Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8⁺ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Abstract

Upon viral infection, antigen-specific CD8 + cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs.Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8 + T cells and to discriminate between naïve and effector cells. Video LinkThe video component of this article can be found at https://www.jove.com/video/58680/ 9 . Also, if working with blood, animals do not need to be sacrificed and minimal amounts of sample are required. One measurement is not limited to a single antigen, but several antigens can be analyzed in one staining when combining tetramers conjugated with different fluorophores. Newly discovered antigens, e.g. from peptide screens, can easily be incorporated in tetramers and used for quantification of the T cell subset.Tetramer staining will not give information about CTL functionality (i.e., cytokine production, effector functions), but only specificity. To gain information about T cell functionality, intracellular cytokine staining (ICS) or Enzyme Linked Immuno Spot (ELISpot) Assay needs to be performed 8,10