Publications & Reports

Use of the 5'-flanking region of the mouse perforin gene to express human Fc gamma receptor I in cytotoxic T lymphocytes.

Smyth MJ, Kershaw MH, Hulett MD, McKenzie IF, Trapani JA
Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia.


Expression of the gene encoding the cytolytic granule protein perforin is restricted to cytotoxic lymphocytes. To undertake a functional analysis of the immediate 5'-promoter region of the mouse perforin gene, we transiently transfected mouse perforin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs into cytotoxic T, T lymphoid, B-lymphoid, and nonlymphoid cell lines. The transcriptional activity of the perforin promoter was restricted to cytotoxic lymphocytes. The perforin promoter was controlled by several positive (in perforin-positive cells) and negative (in perforin-negative cells) cis-acting regions, spread over at least 1.1 kilobases. The most specific expression of the CAT reporter gene in the interleukin-2-dependent cytotoxic T cell line CTLL-R8 was obtained with the mouse perforin promoter encompassing positions -1104 to +1 in relation to the RNA cap site. This construct expressed 65- to 70-fold higher CAT activity than the promoterless CAT construct in perforin-expressing cells but only 1- to 5-fold higher CAT activity than the promoterless construct in nonlymphoid cells. On the basis of these data, we used this most specifically active mouse perforin promoter, -1104 to +1, to express in CTLL-R8, a chimeric human receptor comprising the extracellular domains of human Fc gamma RI and the transmembrane and intracellular domains of TCR zeta. Selection in G418-containing medium produced CTLL-R8 transfectant clones that (1) expressed high levels of human Fc gamma RI mRNA; (2) expressed cell surface Fc gamma RI as demonstrated by immunoprecipitation and their ability to bind the Fc portion of human and mouse monoclonal antibodies (mAbs) in an isotype-specific manner, and (3) bound RBC expressing mucin-1 (Muc-1) peptide in the presence of a chimeric mouse-human anti-Muc-1 mAb. Activation of CTLL-R8 transfectants upon engagement of the human Fc gamma RI was evidenced by their ability to lyse tumor target cells in an mAb isotype-dependent manner. The successful expression of a functional chimeric gene in CTLL-R8 suggests that the mouse perforin promoter represents a novel reagent for expressing exogenous genes in cytotoxic T lymphocytes.


  • Journal: Journal of Leukocyte Biology
  • Published: 01/04/1994
  • Volume: 55
  • Issue: 4
  • Pagination: 514-522