Soluble CD83 Inhibits T Cell Activation by Binding to the TLR4/MD-2 Complex on CD14+ Monocytes.

The transmembrane protein CD83, expressed on APCs, B cells, and T cells, can be expressed as a soluble form generated by alternative splice variants and/or by shedding. Soluble CD83 (sCD83) was shown to be involved in negatively regulating the immune response. sCD83 inhibits T cell proliferation in vitro, supports allograft survival in vivo, prevents corneal transplant rejection, and attenuates the progression and severity of autoimmune diseases and experimental colitis. Although sCD83 binds to human PBMCs, the specific molecules that bind sCD83 have not been identified. In this article, we identify myeloid differentiation factor-2 (MD-2), the coreceptor within the TLR4/MD-2 receptor complex, as the high-affinity sCD83 binding partner. TLR4/MD-2 mediates proinflammatory signal delivery following recognition of bacterial LPSs. However, altering TLR4 signaling can attenuate the proinflammatory cascade, leading to LPS tolerance. Our data show that binding of sCD83 to MD-2 alters this signaling cascade by rapidly degrading IL-1R-associated kinase-1, leading to induction of the anti-inflammatory mediators IDO, IL-10, and PGE2 in a COX-2-dependent manner. sCD83 inhibited T cell proliferation, blocked IL-2 secretion, and rendered T cells unresponsive to further downstream differentiation signals mediated by IL-2. Therefore, we propose the tolerogenic mechanism of action of sCD83 to be dependent on initial interaction with APCs, altering early cytokine signal pathways and leading to T cell unresponsiveness.

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. 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.

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. 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.