RESUMO
Gamma/delta T cells (Vgamma9delta2) contribute to innate immunity and exert natural cytotoxicity against a variety of tumors. Using a synthetic phosphoantigen (Bromohydrin Pyrophosphate, BrHPP), we amplified Vgamma9delta2 T cells in vitro from neuroblastoma patients. In the presence of BrHPP and low doses of IL-2, robust proliferation of Vgamma9delta2 T cells was obtained from peripheral blood mononuclear cells (PBMC) harvested at diagnosis. Moderate proliferation was observed from PBMC harvested after stem cell transplantation, whereas modest levels of Vgamma9delta2 T cells were obtained from PBMC harvested after induction therapy. Proliferation was observed after a single in vitro stimulation with BrHPP. After 21 days in culture, Vgamma9delta2 T cells represented more than 80% of cultured cells (a 50-fold expansion from baseline). Moreover, BrHPP-amplified Vgamma9delta2 T cells from patients-expressed activation markers and were able to lyse allogeneic and autologous neuroblasts. This cytotoxic activity was gammadelta T-cell receptor-dependent. Clinical trials using BrHPP are warranted in patients with poor-prognosis neuroblastoma, either to expand patient-derived Vgamma9delta2 T cells ex vivo or by direct administration to in vivo to boost the pool of resident Vgamma9delta2 T cells in vivo.
Assuntos
Antígenos de Neoplasias/imunologia , Citotoxicidade Imunológica , Difosfatos/imunologia , Neuroblastoma/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas Anticâncer , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Progressão da Doença , Humanos , Ativação Linfocitária , Estadiamento de Neoplasias , Neuroblastoma/patologia , Neuroblastoma/fisiopatologia , Prognóstico , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/patologiaRESUMO
LIGHT (TNFSF14) is a newly identified tumor necrosis factor superfamily member involved in the regulation of immune responses by control of activation, maturation, and survival of immune effector cells. Despite the immunological relevance of the LIGHT protein, little knowledge is available as to how light gene expression is regulated. In T-lymphocytes, most LIGHT surface expression and transcript accumulation occurs after T cell activation. In this study, we have shown that these events are blocked at the transcriptional level by cyclosporin A, an immuno-suppressive drug. Besides, we identified a role for Ca2+ -signaling pathways and NFAT transcription factors in T cell activation-induced LIGHT expression. To further investigate this process, we have identified, cloned, and characterized a 2.1-kilobase 5'-flanking DNA genomic fragment from the human light gene. We have shown the transcriptional activity of the herein-identified minimal 5' regulatory region of human light gene parallels the endogenous expression of light in T cells. Moreover, we demonstrated that induced LIGHT promoter activity can be equally blocked by cyclosporin A treatment or dominant negative NFAT overexpression and further identified by site-directed mutagenesis and electrophoretic mobility supershift analysis of a NFAT transcription factor binding site within the human light minimal promoter. Finally, Sp1 and Ets1 binding sites were identified and shown to regulate light basal promoter activity. Thus, the present study establishes a molecular basis to further understand the mechanisms governing human light gene expression and, consequently, could potentially lead to novel therapeutic manipulations that control the signaling cascade, resulting in LIGHT production in conditions characterized by immunopathologic activation of T cells.