USE OF THE 5'-FLANKING REGION OF THE MOUSE PERFORIN GENE TO EXPRESS HUMAN FC-GAMMA RECEPTOR-I IN CYTOTOXIC T-LYMPHOCYTES

Expression of the gene encoding the cytolytic granule protein perforin is restricted to cytotoxic lymphocytes. To undertake a functional ana lysis of the immediate 5'-promoter region of the mouse perforin gene, we transiently transfected mouse perforin promoter-chloramphenicol ace tyltransferase (CAT) reporter gene constructs into cytotoxic T, T lymp hoid, B-lymphoid, and nonlymphoid cell lines. The transcriptional acti vity of the perforin promoter was restricted to cytotoxic lymphocytes. The perforin promoter tvas controlled by several positive (in perfori n-positive cells) and negative (in perforin-negative cells) cis-acting regions, spread over at least 1.1 kilobases. The most specific expres sion of the CAT reporter gene in the interleukin-2-dependent cytotoxic T cell line CTLL-R8 was obtained with the mouse perforin promoter enc ompassing positions -1104 to +1 in relation to the RNA cap site. This construct expressed 65- to 7O-fold higher CAT activity than the promot erless CAT construct in perforin-expressing cells but only 1- to Ci-fo ld higher CAT activity than the promoterless construct in nonlymphoid cells. On the basis of these data, we used this most specifically acti ve mouse perforin promoter, -1104 to +1, to express in CTLL-R8, a chim eric human receptor comprising the extracellular domains of human Fc g amma RI and the transmembrane and intracellular domains of TCR zeta. S election in G418-containing medium produced CTLL-R8 transfectant clone s that (1) expressed high levels of human Fc gamma RI mRNA; (2) expres sed cell surface Fc gamma RI as demonstrated by immunoprecipitation an d their ability to bind the Fc portion of human and mouse monoclonal a ntibodies (mAbs) in an isotype-specific manner, and (3) bound RBC expr essing mucin-1. (Muc-1) peptide in the presence of a chimeric mouse-hu man anti-Muc-1 mAb. Activation of CTLL-R8 transfectants upon engagemen t of the human Fc gamma RI was evidenced by their ability to lyse tumo r target cells in an mAb isotype-dependent manner. The successful expr ession of a functional chimeric gene in CTLL-R8 suggests that the mous e perforin promoter represents a novel reagent for expressing exogenou s genes in cytotoxic T lymphocytes.