Staining of antigen activated lymphocytes (SAAL): a highly specific method for flow cytometric quantitation of tumor-specific CD8(+) T cells.
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-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-gamma. 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.
The Wistar Institute, Philadelphia, PA 109104, USA.
D W
DW
Kowalczyk
A P
AP
Wlazlo
W
W
Giles-Davis
H C
HC
Ertl
D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology*; D019496:Cancer Vaccines / Q000276:immunology*; D002860:Chromium Radioisotopes; D003601:Cytotoxicity Tests, Immunologic; D005434:Flow Cytometry / Q000379:methods*; D008213:Lymphocyte Activation; D051379:Mice; D013194:Staining and Labeling / Q000379:methods; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D002860:Chromium Radioisotopes