Cytokine Maturation Followed by CD40L mRNA Electroporation Results in a Clinically Relevant Dendritic Cell Product Capable of Inducing a Potent Proinflammatory CTL Response

Abstract

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.

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

“…We have previously published studies showing the relative immunopotency of different DC preparations and the induction of CD8 ϩ CTL responses (36). Additional data are provided herein that support those studies by comparing T cell immunity induced using cytokine mixture-matured DC, considered current "best practice," particularly for use in the clinic, to T cell responses induced by PME-CD40L DC.…”

“…ϩ CD45RA Ϫ CTL is not dependent on IL-12 secretion from PME-CD40L DC when using targets displaying high Ag density Previously, we have shown that PME-CD40L DC produce IL-12 which may serve as a necessary third signal for CTL expansion (36). Therefore, experiments were set up to examine the role played by IL-12 in the induction of lytic activity in MART-1-specific CD28 ϩ CD45RA Ϫ CTL expanded by PME-CD40L DC.…”

“…The MART-1 Ag was chosen as a model system based on the relatively high frequency of MART-1-specific CD8 ϩ T cells present in HLA-A2 healthy donors compared with other tumor Ags (36). RNA-electroporated DC were matured using either the previously reported cytokine mixture (TNF-␣, IL-1␤, PGE 2 , and IL-6.…”

“…It has been reported that the sequential delivery of maturation signals and Ag loading is critical to generating DC with improved immunopotency (34,35). Previously, studies in our laboratory were undertaken to provide a direct head-to-head comparison of four DC preparations to determine the value of the CD40L signal to the immunopotency of the DC matured with cytokines and electroporated with Ag-encoding RNA (36). Results from these studies revealed that only the two processes receiving CD40L RNA could induce high levels of IFN-␥ by MART-1-specific CTL due to the induction of CD8 ϩ T cells with a CD28 ϩ CD45RA Ϫ phenotype.…”

“…This consists of adding TNF-␣, IFN-␥, and PGE 2 to provide proinflammatory signals to immature DC. At this stage, the post matured DC express CD80, CD86, and CD83 costimulatory molecules but are inefficient at priming strong CTL responses to electroporated RNA-encoded Ag (36). A second maturation signal is delivered by coelectroporation with RNA encoding CD40L along with the RNA Ag payload.…”