DNA vaccines can induce impressive specific cellular immune response (IR) when taking advantage of their recognition as pathogen-associated molecular patterns (PAMP) through Toll-like receptors (TLR) expressed on/in cells of the innate immune system. Among the many types of PAMP, immunostimulatory DNA, so-called CpG motifs, was shown to interact specifically with TLR9, which is expressed in plasmacytoid dendritic cells (pDC), a key regulatory cell for the activation of innate and adaptive IR. We now report that CpG motifs, when introduced into the backbone, are a useful adjuvant for plasmid-based DNA (pDNA) vaccines to induce melanoma antigen-specific protective T cell responses in the Cloudman M3/DBA/2 model. The CpG-enriched pDNA vaccine induced protection against subsequent challenge with melanoma cells at significantly higher levels than its parental unmodified vector. Preferential induction of an antigen-specific, protective T cell response could be demonstrated by (i) induction of antigen-dependent tumor cell protection, (ii) complete loss of protection by in vivo CD4+/CD8+T cell- but not NK cell-depletion, and (iii) the detection of antigen-specific T cell responses but not of relevant NK cell activity in vitro. These results demonstrate that employing PAMP in pDNA vaccines improves the induction of protective, antigen-specific, T cell-mediated IR.
The s.c injection of tumor Ag-derived, MHC class I-binding peptides together with cationic poly-amino acids (e.g., poly-l-arginine; pR) has been shown to protect animals against a challenge with tumor cells expressing the respective peptide(s). Given our only restricted knowledge about immunogenic tumor-associated peptides, we sought to determine whether this pR-based vaccination protocol would also induce protective cancer immunity if large proteins were used instead of peptide epitopes. We found that the intracutaneous administration of the model Ag β-galactosidase (β-gal) together with pR (referred to as pR-based protein vaccine; pR-PV) was significantly more potent in protecting mice against the growth of β-gal-expressing RENCA cells than the protein alone. Coadministration of pR enhanced both the β-gal-induced specific humoral and CD8 response. The protective effect required CD8+, but neither CD4+ T lymphocytes nor β-gal-specific Abs. β-Gal priming of protective CD8+ T lymphocytes was found to be CD4+ T cell-independent, to take place within the draining lymph nodes, and to be accomplished by day 5 after vaccination. Ablation of the injection sites as early as 1.5 h after pR-PV administration still led to protection in a large proportion of the animals, indicating that certain protein Ags administered intradermally in the context of polycations are quickly transported to the draining nodes, where they induce molecular and cellular events resulting in the helper-independent priming and expansion of Tc1 cells. However, optimal protection required the prolonged presence of the injection site, suggesting that pR-PV injection facilitates the formation of a cutaneous depot of Ag-charged cells capable of migration and T cell activation.
Using the differentiation antigen Pmel17/gp100 to genetically immunize C57BL/6 mice (H-2(b)), we and colleagues noticed that only mice that had received the human homolog but not animals injected with the murine counterpart were protected against the growth of syngeneic B16 melanoma cells. The goal of this study was to determine whether the state of nonresponsiveness to the autoantigen Pmel17/gp100 can be broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. A construct containing the murine form of Pmel17 was administered intradermally to DBA/2 mice (H-2(d)), which were then investigated for the presence of Pmel17/gp100-specific immunity. We show that administration of plasmid DNA coding for the autologous melanoma-associated antigen Pmel17/gp100 protects DBA/2 mice against the growth of Pmel17-positive M3 melanoma cells but not against Pmel17-negative M3 melanoma cells or unrelated P815 mastocytoma cells. Cell depletion experiments demonstrated that this protective effect is mediated by T lymphocytes. The notion that Pmel17/gp100 represents the biologically relevant target in this system was supported by the observations (i) that recipients of Pmel17/gp100 DNA mount an antigen-specific cytotoxic T lymphocyte response and (ii) that M3 tumors growing in mice immunized with autologous Pmel17/gp100 had lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pmel17/gp100-positive. These results indicate that intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 encoding plasmid DNA can lead to protection against melanoma cells as a result of the induction of a melanoma-associated antigen-specific and protective T-cell-mediated immune response. J Invest Dermatol 115:1082-1087 2000
Subcutaneous injection of GM-CSF-expressing cancer cells into experimental animals results in protective cancer immunity. To delineate the mode of action of such vaccines, we used trinitrophenyl, the antigenic moiety of the contact allergen trinitrochlorobenzene, as surrogate Ag. Trinitrophenyl-derivatized bone marrow-derived dendritic cells were found to elicit a contact hypersensitivity response in syngeneic, but not in allogeneic recipients, compatible with their expected mode of direct Ag presentation. When expressing GM-CSF, haptenized M3 melanoma cells were also able to induce a contact hypersensitivity response but, in contrast to bone marrow-derived dendritic cells, not only in syngeneic but also in allogeneic recipients. This argues for a critical role of host APC. To identify their nature, we introduced the β-galactosidase (βgal) gene into M3-GM cells. Their administration activated βgal-specific, Ld-restricted CTL in syngeneic BALB/c mice. Evaluation of lymph nodes draining M3-GM-βgal injection sites revealed the presence of cells presenting the respective Ld-binding βgal peptide epitope. Based on their capacity to activate βgal-specific CTL, they were identified as being CD11c+ dendritic cells. These experiments provide a rational basis for the use of GM-CSF-based melanoma cell vaccines in an allogeneic setting.
We have established a model for the immunologic rejection of melanoma cells. Using a receptor-mediated, adenovirus-augmented gene delivery system (transferrinfection) we have shown that, upon transfection with an IL-2 gene construct, MHC class I+/class II- murine M-3 cells lose their tumorigenicity in both athymic and euthymic mice. More importantly, we found that these melanoma cells, which produce high levels of IL-2, can be used to induce a long-lasting anti-tumor immune response in syngeneic euthymic DBA/2 mice but not in athymic animals. This immune response, which can also be elicited by coadministration of nonmodified, irradiated M-3 cells and IL-2-transduced fibroblasts, results in the rejection of a subsequent challenge with M-3 cells or, in the elimination of preexisting M-3 cancer cell deposits. We found that transfer of T cell-enriched, but not of T cell-depleted, splenocytes from immunized mice conferred protection against M-3 cells, but not against unrelated KLN 205 cancer cells. Transfer of either CD4+ or CD8+ T cells led to only partial protection against challenge with wild-type M-3 cells. Our further observations that T cell-enriched, but not T cell-depleted splenocytes of immunized animals are capable of tumor-specific lytic activity and that this activity resides in the CD8+ cell population are compatible with the assumption that MHC class I-restricted T cell cytotoxicity is a biologically relevant effector mechanism in this model. That other mechanisms also contribute to melanoma cell destruction is evidenced by the presence of large numbers of macrophages and granulocytes in addition to T cells at the challenge sites of immunized mice.
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