RESUMEN
BACKGROUND: Cancer vaccines reproducibly cure laboratory animals and reveal encouraging trends in brain tumor (glioma) patients. Identifying parameters governing beneficial vaccine-induced responses may lead to the improvement of glioma immunotherapies. CD103(+) CD8 T cells dominate post-vaccine responses in human glioma patients for unknown reasons, but may be related to recent thymic emigrant (RTE) status. Importantly, CD8 RTE metrics correlated with beneficial immune responses in vaccinated glioma patients. METHODS: We show by flow cytometry that murine and human CD103(+) CD8 T cells respond better than their CD103(-) counterparts to tumor peptide-MHC I (pMHC I) stimulation in vitro and to tumor antigens on gliomas in vivo. RESULTS: Glioma responsive T cells from mice and humans both exhibited intrinsic de-sialylation-affecting CD8 beta. Modulation of CD8 T cell sialic acid with neuraminidase and ST3Gal-II revealed de-sialylation was necessary and sufficient for promiscuous binding to and stimulation by tumor pMHC I. Moreover, de-sialylated status was required for adoptive CD8 T cells and lymphocytes to decrease GL26 glioma invasiveness and increase host survival in vivo. Finally, increased tumor ST3Gal-II expression correlated with clinical vaccine failure in a meta-analysis of high-grade glioma patients. CONCLUSIONS: Taken together, these findings suggest that de-sialylation of CD8 is required for hyper-responsiveness and beneficial anti-glioma activity by CD8 T cells. Because CD8 de-sialylation can be induced with exogenous enzymes (and appears particularly scarce on human T cells), it represents a promising target for clinical glioma vaccine improvement.
Asunto(s)
Antígenos CD/inmunología , Neoplasias Encefálicas/terapia , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/farmacología , Células Dendríticas/inmunología , Glioma/terapia , Cadenas alfa de Integrinas/inmunología , Animales , Antígenos CD/metabolismo , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/inmunología , Femenino , Glioblastoma/inmunología , Glioblastoma/metabolismo , Glioblastoma/terapia , Glioma/inmunología , Glioma/metabolismo , Humanos , Inmunoterapia Adoptiva/métodos , Cadenas alfa de Integrinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuraminidasa/metabolismo , Neuraminidasa/farmacología , Sialiltransferasas/metabolismo , Sialiltransferasas/farmacología , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding beta-galactosidase (beta-gal) driven by a TetOn system containing the rtTAS(s)M2 transactivator and the tTS(Kid) repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-beta-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-beta-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-beta-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-beta-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.
Asunto(s)
Adenovirus Humanos/fisiología , Antígenos Virales/metabolismo , Vectores Genéticos , Tetraciclina/farmacología , Transgenes/fisiología , Adenovirus Humanos/genética , Animales , Antígenos Virales/genética , Regulación de la Expresión Génica , Humanos , Ratas , Transgenes/efectos de los fármacos , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Glioblastoma multiforme is an intracranial tumor that has very poor prognosis. Patients usually succumb to their disease 6 to 12 months after they are diagnosed despite very aggressive treatment modalities. We tested the efficacy of a potent differentiation and proliferation factor for the professional antigen-presenting dendritic cells (DCs), i.e., Flt3L, for its potential role as a novel therapy for gliomas. We investigated the ability of recombinant adenoviral vectors encoding human soluble Flt3L (hsFlt3L) to improve the survival of Lewis rats bearing intracranial syngeneic CNS-1 gliomas. We show that RAdhsFlt3L can improve survival in a dose-dependent manner. Seventy percent of rats survive when treated with 8 x 10(7) pfu RAdhsFlt3L (P < 0.0005). In addition we demonstrate in both naive Lewis rats and C57BL/6 mice the presence of increased numbers of cells bearing DC markers (OX62 and MHCII, in rats, or CD11C, 33D1, MHCII, and F4/80, but not DEC205, in mice) in sites of brain delivery of RAdhsFlt3L. These results show that expression of hsFlt3L in the brain leads to the presence of cells displaying DC markers. We demonstrate that treatment with hsFlt3L leads to inhibition of tumor growth and significantly increased life span of animals implanted with syngeneic CNS-1 glioma cells. Animals that had survived for long periods, i.e., 6 months, had eliminated the implanted tumors after neuropathological analysis; on the other hand, some of the 3-month survivors still appeared to harbor brain tumors. Our results have profound implications for immune-mediated brain tumor therapy and also suggest the ability to recruit DC-like cells within the brain parenchyma in response to the local expression of Flt3L from adenoviral vectors.
Asunto(s)
Adenoviridae/genética , Neoplasias de los Nervios Craneales/genética , Neoplasias de los Nervios Craneales/terapia , Glioma/genética , Glioma/terapia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Animales , Neoplasias de los Nervios Craneales/metabolismo , Neoplasias de los Nervios Craneales/patología , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Terapia Genética , Glioma/metabolismo , Glioma/patología , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Trasplante de Neoplasias , Ratas , Solubilidad , Tasa de Supervivencia , Factores de TiempoAsunto(s)
Adenoviridae/genética , Encefalopatías/terapia , Terapia Genética/métodos , Vectores Genéticos , Animales , HumanosRESUMEN
Gene therapy, which entails the use of nucleic acids as drugs, is a new approach to treat disease. Gene therapy has been successfully implemented in several preclinical animal models, including several paradigms of experimental pituitary tumors. In spite of these successes, several critical issues need to be addressed before gene therapy can become a clinical reality for the treatment of pituitary tumors. These include the development of safer and more effective gene delivery vectors, the uncovering of novel therapeutic targets, the development of molecular switches which will allow turning therapeutic transgene expression "on" and "off" as and when it is needed, and the ability to scale up the vector preparations devoid of any putative contaminants. There are still many basic science developments that must take place in order to allow this new therapeutic technology to make its way successfully into the clinical arena to treat pituitary disease. We envisage these developments taking place within the next five years, gene therapy for pituitary tumors will then form part of the armamentarium available to better treat and manage pituitary tumors.