An Assay to Evaluate Immunodominance in Cytotoxic T Lymphocyte Responses

In This Article

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% heat-inactivated fetal bovine serum (FBS) in tissue culture-treated flasks at 37 °C in humidified atmosphere containing 10% carbon dioxide (CO₂).

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Results

CFSE fluorescence histogram, cell proliferation analysis, cytometry experiment, Naive-PBS-PC61 comparison.
Figure 1: Representative cytofluorimetric analysis of T_CD8-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 dif.......