[CANCER RESEARCH 60, 6427– 6433, November 15, 2000] Superior Tumor Protection Induced by a Cellular Vaccine Carrying a Tumor- specific T Helper Epitope by Genetic Exchange of the Class II-associated Invariant Chain Peptide 1 Jeroen van Bergen, 2 Marcel Camps, Rienk Offringa, Cornelis J. M. Melief, Ferry Ossendorp, and Frits Koning Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, the Netherlands ABSTRACT Efficient loading of MHC class II molecules with a T helper epitope of choice can be achieved through genetic exchange of the MHC class II-associated invariant chain peptide (CLIP) sequence with a sequence encoding the helper peptide. We have now used this method to engineer a cellular vaccine that continuously expresses a tumor-specific helper epitope in a defined costimulatory context. We provide evidence (a) that this cellular vaccine induces peptide-specific helper T cells in vivo that are functional in protecting mice from challenge with a highly aggressive tumor, (b) that this vaccine can directly prime tumor-specific helper T cells in vivo, and (c) that this cellular vaccine is superior compared with similar cells loaded with synthetic T helper peptide in inducing tumor protection. In conclusion, cellular vaccines for activation of antigen-spe- cific helper T cells can be greatly improved by the introduction of invari- ant chain constructs containing a T helper epitope by class II-associated invariant chain peptide exchange. INTRODUCTION The activation of CD4+ helper T cells is essential to obtain specific systemic immunity. Helper T cells provide specific help to cytolytic T lymphocytes, antibody-producing B lymphocytes, and phagocytic cells. All of these forms of “help” are potentially involved in the eradication of tumor cells by the immune system (reviewed in Refs. 1–3). Thus, tumor immunotherapy protocols benefit from the con- comitant induction of tumor-specific helper T cells, even in the case of class II-negative tumors (4, 5). In fact, in two mouse models the sole induction of tumor-specific helper T cells was sufficient to protect animals from subsequent tumor challenge (5, 6). A central role for CD4 + T cells in tumor immunity emerged from studies of FMR 3 MuLV type tumors (4). Protective immunity toward the MHC class II-negative FBL tumor cell line (a Friend MuLV- induced erythroleukemia cell line) could be transferred from immune mice to naive mice by purified CD4 + T cells. In the Rauscher MuLV model, a single s.c. vaccination with a synthetic Rauscher env/gp70- derived helper peptide in IFA protected, on average, 50% of the mice against subsequent challenge with the class II-negative tumor cell line RMA (5). Optimal presentation of an epitope of choice for activation of helper T cells in vitro can be achieved by genetic exchange of CLIP with the helper peptide (7). This approach guarantees continuous and high- density expression of the T helper epitope on the surface of class II-positive APCs. We hypothesized that such a cell, provided it expresses the proper costimulatory signals, would be an efficient inducer of peptide-specific helper T cells in vivo. A well-controlled costimulatory context of the MHC class II-peptide complex is of importance, because antigen presentation in the absence of costimu- lation could cause T-cell tolerance. To this end, a cellular vaccine was created by transfecting a B-cell line expressing I-A b , CD40, CD80, and CD86 with an Ii vector encoding the Rauscher MuLV T helper epitope in the position of the CLIP sequence. Mice were injected with this cellular vaccine, and the induction of peptide-specific helper T cells as well as the induction of tumor protection was evaluated. We show that the CLIP-engineered cellular vaccine directly primes tu- mor-specific helper T cells that protect animals from a lethal tumor challenge. Moreover, the level of protection is higher than that in- duced by the same cells loaded exogenously with the synthetic helper peptide. MATERIALS AND METHODS Mice and Cell Lines. C57BL/6 (H-2 b ) mice were obtained from IFA/ CREDO (Rijswijk, the Netherlands) and bred under specific pathogen-free conditions at the Leiden University Medical Center animal facility. Female mice, 6 –10 weeks of age, were used for all experiments. The nontumorigenic 771 B-cell lymphoma was derived from a C57BL/10 (H-2 b ) mouse that had been inoculated neonatally with MCF1233 MuLV (8, 9). MCF is immunolog- ically distinct from FMR MuLV types and does not share any CTL or helper epitope with Rauscher MuLV (10). Transfectants of 771 were maintained in medium supplemented with 0.5 mg/ml hygromycin B (Boehringer Mannheim, Mannheim, Germany). RMA is a mutagenized derivative of RBL-5, a Raus- cher MuLV-induced T-cell lymphoma cell line of C57BL/6 origin (11). The 3A12 helper T-cell clone was obtained from a C57BL/6 mouse vaccinated with the MuLV env/gp70-derived helper epitope (5). The LacZ inducible T-cell hybridoma BWZ36.1x3A12 was produced from 3A12 as described previously (12). All cell lines and bulk splenocytes were cultured in Iscove’s modified Dulbecco’s medium (Bio-Whittaker Europe, Verviers, Belgium) supplemented with 5% FCS (Greiner, Frickenhausen, Germany) and penicillin (100 units/ ml), unless otherwise indicated. Genetic Constructs and Transfections. A mouse Ii cassette vector was constructed in which the CLIP sequence can be replaced with sequences of choice (13). With reverse-transcribed cDNA from the 771 cell line as a template, the regions upstream and downstream of CLIP were amplified separately using primer pairs 5'-AAACTGGATCCTAGAGCCATGGATGA- CCAACG-3'/5'-GGCATGAATTCCTTCGAAACAGGTTTGGCAGATTTC- GGAAGC-3' and 5'-CCTTGGAATTCCGGCCGATGTCCATGGATAAC- ATGCTCCTTG-3'/5'-GTCCTCTCGAGAGCTGGCCTCTGTCTTCACA-3'. The products of these PCRs were blunted and phosphorylated and subse- quently ligated into the pIc20H vector. From these plasmids, the upstream region was isolated as a BamHI/EcoRI fragment, whereas the downstream region was isolated as an EcoRI/XhoI fragment. Both fragments were ligated into the multiple cloning site of pcDNAI/Amp (Invitrogen, Leek, the Nether- lands). The resulting gene construct encodes a modified Ii, which carries unique cloning sites SfuI and EagI in place of the CLIP-encoding sequence. Double-stranded oligonucleotides with sequences encoding either CLIP (QM- RMATPLLMR) or the antigenic core of the MuLV env/gp70-derived helper peptide (SLTPRCNTAWNR) were ligated into this cassette. The sequences of these oligonucleotides were as follows: (a) CLIP, 5'-CGCAGATGCGGA- Received 2/24/00; accepted 9/20/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Pioneer Grant 900-93-001 (to J. v. B. and F. K.) from the Netherlands Organization for Scientific Research and the Netherlands Cancer Foundation Grant 97-1451 (to M. C., C. J. M. M., and F. O.). F. O. and F. K. contributed equally to this study. 2 To whom requests for reprints should be addressed. Present address: Division of Immunology, Department of Pathology, University of Cambridge, Tennis Court Road, CB2 1QP Cambridge, United Kingdom. Phone: 44-0-1223-333921; Fax: 44-0-1223- 333875; E-mail: jv228@cam.ac.uk. 3 The abbreviations used are: FMR, Friend, Moloney, Rauscher; MuLV, murine leukemia virus; Ii, invariant chain; CLIP, class II-associated invariant chain peptide; DC, dendritic cell; IFA, incomplete Freund’s adjuvant; APC, antigen-presenting cell; wt, wild-type; gag-L, gag-leader. 6427 Research. on September 28, 2021. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from