Potency of mature CD40L RNA electroporated dendritic cells correlates with IL-12 secretion by tracking multifunctional CD8(+)/CD28(+) cytotoxic T-cell responses in vitro.

Electroporation of mature dendritic cells (DC) with RNA-encoding CD40L greatly enhances the production of interleukin (IL)-12, a proinflammatory cytokine necessary for the induction of T-cell immunity. Results presented herein reveal a correlation between the priming of CD28(+) antigen-reactive effector memory cytotoxic T lymphocytes (CTL) displaying 3 or 4 simultaneous effector functions and the quantity of IL-12 produced by postmaturation electroporation-CD40L DC. By using multiparameter flow cytometry, the quantities of IL-12 needed to prime naive antigen-reactive T cells to simultaneously produce interferon-γ and tumor necrosis factor-α in the presence or absence of IL-2 secretion in conjunction with lytic activity defined by CD107a expression can be used to determine the overall potency of a DC product.

The immunosuppressive properties of the HIV Vpr protein are linked to a single highly conserved residue, R90.

Background: A hallmark of AIDS progression is a switch of cytokines from Th1 to Th2 in the plasma of patients. IL-12, a critical Th1 cytokine secreted by antigen presenting cells (APCs) is suppressed by Vpr, implicating it as an important virulence factor. We hypothesize that Vpr protein packaged in the virion may be required for disabling APCs of the first infected mucosal tissues.

Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion.

Background: RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade.

Priming of a novel subset of CD28+ rapidly expanding high-avidity effector memory CTL by post maturation electroporation-CD40L dendritic cells is IL-12 dependent.

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.

Cytokine maturation followed by CD40L mRNA electroporation results in a clinically relevant dendritic cell product capable of inducing a potent proinflammatory CTL response.

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.

Multiplex RT-PCR amplification of HIV genes to create a completely autologous DC-based immunotherapy for the treatment of HIV infection.

Background: Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens.

Methodology/principal Findings: The present study describes a novel approach for RT-PCR amplification of HIV antigens.

Nonmyeloablative conditioning is sufficient to allow engraftment of EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic skin grafts in mice.

Immunologic reactions against gene therapy products may prove to be a frequent problem in clinical gene therapy protocols. Enhanced green fluorescence protein (EGFP) is commonly used as a marker in gene transfer protocols, and immune responses against EGFP-expressing cells have been documented. The present study was designed to investigate the effect of a pharmacologic, nonmyeloablative, conditioning regimen on the development of EGFP+ donor/recipient mixed bone marrow chimerism and ensuing tolerance to EGFP-expressing transplants.