B7-1, IFN AND ANTI-CTLA-4 CO-OPERATE TO PREVENT T-CELL TOLERIZATION DURING IMMUNOTHERAPY AGAINST A MURINE T-LYMPHOMA Jo A. VAN GINDERACHTER 1 * , YuanQing LIU 1 , Anja B. GELDHOF 1 , Lea BRIJS 1 , Kris THIELEMANS 2 , Patrick DE BAETSELIER 1 AND Geert RAES 1 1 Department of Cellular Immunology, Flanders Interuniversity Institute For Biotechnology, Vrije Universiteit Brussel, Sint-Genesius-Rode, Belgium 2 Laboratory of Hematology, Academic Hospital, Vrije Universiteit Brussel, Jette, Belgium We previously reported on a murine T lymphoma cell line, BW-Sp3, with inherent immunogenicity. BW-Sp3 tumors can elicit an anti-tumor CD8  CTL response capable of me- diating a regression of subcutaneous tumors. However, this immune response is inadequate to eliminate cancer cells completely in a significant percentage of the recipients, re- sulting in progressing tumors. In this tumor model, tumor progression correlated with a tolerization of tumor-reactive T cells and cellular immunotherapy of tumor bearing ani- mals, with or without B7-mediated costimulation, even in- creased tumor progression (Raes et al, 1998). In the present study, we investigated whether the co-expression of IFN, together with B7-1, could have beneficial effects on immu- notherapy. Although immunotherapy with IFN and B7-1 single transfectants tended to tolerize anti-tumor T-cells and consequently increased tumor growth, the B7-1/IFN double transfectants resulted in a more beneficial outcome. This phenomenon correlated with an increased CTL-inducing po- tential of the double transfectants. Secondly, we wondered whether CTLA-4 signalling was involved in the down-regula- tion of the anti-tumor response. Indeed, when immunother- apy was provided along with anti-CTLA-4, the protection by B71/IFN double transfectants was further improved and the tumor-promoting effect of BW-Sp3(B7-1) was compensated for. Our results indicate that B7-1, IFN and the blockade of CTLA-4 cooperate to tilt the balance in favour of tumor elimination, while either factor alone fails to do so or even promotes tumor growth. Int. J. Cancer 87:539 –547, 2000. © 2000 Wiley-Liss, Inc. CD8 + cytotoxic T-lymphocytes are considered to be a major cell population involved in the specific anti-tumor response elic- ited by immunogenic tumors. It has been widly accepted that at least two distinct and combined signals are required for the initi- ation of an optimal T-cell response. “Signal one” consists of the interaction between an antigen-specific receptor on T-cells and antigenic peptide-MHC complexes on cancer cells or professional antigen-presenting cells (APC). The second signal is delivered by costimulatory molecules through their counter-receptors on the T-cells. Engagement of the T-cell receptor in the absence of costimulation may even cause unresponsiveness or anergy (Muel- ler et al, 1989). It has now become clear that the major costimu- latory receptor on T-cells is CD28 and that its ligands on APCs are B7-1 (CD80) and B7-2 (CD86) (Allison, 1994). However, B7 molecules are usually not expressed on the majority of human and murine cancers. Hence, even if a cancer cell expresses normal levels of MHC class I antigens and presents tumor-associated antigenic peptides, the lack of costimulatory signals may prevent them from inducing CTL responses directly. Transfection of either the B7-1 or B7-2 gene into immunogenic but not non-immuno- genic cancer cells has resulted in the induction of protective anti-tumor T cell responses in several murine models (Allison et al, 1995). Engineering cancer cells to secrete immunostimulatory cyto- kines represents another approach to enhance tumor immunoge- nicity. A multitude of cytokines has been used for this purpose (Colombo and Forni, 1994). In this context, IFN may directly activate tumor-reactive CTLs and antigen-presenting cells, but can also potentiate the antigen presenting capacity of the tumor cells themselves, by inducing the MHC class I antigen expression (Wallach et al., 1982) and reverting antigen processing defects (Sibille et al., 1995). Recently, blocking CTLA-4 signalling was shown to be a novel approach to promote tumor rejection (Leach et al., 1996). The rationale behind this comes from the concept that CTLA-4, a second T-cell receptor for B7, is a negative regulator for T-cell activation (Krummel and Allison, 1995). Several mechanisms may account for the T-cell attenuating role of CTLA-4. Engagement of CTLA-4 induces TGF- production by murine CD4 + T cells, which in turn partially suppresses the T cell proliferative response (Chen et al., 1998). On the other hand, upon TCR ligation and CD28 costimulation, CTLA-4 associates with the CD3 chain and recruits the tyrosine phosphatase SHP-2, which then antagonizes TCR signal transduction (Lee et al., 1998). Inherent immunogenicity of the cancer cells is a prerequisite for the successful application of immune-based therapies. However, the applicability of anti-cancer immunotherapy can be hampered by the existence of tumor-induced antigen-specific T-cell tolerance (Sotomayor et al., 1996). We focused on the immunogenic murine BW-Sp3 T-cell lymphoma, which induces T-cell tolerance in a significant percentage of mice. In this model, T-cell tolerization is exacerbated when applying cell-based immunotherapy with B7-1 transfectants (Raes et al., 1998). IFN expressing BW-Sp3 cells were documented to be highly immunogenic, forming no visible tumors at all upon s.c. inocula- tion and eliciting strong CTL-responses against the parental cells (Raes et al., 1995). Therefore, in the present study, we wanted to evaluate the use of IFN transfected cells, either alone or in combination with B7-1, in immunotherapy. Secondly, the possible role of CTLA-4 mediated negative signalling in the tumor-promot- ing effect of immunotherapy with BWSp3(B7-1) cells was exam- ined. Our results will demonstrate that a combination of B7-1 costimulation, IFN immunogenization and CTLA-4 blockade reduces the tumor-promoting effects of cell-based immunotherapy in nearly all animals, suggesting the need for multiple immunos- timulatory signals to prevent a shutdown of the anti-tumor re- sponse by highly immunogenic tumors. MATERIAL AND METHODS Media and culture conditions All cancer cell lines were cultured in RPMI complete medium: RPMI-1640 medium supplemented with 10% heat-inactivated fe- Jo A. Van Ginderachter and YuanQing Liu contributed equally to this work. *Correspondence to: Department of Cellular Immunology, Flanders Interuniversity Institute for Biotechnology, IMOL, Vrije Universiteit Brus- sel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium. Fax: 0032-2- 359.03.59. E-mail: jvangind@vub.ac.be Received 18 October 1999; Revised 10 March 2000; Accepted 24 March 2000 Int. J. Cancer: 87, 539 –547 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer