Use of the 5′ -flanking region of the mouse perforin gene to express human Fcγ receptor I in cytotoxic T lymphocytes

Abstract

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.

Noninvasive Imaging of Cell-Mediated Therapy for Treatment of Cancer

“…Regulatory elements for other intracellular proteins specific for lymphocyte subsets have been identified and used in transgenic mouse systems for the tissue-specific expression of Cre recombinase and imaging reporter genes (117). Theoretically, the use of these regulatory elements to couple the expression of optical or PET imaging reporter genes to that of subset-specific transcription factors (118), signaling molecules such as lck (119), cytolytic granule contents such as perforin (120) and granzymes (121), or effector cell cytokines (122) such as interferon-g and interleukins (123,124) to monitor various stages of T-cell activation is possible (Fig. 11).…”

Expression in cytotoxic T lymphocytes of a single-chain anti-carcinoembryonic antigen antibody. Redirected Fas ligand-mediated lysis of colon carcinoma

“…This redirection of CTL can loosely be classified into three categories: (a) mAbmediated [1]; (b) bispecific antibody (bsAb)-mediated [2]; and (c) genetic modification of CTL with receptors reactive with TAA. Tumor recognition occurs by: scFv of antitumor mAb [3][4][5][6], ligands to TAA [7] and FcR [8,9]. The affinity between tumor antigen-ligand pairs is often very high [10], which could be an advantage; however, an appropriate antigen-ligand pair is not defined for every TAA.…”