Flow cytometric enumeration of antigen-specific T lymphocytes.

Abstract

Until the second half of the 1990s, quantification of MHC-restricted, peptide-specific T lymphocytes required cell culture– based techniques of mononuclear cell suspensions that were often laborious, cumbersome, and prone to bias towards cellular subsets with growth advantage. Initially, limiting dilution assays were used. These assays include multiple days of culturing T lymphocytes in the presence of antigen and antigen-presenting cells, and the functional read-outs produced were T-cell proliferation (1) or cytolytic activity (2). However, such assays may not accurately reflect the function and frequency of T cells in vivo, due to selective proliferation and apoptosis that occur over time in culture. Alternatively, assays for T-cell activation have been developed based on the upregulation of the early activation marker CD69 (3) and/or the production of cytokines upon specific stimulation. The cytokines thus being secreted by the T cells can be measured