OPTIMIZATION OF A PEPTIDE-BASED PROTOCOL EMPLOYING IL-7 FOR IN-VITRO RESTIMULATION OF HUMAN CYTOTOXIC T-LYMPHOCYTE PRECURSORS

A variety of different methods for the in vitro restimulation of human cytotoxic T lymphocyte (CTL) precursors (CTLp) are in use. Our aim wa s to enhance the detection of circulating human CTLp in peripheral blo od. We have developed a standardized and highly efficient method for r estimulating CTLp. Synthetic peptides were used to restimulate cognate CTLp from peripheral blood mononuclear cells (PBMC), acid effector CT L capable of lysine peptide-pulsed and virus infected targets were gen erated. The effects of several parameters on CTL specific for influenz a A, EBV and HIV-1 were evaluated, and the optimum peptide concentrati on for CTL generation was established. Supplementation of initial cult ures with IL-7 greatly enhanced peptide-specific lyric activity for al l peptides tested and the dose-response relationship for IL-7 was deli neated. A novel technique using peptide-MHC class I molecule tetramers to stain T cells bearing cognate T cell receptors permitted enumerati on of antigen-specific CD8 + CTL during in vitro restimulation; IL-7 s upplementation selectively expanded the population of peptide-specific CD8 + CTL. Importantly, this protocol, whilst enhancing the restimula tion and lyric activity of secondary CTL, does not induce primary CTL in vitro. The improved efficiency with which CTL are generated in this system substantially enhances the sensitivity of CTL culture and the Cr-51 release assay to detect low levels of CTL activity. (C) 1997 Els evier Science B.V.