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

A novel method for quantitative analysis of tumor-specific CD8(+) T lymphoc ytes was developed. Lymphocytes from mice vaccinated with tumor-associated antigens (TAAs) were expanded for 5 days in tissue culture and then stimula ted in vitro for 5 h with tumor cells. They were subsequently surface-stain ed for CD8 and for intracellular interferon gamma (IFN-gamma) 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-gamm a. The assay did not exhibit the high background activity of traditional Cr -51-release assays that without elaborate effector cell purifications commo nly fail to distinguish between T cell-mediated antigen-specific cytolysis and non-specific lysis by lymphokine-activated killer (LAK) cells. The desc ribed method, which does not require prior identification of individual TAA s and their T cell epitopes nor access to specific reagents such as MHC-pep tide tetramers, represents a simple yet useful technique for studying tumor -specific cytolytic T cell responses. (C) 2000 Elsevier Science B.V. All ri ghts reserved.