Bone marrow-derived dendritic cells pulsed with tumor homogenate induce immunity against syngeneic intracerebral glioma
Introduction
Dendritic cells (DCs) are specialized antigen-presenting cells that play a pivotal role in the induction of T and B cell immunity (Steinman, 1991). These cells have the exceptional ability to activate naive CD4+ and CD8+ T cells in vitro and in vivo. DCs require the presence of an antigen in order to initiate cytotoxic responses (Porgador and Gilboa, 1995). When the DCs are pulsed with peptide (Nair et al., 1997a), tumor homogenate (Nair et al., 1997b) or ribonucleic acid (RNA) (Boczkowski et al., 1996) they are able to elicit a primary cytotoxic T lymphocyte (CTL) response in vitro. Subsequent studies in vivo demonstrated that immunization of mice with DCs pulsed with an antigen can prime a CTL response that is tumor-specific and provides protective immunity in treated mice (Paglia et al., 1996; Porgador et al., 1996) mediated by CD4+ and CD8+ cells (Zitvogel et al., 1996). DC-based therapy is currently being evaluated in clinical trials for a variety of tumors.
A number of features limit the potential use of DC-based vaccines against primary intracerebral tumors. First, the central nervous system (CNS) has been characterized as being “immunologically privileged” based on the fact that tissue or tumor allografts survive better in the brain than in extracerebral locations (Medawar, 1948). Second, patients with malignant gliomas are in a state of immunosuppression characterized by reduced delayed-type hypersensitivity reactions to recall antigens, impaired blastogenic responses (Bullard et al., 1986) and a functional deficit of T cells (Fontana et al., 1984; Morford et al., 1997). Finally, vaccination with DCs pulsed with antigens derived from intracerebral tumors risks the inadvertent induction of a deleterious immune response against normal CNS antigens (Wikstrand and Bigner, 1981).
Subcutaneous vaccination with genetically modified cytokine-secreting tumor cells have been demonstrated to be efficacious against intracranial tumors in a murine model and supports the notion that an efficacious immune response can be generated against intracerebral tumors (Sampson et al., 1996). In order to evaluate DC-based therapies for intracerebral tumors in a model system that closely recapitulates the clinical environment in which such vaccines will be utilized, we developed a syngeneic-murine glioma that is unlike chemically induced tumor models that cannot mirror the antigenic properties of human tumors that arise “spontaneously”. The transplantable 560-glioma cell line was derived from an astrocytoma that arose spontaneously within an inbred mouse of the VM/Dk (H-2b) strain and is therefore syngeneic. Additionally, the 560 cell line secretes a biologically active form of the immunosuppressive cytokine TGF-β (Sampson et al., 1997), a cytokine that contributes to the profound immunosuppression in patients with glioblastoma multiforme.
The majority of human glioblastoma multiforme overexpress the mutated epidermal growth factor receptor (EGFRvIII) (Wikstrand et al., 1997). The mutated EGFRvIII exerts a pronounced enhancement of glioblastoma tumorigenicity by stimulating proliferation and inhibiting apoptosis (Nagane et al., 1996). Furthermore, expression of the EGFRvIII may mediate glioma cell invasion in vivo (Mishima et al., 1999). Therefore, in order to verify the veracity of our vaccine we recapitulated the human scenario of a spontaneously glioblastoma multiforme as closely as possible by transfecting our glioma cell line with the murine equivalent of the human EGFRvIII.
Within this model system we have demonstrated that systemic vaccination with bone marrow-derived DCs pulsed with 560 glioma homogenate is a safe and effective therapy for a syngeneic spontaneously arising glioma, which is highly tumorigenic, of clear astrocytic lineage and known to secrete immunosuppressive cytokines, similar to human gliomas. Vaccination with DCs pulsed with 560 glioma homogenate generated both a humoral and cell-mediated immune response and induced long-term protection against subsequent rechallenge with tumor. Finally, no evidence of autoimmune encephalomyelitis was seen on microscopic pathological examination as a result of utilizing this form of highly effective immunotherapy.
Section snippets
Tumor cell lines and animal models
The 560, 492 and 497 glioma cell lines were derived from intracerebral astrocytomas that arose spontaneously in the VM/Dk mice (Serano et al., 1980); the fibroblast cell line, 3T3, was also derived from the VM/Dk mice (H-2b). The levels of MHC I and II expression on the 560 cell lines have been previously published (Sampson et al., 1997). I. Fidler (M.D. Anderson Cancer Center, Houston, TX) provided the B16/F10 murine melanoma cell line (B16), which was derived from a spontaneously arising
DC phenotype
Prior to vaccination the phenotype of DCs was confirmed. DCs demonstrated increased expression compared with isotype controls of CD80, CD86, H-2Kb, I-Ab, and no expression of F4/80 or RB6. The DC phenotype and purity is similar to previously published reports and is demonstrated in Fig. 1.
I.p. vaccination with DCs pulsed with glioma tumor homogenate protects against subsequent intracerebral tumor challenge
To determine whether vaccination with 560EGFRvIII-pulsed DCs generated protective immunity against intracerebral tumors, VM/Dk mice (n=10 for first experiment; n=8 for repeat) received four i.p. vaccinations,
Discussion
The data presented here demonstrates that systemic immunotherapy using DCs pulsed with glioma tumor homogenates is capable of inducing an antitumor response against a syngeneic, TGF-β-secreting murine glioma within the immunologically privileged brain confirming that the CNS may not be an absolute barrier to DC-based immunotherapy. Although a limited number of previous experimental studies have been able to demonstrate that systemic vaccination can induce an efficacious response against
Acknowledgements
The authors acknowledge the expert technical assistance of Catherine McLaughlin and the kind advisement of Dr. Smita Nair and Dr. Eli Gilboa. Immunex kindly provided the GM-CSF. This work was supported by grants from American Association of Neurological Surgeons (A.B. Heimberger) and National Institute of Health CA11898 and NS20023 (D.D. Bigner). Amy B. Heimberger is the recipient of the National Institute of Health Neurooncology Research Fellowship.
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