IN-VITRO INDUCTION OF PRIMARY, ANTIGEN-SP ECIFIC CTL FROM HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS STIMULATED WITH SYNTHETIC PEPTIDES

A protocol for in vitro induction of primary, antigen-specific CTL fro m human peripheral blood mononuclear cells (PBMCs) was developed. Anti gen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAG-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which cou ld be subsequently stabilized with peptide and beta(2)-microglobulin ( beta(2)m) SAC-I activated PBMCs from HLA-A2.1 normal donors loaded wit h HBV core 18-27 peptide following acid treatment were used to stimula te PBMCs depleted of CD4 + T cells, in the presence of recombinant int erleukin-7 (rIL-7). After 12 days, cells were restimulated with autolo gous, peptide-pulsed, adherent cells and tested for CTL activity 7 day s later. In 23 independent experiments from 13 different HLA-A2.1 dono rs, this protocol resulted in induction of primary CTL more than 90 % of the time. As indicated by both the frequency and magnitude of the r esponse against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen pe r cent of the cultures generated were capable of lysing target cells t ransfected with the HBV core antigen and, in general, these CTL cultur es exhibited high avidity for the HBV core peptide. This protocol is g enerally applicable to different antigens and class I alleles, and thu s, may be utilized to screen large numbers of peptides to identify hum an CTL epitopes.