RESUMO
Dendritic cell (DC)-based immunotherapeutics must induce robust CTL capable of killing tumor or virally infected cells in vivo. In this study, we show that RNA electroporated post maturation and coelectroporated with CD40L mRNA (post maturation electroporation (PME)-CD40L DC) generate high-avidity CTL in vitro that lyse naturally processed and presented tumor Ag. Unlike cytokine mixture-matured DC which induce predominantly nonproliferative effector memory CD45RA(+) CTL, PME-CD40L DC prime a novel subset of Ag-specific CTL that can be expanded to large numbers upon sequential DC stimulation in vitro. We have defined these cells as rapidly expanding high-avidity (REHA) CTL based on: 1) the maintenance of CD28 expression, 2) production of high levels of IFN-gamma and IL-2 in response to Ag, and 3) the demonstration of high-avidity TCR that exhibit strong cytolytic activity toward limiting amounts of native Ag. We demonstrate that induction of REHA CTL is dependent at least in part on the production of IL-12. Interestingly, neutralization of IL-12 did not effect cytolytic activity of REHA CTL when Ag is not limiting, but did result in lower TCR avidity of Ag-reactive CTL. These results suggest that PME-CD40L DC are uniquely capable of delivering the complex array of signals needed to generate stable CD28(+) REHA CTL, which if generated in vivo may have significant clinical benefit for the treatment of infectious disease and cancer.
Assuntos
Antígenos de Neoplasias/imunologia , Ligante de CD40/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos de Neoplasias/genética , Antígenos CD28/genética , Antígenos CD28/imunologia , Ligante de CD40/genética , Linhagem Celular Tumoral , Células Dendríticas/citologia , Eletroporação/métodos , Regulação da Expressão Gênica/genética , Humanos , Memória Imunológica/genética , Imunoterapia/métodos , Infecções/genética , Infecções/imunologia , Infecções/terapia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , RNA/genética , RNA/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/citologiaRESUMO
Dendritic cells (DC) for the immunotherapy of cancer and infectious disease require the appropriate maturation and activation signals to effectively present antigen to drive a proinflammatory response. Here we present a comparison of 4 different maturation protocols for antigen-encoded mRNA electroporated DC. Two protocols rely on cytokine-induced maturation given either preelectroporation or postelectroporation. In addition to the cytokine treatment, 2 further maturation protocols use coelectroporation of CD40L mRNA, with antigen-encoding RNA, to deliver CD40 signals. There were no significant differences in expression of costimulatory molecules such as CD80, CD83, and CD86 or the levels of expression of major histocompatibility complexes. However, results indicate that delivery of an inflammatory signal that includes interferon-gamma before the CD40 signal results in high levels of expression of interleukin-12 that was not seen in the absence of CD40L mRNA. All 4 preparations could induce expansion of primary MART-1-specific CD8+ T cells from healthy donors in vitro, but only the 2 processes receiving CD40L could induce interferon-gamma expression by those responder cells. Only DC electroporated with CD40L RNA after delivery of the inflammatory signal (PME-CD40L DC), could drive the long-term expansion of MART-1-reactive cells that displayed a CD28+/CD45RA- effector/memory phenotype with strong cytolytic activity.