514 Journal of Leukocyte Biology Volume 55, April 1994 Use of the 5’ -flanking region of the mouse perform gene to express human Fcy receptor I in cytotoxic T lymphocytes Mark J. Smyth, Michael H. Kershaw, Mark D. Hulett, Ian F.C. Mckenzie, and Joseph A. Trapani Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelber& Victoria, Australia Abstract: Expression of the gene encoding the cytolytic granule protein perform is restricted to cytotoxic lym- phocytes. To undertake a functional analysis of the im- mediate 5 ‘ -promoter region of the mouse perform gene, we transiently transfected mouse perform promoter-chlo- ramphenicol acetyltransferase (CAT) reporter gene con- structs into cytotoxic T, T lymphoid, B-lymphoid, and nonlymphoid cell lines. The transcriptional activity of the perform promoter was restricted to cytotoxic lympho- cytes. The perform promoter was controlled by several positive (in perform-positive cells) and negative (in perform-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 cyto- toxic T cell line CTLL-R8 was obtained with the mouse perform 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 perform-expressing cells but only 1- to 5-fold higher CAT activity than the promoterless con- struct in nonlymphoid cells. On the basis of these data, we used this most specifically active mouse perform promoter, -1104 to + 1, to express in CTLL-R8, a chi- meric human receptor comprising the extracellular do- mains of human Fc-yRI and the transmembrane and in- tracellular domains of 1XJR . Selection in G418-containing medium produced CTLL-R8 transfectant clones that (1) expressed high levels of human FcyRI mRNA; (2) cx- pressed cell surface Fc-yRI as demonstrated by im- munoprecipitation and their ability to bind the Fc por- tion of human and mouse monoclonal antibodies (mAbs) in an isotype-specific manner, and (3) bound RBC cx- pressing mucin-1 (Muc-1) peptide in the presence of a chi- meric mouse-human anti-Muc-1 mAb. Activation of CTLL-R8 transfectants upon engagement of the human Fc7RI was evidenced by their ability to lyse tumor target cells in an mAb isotype-dependent manner. The success- ful expression of a functional chimeric gene in CTLL-R8 suggests that the mouse perform promoter represents a novel reagent for expressing exogenous genes in cytotoxic T lymphocytes. J. Leukoc. Biol. 55: 514-522; 1994. Key Words: cytotoxic T lymphocyte . immunoglobulin G . 5’ promoter . transcriptional control chimeric . CAT INTRODUCTION Perform is one of the effector molecules that participates in the granule exocytosis mechanism of target cell lysis by cyto- toxic T lymphocytes (CTLs) and large granular lymphocytes (LGLs) [1, 2]. Perform is located in specialized cytolytic granules that are vectorially secreted onto the target cell dur- ing the effector-target cell interaction. Perform release in the presence of calcium ions enables polymerization in the target cell membrane leading to the formation of transmembrane pores and often subsequent osmotic lysis. Although perform plays a central role in some forms of lymphocyte-mediated lysis, the expression, rather than the function of perform, has been most comprehensively studied. A number of studies highlight a correlation between perform expression and cyto- lytic activity in lymphocyte populations [3-10]. In vitro and in vivo, perform mRNA and protein expression is tightly regulated and specific for CTLs and LGLs [6-10]. Perform gene expression has not been detected in B cells, monocyte- or macrophage-like cells, polymorphonuclear leukocytes, or nonleukocyte cells [1, 6, 7]. To date, all class I-restricted CD8 CTL clones, natural killer (NK) cell clones, and some class Il-restricted CD4 CTL clones express perform [6, 7, 11]. Analysis ofhuman peripheral blood lymphocytes (PBLs) reveals a tight regulation of perform expression. Perform ex- pression is constitutive in unstimulated CD3 LGLs, T cells, and CD8 CDi1b T cells [1, 3, 4], but can be induced by interleukin-2 (IL-2) in CD8 CD11b T cells indepen- dently of T cell receptor (‘TCR)-CD3 ligation [3]. In addi- tion, IL-6 may costimulate the induction of perform tran- scription in CD8 T cells by low doses of IL-2 ( > 5 U/ml) [5]. Although resting CD4 T cells do not express signal- transducing IL-2 receptor or respond to IL-2 alone, CD4 T cells can express perform in response to TCR-CD3 ligation (via anti-CD3 mAb) and IL-2 [12]. In vivo, perform gene ex- pression has been detected in CD3 LGLs and CD3 CTLs responding to viral, transplantation, and tumor antigen and in T cells during autoimmune disease [8-10, 13, 14]. Tran- scriptional control appears to be partially responsible for the regulated expression of perform in CTLs [15]. We and others [16-18] have previously elucidated the genomic organization of human and mouse perform and characterized the mouse perform promoter and upstream region. Analysis of the first 1200 base pairs of the promoter revealed six regions within 0.8 kilobases of the RNA cap site that were highly conserved (up to 87% sequence identity) between human and mouse perform genes. However, most of the conserved sequences lacked consensus to recognized eu- Abbreviations: BLT, a-N-benzyloxycarbonyl-i.-lysine-thiobenzylester; CAT, chloramphenicol acetyltransferase; cBC2, chimeric BC2; CTL, cyto- toxic T lymphocyte; Hu-FcR-yRI, human Fe receptor I for IgG; IL-2R, interleukin-2 receptor; LAK, lymphokine-activated killer; LGL, large granular lymphocyte; mAb, monoclonal antibody; Muc-1, mucin-l; PBL, peripheral blood lymphocyte; PBS, phosphate-buffered saline; PCR, poly- merase chain reaction; SP, serine protease; SDS-PAGE, sodium dodecyl sul- fate-polyacrylamide gel electrophoresis; TCR, T cell receptor. Reprint requests: Mark J. Smyth, Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, 3084 Victoria, Aus- tralia. Received September 15, 1993; accepted November 29, 1993.