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Updated: Oct 19, 2025
Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses
Published on: May 6, 2019
In-vivo gp100-specific Cytotoxic CD8+ T Cell Killing Assay
Mahdia Benkhoucha1, Nicolas Molnarfi1, Elodie Belnoue2
1Department of Pathology and Immunology, School of Medicine, University of Geneva, Geneva, Switzerland.
Insights
This study details the in vivo killing assay (IVKA) to measure antigen-specific CD8+ T cell activity against melanoma cells. The assay quantifies cytotoxic T lymphocyte (CTL) responses, crucial for anti-tumor immunity.
Area of Science:
- Immunology
- Cellular Biology
- Cancer Research
Background:
- Cytotoxic CD8+ T lymphocytes (CTLs) are vital for adaptive immunity and anti-tumor responses.
- CTLs eliminate infected or mutated cells presenting specific antigens via MHC class I.
- Quantifying CTL killing activity in vivo is essential for evaluating immune responses.
Purpose of the Study:
- To describe a protocol for the in vivo killing assay (IVKA).
- To quantify antigen-specific CD8+ T cell lysis of melanoma cells.
- To assess the adaptive cytotoxic response against tumor antigens.
Main Methods:
- The in vivo killing assay (IVKA) protocol is described.
- Target cells expressing a melanoma epitope (hgp10025-33) are prepared and labeled.
- Recipient mice receive labeled target cells, and spleen cells are analyzed by FACS to measure lysis.
Main Results:
- The IVKA protocol allows for the measurement of antigen-specific CD8+ T cell killing.
- The percentage of specific cell lysis is calculated by comparing CFSE-labeled peptide-pulsed cells to Brilliant violet-labeled control cells.
- The assay effectively quantifies the cytotoxic T lymphocyte response.
Conclusions:
- The described IVKA protocol is effective for measuring in vivo antigen-specific CD8+ T cell activity.
- This method is crucial for evaluating the adaptive cytotoxic immune response against pathogens and tumor antigens.
- The assay provides a quantitative measure of CTL-mediated cell lysis.
Abstract:
Cytotoxic CD8+ T lymphocytes (CTLs) represent a crucial component of the adaptive immune system and play a prominent role in the anti-tumor immune responses of both mice and humans. Cytotoxic CD8+ T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantify in vivo this antigen-specific CD8+ T cell killing activity, we use the in vivo killing assay (IVKA). Here, we describe the protocol for the lysis of cells expressing a CD8+ T cell melanoma epitope of the hgp10025-33 protein (KVPRNQDWL). C57BL/6 recipient mice, receive first target cells, prepared from naive congenic (CD45.1) C57BL/6 spleen cells pulsed with the hgp10025-33 peptide and labeled with CFSE and of non-pulsed control cells labeled with Brilliant violet. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSE cells and Brillant Violet (BV) cells. The percentage of lysis is calculated by the difference between CFSE versus BV. Measuring the ability of antigen-specific CD8+ T cells to lyse their antigen in vivo is very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.
