[CANCER RESEARCH 60, 3247–3253, June 15, 2000] Genetically Modified Dendritic Cells Prime Autoreactive T Cells through a Pathway Independent of CD40L and Interleukin 12: Implications for Cancer Vaccines 1 Yonghong Wan, 2 Jonathan Bramson, Andrew Pilon, Qing Zhu, and Jack Gauldie Department of Pathology and Molecular Medicine, Center for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 ABSTRACT Genetic immunization through ex vivo transduction of dendritic cells has been suggested as an effective approach to enhance antitumor immu- nity by activating both CD4  and CD8  T cells. Immunizing mice with dendritic cells transduced with an adenovirus expressing the human melanoma antigen glycoprotein 100 (DCAdhgp100) as a cancer vaccine, we demonstrated complete protective immunity and a potent CTL re- sponse against melanomas expressing murine glycoprotein 100 in a CD4  cell-dependent manner. Surprisingly, however, effective tumor rejection was not the result of cooperation between CD4  and CD8  T cells. Protective immunity was completely lost when CD4  cells were depleted immediately before tumor challenge, whereas it was unaffected by re- moval of CD8  cells, establishing a principal role for CD4  cells in the effector phase of tumor rejection. Neither protective immunity nor CTL generation in this model required interleukin 12, in spite of high levels of IFN- secretion by tumor-reactive T cells. Most notably, the DCAdhgp100 vaccine could elicit protective antitumor CD4  cells in the absence of CD40 ligand, although it does not bypass the need for CD40- mediated signals to generate melanoma-reactive CTLs. Thus, in contrast to the current thinking that the optimal cancer vaccine should include determinants for both CD4  and CD8  cells, the potency of the DCAdhgp100 vaccine appears to be a result of its ability to directly prime autoreactive CD4  cells through a process that does not require interleu- kin 12 and CD40 signals. INTRODUCTION Cancer vaccines offer the promise of a new generation of therapies using the immune system to cure tumors. This approach is expected to have the capacity to eliminate metastatic disease without the side effects of current cytotoxic approaches. Evidence that the immune system is stimulated by determinants expressed on malignant tissue comes from studies with tumor-reactive T-cell lines generated from the tumor-infiltrating lymphocyte populations and peripheral blood of diseased patients. These T-cell lines have been used to identify and clone antigenic targets on tumors, providing a foundation for the development of cancer vaccines (1, 2). One aim of vaccinologists is to build on the preexisting immune repertoire and raise the T-cell reac- tivity to a level capable of tumor rejection. Unfortunately, many potential vaccine targets in tumors are nonmutated self-proteins, and, as such, the immunization process is limited by self-tolerance (3–5). Two similar strategies have been developed to overcome self- tolerance to tumor antigen: (a) immunization with heteroclitic pep- tides (through protein engineering of key MHC/TCR contact residues; Ref. 6); and (b) xenoimmunization (using homologous proteins from different species; Refs. 7–9). Both strategies operate by activating T cells with low affinity for self-peptide using strong agonist peptide variants, which ultimately leads to cross-reactivity with the natural peptides expressed on the tumor cell. However, despite enhanced T-cell activation, tolerance to self-proteins, such as the melanoma antigen gp100, 3 is not readily overcome. Genetic vaccination of mice with the xenoantigen, hgp100 generated cross-reactive CD8 + T cells responsive to either hgp100 or murine gp100. However, whereas immunized animals could resist challenge with tumors engineered to express hgp100, they eventually succumbed to tumor cells naturally expressing murine gp100 (8, 10 –13). On the other hand, recent reports from our laboratory (14) and Kaplan et al. (15) demonstrated that vaccination using bone marrow-derived DCs genetically modified with an Ad expressing hgp100 (DCAdhgp100) could produce almost complete protective immunity in a CD4 + cell-dependent manner. These data suggested that the DC/Ad vaccine approach might activate an alternate set of autoreactive T cells, thereby offering a unique advantage for raising immunity against weak tumor antigens. Until recently, most tumor vaccines have been designed to maxi- mize the CTL response. New evidence, however, points to the central role of CD4 + T cells in directing both innate and adaptive antitumor immune responses (16 –18). In fact, a recent report has demonstrated that immunization with a CD4 + T-cell epitope alone can effectively protect mice from virally induced tumors (19). Furthermore, in addi- tion to providing “help” through paracrine cytokine secretion, CD4 + cells play a critical role in CTL priming by stimulating the antigen- presenting function of DCs through the interaction of CD40 and CD40L (20 –22). Interestingly, virus infection of DCs could bypass the requirement of CD40L for CD8 + CTL activation, but activation of CD4 + cells was still dependent on CD40L, indicating that CD40 signaling may be necessary for priming both CD4 + and CD8 + T cells (23, 24). Using DCAdhgp100 immunization against B16 murine melanoma as a model, we have investigated the role of T cells, MHC presenta- tion, IL-12 production, and CD40 signaling after immunization of genetically deficient mice. Our results indicate that the DC/Ad vac- cine directly stimulates autoreactive CD4 + T cells, leading to tumor rejection through a pathway that is independent of CD8 + T cells and activation signals from IL-12 and CD40 ligation. MATERIALS AND METHODS Animals and Cell Culture. Six- to eight-week-old C57BL/6 and BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA). CD8 -/- and CD4 -/- mice were kindly provided by Tak Mak (Ontario Cancer Institute, Toronto, Canada). IL-12 p40 -/- mice were kindly provided by Jeanne Magram (Hoffmann-La Roche, Inc.). C57BL/6J-B2m tmlUnc (2m-defi- cient; 2m -/- ), B6, 129S-H2 dlAbl-Ea (MHC class II -/- ), C57BL/6J- Tnfsf5 tmlImx (CD40L -/- ), and C57BL/6J-Lyst bg-J (beige) mice were purchased from Jackson Laboratories (Bar Harbor, ME). All the tumor cell lines were derived from C57BL/6 mice; B16F10 is a subclone of the spontaneous murine melanoma B16, MCA207 is a methylcholanthene-induced fibrosarcoma, and EL4 is a lymphoma. B16F10 and MCA207 were cultured in DMEM supple- mented with 10% fetal bovine serum, 2 mML-glutamine, 100 units/ml peni- cillin, and 100 g/ml streptomycin. EL4 cells were cultured in RPMI 1640 supplemented with the same additives as described above. Received 1/7/00, accepted 4/17/00. 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 in part by funds from the Medical Research Council of Canada, the Hamilton Health Science Corporation, and St. Joseph’s Hospital. 2 To whom requests for reprints should be addressed, at the Department of Pathology and Molecular Medicine, Center for Gene Therapeutics, McMaster University, HSC/ 4H21B, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. 3 The abbreviations used in this paper are: gp, glycoprotein; hgp100, human glyco- protein 100; DC, dendritic cell; Ad, adenovirus, 2m, 2 microglobulin; PKO, perforin knockout; IL, interleukin; CD40L, CD40 ligand; NK, natural killer; MAb, monoclonal antibody; ATCC, American Type Culture Collection; GM-CSF, granulocyte macrophage colony-stimulating factor; Th, T helper; NKT, natural killer T. 3247 on July 1, 2015. © 2000 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from