Source: Choi, J. et al., Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses. J. Vis. Exp. (2019)
The video showcases a flow cytometry-based assay for assaying immunodominance in cytotoxic T lymphocytes or CTL responses. Injected fluorescent target cells exhibit varying fluorescence corresponding to immunogenic epitopes. Post-spleen collection and sorting reveal a reduction in high-intensity fluorescent cells, confirming immunodominance in CTL 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
2. Treatment Regimens
3. Preparation of Target Splenocytes
4. Coating Target Splenocytes with Irrelevant and Cognate Peptides
5. Labeling target splenocytes with CFSE
6. Examination of Adequate/Equal CFSE Labeling of Target Splenocyte Populations
7. Injection of CFSE-labeled Target Cells into Naïve and T-Ag-primed Recipients
8. Data Acquisition
9. Data Analysis
Table 1. Peptides introduced in this protocol.
Protein Antigen Source | Peptide Epitope | Designation | Sequence | MHC I Restriction |
SV401 Large T Ag2 | T Ag206-215 | Site I | SAINNYAQKL | H-2Db |
SV40 Large T Ag | T Ag223-231 | Site II/III | CKGVNKEYL | H-2Db |
SV40 Large T Ag | T Ag404-411 | Site IV | VVYDFLKC | H-2Kb |
SV40 Large T Ag | T Ag489-497 | Site V | QGINNLDNL | H-2Db |
HSV-13 Glycoprotein B | gB498-505* | gB498-505 | SSIEFARL | H-2Kb |
LCMV4 Glycoprotein | GP33-41* | GP33-41 | KAVYNFATC | H-2Db |
1Simian Virus 40 | ||||
2Large Tumor Antigen | ||||
3Herpes Simplex Virus type 1 | ||||
4Lymphocytic Choriomeningitis Virus | ||||
*used as an irrelevant peptide |
Figure 1: Representative cytofluorimetric analysis of TCD8-mediated cytotoxicity against T Ag-derived epitopes in the presence or absence of nTreg cells. Target splenocytes pulsed with control peptides, site I or site IV, which were differentially labeled with CFSE, were tracked by flow cytometry in the spleen of a naïve mouse (left panel), a PBS-injected T Ag-primed mouse (middle panel), and a PC61 (nti-CD25)-injected (nTreg-depleted) T Ag-primed mouse (right panel). Percent specific killing of target cells was calculated using the formula described in the protocol, and representative numbers are shown. This figure is adopted, with permission, from Haeryfar et al. Copyright 2005. The American Association of Immunologists, Inc.
Figure 2: In vivo cytotoxicity of T Ag-specific TCD8 in anti-PD-1-treated mice. (A) Representative histogram plots demonstrate CFSE peaks corresponding to target splenocytes pulsed with an irrelevant peptide (CFSElow), site II/III (CFSEint), and site I (CFSEhigh) in T Ag-primed mice that received an isotype (left panel) or a PD-1-blocking mAb (right panel). (B) Percent specific killing of each cognate target cell population was calculated using CFSE+ event numbers in T Ag-primed mice (n = 3 per group) and naïve recipients (not shown) and the formula described in the protocol. Error bars represent standard errors of the mean (SEM), and ** denotes a statistical difference with p < 0.01 by unpaired Student's t-tests. This figure is adopted, with permission, from Memarnejadian et al. Copyright 2017. The American Association of Immunologists, Inc.
The authors have nothing to disclose.
0.25% Trypsin-EDTA (1X) | Thermo Fisher Scientific | 25200-056 | |
ACK Lysing Buffer | Thermo Fisher Scientific | A1049201 | |
Anti-mouse CD25 (clone PC-61.5.3) | Bio X Cell | BE0012 | |
Anti-mouse PD-1 (clone RMP1-14) | Bio X Cell | BE0146 | |
CFSE | Thermo Fisher Scientific | C34554 | |
DMEM (1X) | Thermo Fisher Scientific | 11965-092 | |
Fetal bovine serum (FBS) | Wisent Bioproducts | 080-150 | Heat-inactivate prior to use |
GlutaMAX (100X) | Thermo Fisher Scientific | 35050-061 | |
HEPES (1M) | Thermo Fisher Scientific | 15630080 | 10 mM final concentration |
MEM Non-Essential Amino Acids Solution (100X) | Thermo Fisher Scientific | 11140-050 | |
Penicillin/Streptomycin | Sigma-Aldrich | P0781 | Stock is 100X |
Rat IgG1 (clone KLH/G1-2-2) | SouthernBiotech | 0116-01 | Isotype control |
Rat IgG1 (clone HRPN) | Bio X Cell | BE0088 | Isotype control |
Rat IgG1 (clone TNP6A7) | Bio X Cell | BP0290 | Isotype control |
Rat IgG2a (clone 2A3) | Bio X Cell | BP0089 | Isotype control |
RPMI 1640 (1X) | Thermo Fisher Scientific | 11875-093 | |
Sodium Pyruvate (100 mM) | Thermo Fisher Scientific | 11360-070 | 1 mM final concentration |