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.
Keywords: Cytotoxic T cells, CD8 T cells, In vivo killing assay, Antigen specific CTL, Tumor immunity, Cancer
Background
The advantage to use the two types of dye (CFSE and BV) is for their high stability in vivo, both dyes could be detectable even more than one week in vivo (based on our lab experience), on the other hand, as we used Galios cytometer, CFSE and BV are read respectively in fl1 vs. fl9 (fl1 and fl9 are two different lasers used by the FACS machine) which is very practical for the settings and compensations.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
A similar result was obtained when target cells pulsed with the hgp100 peptide were stained with BV and target cells not pulsed with any peptide were stained with CFSE.
Recipes
Acknowledgments
This work was supported by grants to P.H.L. from the Swiss National Science Foundation (No. 310030_153164) and the SMSS.
Competing interests
The authors declare no conflicts of interest or competing interests
Ethics
All mice were 8-10 weeks old. Animals were housed in a specific pathogen-free barrier facility at the University Medical Center, Faculty of Medicine (Geneva, Switzerland). All experimental protocols and procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Geneva University School of Medicine. Animal care and experimental procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Geneva University School of Medicine.
References
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