CTLA-4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction Elisabetta Contardi 1 , Giulio L. Palmisano 2 , Pier Luigi Tazzari 3 , Alberto M. Martelli 4 , Federica Fala ` 4 , Marina Fabbi 5 , Tomohiro Kato 8 , Enrico Lucarelli 9 , Davide Donati 9 , Letizia Polito 10 , Andrea Bolognesi 10 , Francesca Ricci 3 , Sandra Salvi 6 , Vittoria Gargaglione 1 , Stefano Mantero 11 , Marco Alberghini 9 , Giovanni Battista Ferrara 2 and Maria Pia Pistillo 7 * 1 Department of Oncology, Biology and Genetics (DOBIG), University of Genova, Genova, Italy 2 Department of Biology, University of Genova, Genova, Italy 3 Service of Transfusion Medicine, S.Orsola-Malpighi Hospital, Bologna, Italy 4 Department of Human Anatomical Sciences, University of Bologna, Bologna, Italy 5 Laboratory of Immunopharmacology, National Cancer Research Institute, Genova, Italy 6 Laboratory of Anatomic Pathology, National Cancer Research Institute, Genova, Italy 7 Laboratory of Translational Research A, National Cancer Research Institute, Genova, Italy 8 Department of Bioregulation, Institute of Medical Sciences, St. Marianna University School of Medicine, Kanagawa, Japan 9 Istituti Ortopedici Rizzoli, Regeneration and Tissue Engineering Laboratory and Department of Surgical Pathology, Bologna, Italy 10 Department of Experimental Pathology, University of Bologna, Bologna, Italy 11 Dulbecco Telethon Institute, CNR-ITB, Milano, Italy CTLA-4 (CD152) is a cell surface receptor that behaves as a nega- tive regulator of the proliferation and the effector function of T cells. We have previously shown that CTLA-4 is also expressed on neoplastic lymphoid and myeloid cells, and it can be targeted to induce apoptosis. In our study, we have extended our analysis and have discovered that surface expression of CTLA-4 is detectable by flow cytometry on 30 of 34 (88%) cell lines derived from a vari- ety of human malignant solid tumors including carcinoma, mela- noma, neuroblastoma, rhabdomyosarcoma and osteosarcoma (but not in primary osteoblast-like cultures). However, by reverse tran- scriptase-PCR, CTLA-4 expression was detected in all cell lines. We have also found, by immunohistochemistry, cytoplasmic and surface expression of CTLA-4 in the tumor cells of all 6 osteosar- coma specimens examined and in the tumour cells of all 5 cases (but only weakly or no positivity at all in neighbouring nontumor cells) of ductal breast carcinomas. Treatment of cells from CTLA- 4-expressing tumor lines with recombinant forms of the CTLA-4- ligands CD80 and CD86 induced apoptosis associated with sequen- tial activation of caspase-8 and caspase-3. The level of apoptosis was reduced by soluble CTLA-4 and by anti-CTLA-4 scFvs anti- bodies. The novel finding that CTLA-4 molecule is expressed and functional on human tumor cells opens up the possibility of antitu- mor therapeutic intervention based on targeting this molecule. ' 2005 Wiley-Liss, Inc. Key words: CTLA-4; tumors; ligands; apoptosis Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152) is a homodimeric glycoprotein belonging to the human Ig gene superfamily originally described on the surface of murine and human activated T cells. 1 The vast majority of in vitro and in vivo studies on CTLA-4 support its negative role on T-cell activation contributing to the physiologic termination of the immune response. 2,3 CTLA-4 inhibitory function occurs upon interaction with its ligands, CD80 (B7.1) and CD86 (B7.2), expressed on anti- gen-presenting cells (APCs), resulting in inhibition of IL-2, IFN-g, IL-4 cytokines production, IL-2 receptor expression and cell cycle progression. 4,5 Several mechanisms of CTLA-4 function have been proposed including ligand competition with the positive T-cell costimulatory CD28 molecule, 6 interference of TCR signal- ling 7 and inhibition of cyclin D3 and cyclin-dependent kinases (cdk4/cdk6) production. 8 A possible function of CTLA-4 in the regulatory role of suppressor CD41CD251 T cells has generated widespread interest indicating another mechanism by which CTLA-4 might downregulate immune responses 9 and also pro- mote peripheral tolerance. 10 We and others have previously shown that CTLA-4 is also expressed on nonlymphoid cells including placental fibroblasts, 11 cultured muscle cells, 12 monocytes 13 and a variety of leukemia cells, 14 suggesting that this molecule might be involved in control- ling functions other than the widely described T-cell response inactivation. For example, maintenance of pregnancy, 15 autoim- mune myositis development 12 and regulation of monocyte func- tion 13 have been proposed. Although we have previously demon- strated that CTLA-4 is expressed on neoplastic cells of hemato- poietic origin, its expression on solid tumor-derived cells has not yet been examined. Tumor cells have developed multiple mechanisms to evade the immune system, including immunosuppressive properties and poor immunogenicity. Tumor cells can secrete soluble immuno- suppressive factors 16,17 or promote the generation of ‘‘suppres- sor’’ T cells. 18 Conversely, most tumor cells are poor antigen-pre- senting cells (APCs) due to the low (or absent) expression of HLA molecules 19 or immunostimulatory cytokines 20 as well as costimu- latory molecules 21 whose interaction with specific counter recep- tors on T cells is essential to efficiently elicit T-cell activation. 22 To increase antitumor immunity, new approaches have been developed based on combination of CTLA-4 function block- ade 23,24 with enhancement of tumor APCs function. 25 To this regard, expression of costimulatory molecules has been induced on tumor cells by gene transfer, resulting in enhanced antitumor response and tumor rejection not only in animal models but also in a number of clinical trials. 26,27 Since CTLA-4 appears to have a physiologic role in inducing downregulation in responding T cells, it might be important to determine a possible expression in tumour cells and to investigate its role in initiating and maintaining the neoplastic process. In this Grant sponsor: Ministero Istruzione Universita ` e Ricerca, Progetto Stra- tegico L449/97 Settore Oncologia D.D. 21/Ric; Grant sponsor: CIPE (02/ 07/2004, CBA project); Grant sponsor: Ministero della Salute RF2003; Grant sponsor: Fondazione Cassa di Risparmio di Bologna, Italy; AIRC. *Correspondence to: Laboratory of Translational Research A, National Cancer Research Institute, Largo R. Benzi, 10, 16132 Genova, Italy. Fax: 139-010-5737237. E-mail: mariapia.pistillo@istge.it Received 29 July 2004; Accepted after revision 17 February 2005 DOI 10.1002/ijc.21155 Published online 23 May 2005 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: BR, breast; CTLA-4, cytotoxic T-lymphocyte-associated antigen-4; FITC, fluorescein isothiocyanate; HSSCs, human stromal stem cells; IHC, immunohistochemistry; OS, osteosarcoma; PBMCs, peripheral blood mononuclear cells; PE, phycoerythrin; PHA, phytohemagglutinin; PI, propidium iodide; PMA, phorbol ester; RT-PCR, reverse transcriptase- polymerase chain reaction; scFvs, single-chain antibody fragments. Int. J. Cancer: 117, 538–550 (2005) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer