In vivo OVA-specific Cytotoxic CD8+ T Cell Killing Assay

Abstract

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 (IVK). Here we describe the protocol for the lysis of cells expressing a CD8+ T cell epitope of the OVA protein (SIINFEKL). Mice are previously immunized with the OVA protein and 7 days after immunization, they receive a mix of target cells, prepared from naive C57BL/6 spleen cells pulsed with the SIINFEKL peptide and labeled with high level of CFSE and of non-pulsed control cells labeled with low level of CFSE. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSEhigh cells and CFSElow cells. The percentage of lysis is calculated by the difference between CFSE high versus low in immunized vs non-immunized mice.Measuring the ability of antigen-specific CD8+ T cell to lyse their antigen in vivo is very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.