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).

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