An Assay to Evaluate Immunodominance in Cytotoxic T Lymphocyte Responses
Published: March 29, 2024
Abstract
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.
Protocol
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 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% h…
Representative Results
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 (m…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
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Cite This Article
An Assay to Evaluate Immunodominance in Cytotoxic T Lymphocyte Responses. J. Vis. Exp. (Pending Publication), e22013, doi: (2024).