Staining of antigen activated lymphocytes (SAAL): a highly specific method for flow cytometric quantitation of tumor-specific CD8 + T cells

Abstract

A novel method for quantitative analysis of tumor-specific CD8 + T lymphocytes was developed. Lymphocytes from mice vaccinated with tumor-associated antigens (TAAs) were expanded for 5 days in tissue culture and then stimulated in vitro for 5 h with tumor cells. They were subsequently surface-stained for CD8 and for intracellular interferon gamma (IFN-γ) and analyzed by flow cytometry. The specificity and sensitivity of this assay, staining of antigen-activated lymphocytes (SAAL), was comparable to that of surface staining with major histocompatibily class (MHC) I-peptide tetramers or of staining of peptide re-stimulated CD8 + T cells for intracellular IFN-γ. The assay did not exhibit the high background activity of traditional 51Cr-release assays that without elaborate effector cell purifications commonly fail to distinguish between T cell-mediated antigen-specific cytolysis and non-specific lysis by lymphokine-activated killer (LAK) cells. The described method, which does not require prior identification of individual TAAs and their T cell epitopes nor access to specific reagents such as MHC-peptide tetramers, represents a simple yet useful technique for studying tumor-specific cytolytic T cell responses.