize the recipient mice by cervical dislocation.   
   NOTE: The duration of in vivo cytotoxicity can vary depending on the experimental system employed, the immunogenicity of target Ags, the anticipated abundance of peptide antigen-specific T_CD8 in the spleen, and the robustness of their lytic function among other factors.
2. Remove and process each spleen separately as in steps 3.2−3.9.
3. Discard the supernatant and resuspend the pelleted cells in 3 mL of PBS.
   NOTE: Take extra care to handle the splenic tissue and cell preparations at 4 °C or on ice before cytofluorimetric analyses. This is to prevent continued cytotoxicity ex vivo.
4. Transfer approximately 1×10⁷ cells from each processed spleen into a clean FACS tube.
5. Interrogate cells immediately using a flow cytometer equipped with a 488-nm laser. Draw a lymphocyte gate based on FSC and SSC properties of the cells.
6. Identify CFSE^– recipient's splenocytes and CFSE⁺ transferred target cells. Draw additional gates accommodating distinct CFSE^low, CFSE^int, and CFSE^high target cell populations.
7. Acquire a total of 2000 CFSE^low events in the FL-1 channel.

9. Data Analysis

1. Calculate the specific lysis of each cognate target cell population using the following formula:
    % Specific cytotoxicity =  
   where x = CFSE^int/high event number in T Ag-primed mouse, y = CFSE^low event number in T Ag-primed mouse, a = CFSE^int/high event number in naïve mouse, and b = CFSE^low event number in naive mouse.         
   NOTE: In ‘three-peak' cytotoxicity assays in which the specific lysis of more than one cognate target population is evaluated, it is not appropriate to use target cell frequencies. This is simply because the frequency of a cognate target cell population is influenced not only by the percentage of the irrelevant controls but also by that of the other cognate target splenocytes. Therefore, event numbers within each gate should be used in the above formula to accurately calculate the lysis of each cognate target cell population (either CFSE^int or CFSE^high cells) against CFSE^low controls.

Table 1. Peptides introduced in this protocol.