Debra Tross

Synthetic oligonucleotides as modulators of inflammation

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs mimic the immunostimulatory activity of bacterial DNA. CpG ODN directly stimulate human B cells and plasmacytoid dendritic cells, promote the production of Th1 and proinflammatory cytokines, and trigger the maturation/activation of professional APC. CpG ODN are finding use in the treatment of cancer, allergy, and infection. In contrast, ODN containing multiple TTAGGG motifs mimic the immunosuppressive activity of self‐DNA, down‐regulating the production of proinflammatory and Th1 cytokines. Preclinical studies suggest that “suppressive” ODN may slow or prevent diseases characterized by pathologic immune stimulation, including autoimmunity and septic shock. Extensive studies in animal models suggest that the therapeutic value of CpG and TTAGGG ODN may be optimized by early administration.

FDA Guidance on Prophylactic DNA Vaccines: Analysis and Recommendations

The FDA has been regulating the conduct of prophylactic DNA vaccine trials in the US for nearly 15 years. This work describes the evolution of FDA policy over that period, the status of current regulatory guidance, and provides recommendations for further changes to facilitate development in this field.

Effect of CpG Oligonucleotides on Vaccine-Induced B Cell Memory

Adding synthetic oligodeoxynucleotides containing unmethylated CpG motifs to Anthrax vaccine adsorbed (AVA, the licensed human vaccine) increases the speed and magnitude of the resultant Ab response. Ab titers persist in the protective range for >1 year, significantly longer than in animals vaccinated with AVA alone. Unexpectedly, a majority of mice immunized with CpG-adjuvanted AVA maintained resistance to anthrax infection even after their Ab titers had declined into the subprotective range. The survival of these animals was mediated by the de novo production of protective Abs by high affinity memory B cells re-stimulated immediately after challenge. Thus, a previously unrecognized benefit of CpG oligodeoxynucleotides adjuvants is their ability to expand the long-lived memory B cell population. Current findings demonstrate that CpG-adjuvanted AVA mediates protection both by stimulating a strong/persistent serum Ab response and by generating a high-affinity long-lived pool of memory B cells.

Short- and long-term changes in gene expression mediated by the activation of TLR9.

CpG DNA binds to Toll-like receptor 9 to stimulate a strong innate immune response. The magnitude, duration and scope of CpG-induced changes in gene expression are incompletely understood despite extensive studies of TLR9 mediated signal transduction pathways. In particular, the prolonged effects of CpG DNA on gene activation have not been investigated despite evidence that a single dose of CpG DNA alters immune reactivity for several weeks. This study used gene expression analysis to monitor changes in mRNA levels for 14 days, and identified the genes, pathways and functional groups triggered in vivo following CpG DNA administration. Two discrete peaks of gene activation (at 3h and 5 days) were observed after CpG injection. Both the behavior and function of genes activated during the second peak differed from those triggered shortly after CpG administration. Initial gene up-regulation corresponded to a period when TLR9 ligation stimulated genes functionally associated with the generation of innate and adaptive immune responses (e.g. the NF-kappaB and B-cell receptor pathways). The second peak reflected processes associated with cell division (e.g. cell cycle and DNA replication and repair). The complex bimodal pattern of gene expression elicited by CpG DNA administration provides novel insights into the long-term effects of TLR9 engagement on genes associated with immunity and cell proliferation.

Inductive and suppressive networks regulate TLR9‐dependent gene expression in vivo

Bacterial DNA expressing unmethylated CpG motifs binds to TLR9, thereby stimulating a broadly protective, innate immune response. Although CpG‐mediated signal transduction has been studied, the scope of TLR9‐dependent gene expression is incompletely understood. To resolve these issues, mice were treated with immunostimulatory CpG oligonucleotides (ODN) and splenic mRNA levels monitored from 30 min through 3 days by microarray. Through the unique application of bioinformatic analysis to these experimental data, this study is the first to describe the complex regulatory networks responsible for TLR9‐mediated gene expression. Current results are the first to establish that CpG‐induced stimulation of the innate immune system proceeds in multiple waves over time, and gene up‐regulation is mediated by a small number of temporally activated “major inducers” and “minor inducers”. An additional study of TNF knockout mice supports the conclusion that the regulatory networks identified by our bioinformatic analysis accurately identified CpG ODN‐driven gene–gene interactions in vivo. Equally important, this work identifies the counter‐regulatory mechanisms embedded within the signaling cascade that suppresses the proinflammatory response triggered in vivo by CpG DNA stimulation. Identifying these network interactions provides novel and global insights into the regulation of TLR9‐mediated gene activation, improves our understanding of TLR‐mediated host defense, and facilitates the development of interventions designed to optimize the nature and duration of the ensuing response.

Global changes in gene expression and synergistic interactions induced by TLR9 and TLR3

The innate immune system is triggered when pathogen-associated molecular patterns (PAMPs) expressed by infectious microorganisms interact with toll-like receptors (TLR) present on immune cells. Individual TLRs signal through distinct molecular pathways. For example, TLR9 interacts with unmethylated CpG motifs expressed by bacterial DNA and triggers via a MyD88 dependent pathway whereas TLR3 recognizes viral RNA through a MyD88-independent pathway. Bioinformatic analysis of microarray data was used to identify the regulatory patterns underlying changes in gene expression induced when RAW 264.7 macrophages were stimulated via TLR9 by CpG oligonucleotides (ODN) and/or via TLR3 by poly (I:C). While the genes activated by each ligand mediated similar functions, poly (I:C) elicited a larger and more diverse change in gene expression. Co-stimulation with both ligands accelerated gene expression and synergistically activated genes primarily associated with immune function. This is the first work to compare global changes in gene regulation triggered by distinct TLR pathways and clarify their impact on gene expression.

CpG Oligonucleotides as Cancer Vaccine Adjuvants.

Adjuvants improve host responsiveness to co-delivered vaccines through a variety of mechanisms. Agents that trigger cells expressing Toll-like receptors (TLR) activate an innate immune response that enhances the induction of vaccine-specific immunity. When administered in combination with vaccines designed to prevent or slow tumor growth, TLR agonists have significantly improved the generation of cytotoxic T lymphocytes. Unfortunately, vaccines containing TLR agonists have rarely been able to eliminate large established tumors when administered systemically. To improve efficacy, attention has focused on delivering TLR agonists intra-tumorally with the intent of altering the tumor microenvironment. Agonists targeting TLRs 7/8 or 9 can reduce the frequency of Tregs while causing immunosuppressive MDSC in the tumor bed to differentiate into tumoricidal macrophages thereby enhancing tumor elimination. This work reviews pre-clinical and clinical studies concerning the utility of TLR 7/8/9 agonists as adjuvants for tumor vaccines.

Effect of TLR Agonists on the Differentiation and Function of Human Monocytic Myeloid-Derived Suppressor Cells

Tumors persist by occupying immunosuppressive microenvironments that inhibit the activity of tumoricidal T and NK cells. Monocytic myeloid-derived suppressor cells (mMDSC) are an important component of this immunosuppressive milieu. We find that the suppressive activity of mMDSC isolated from cancer patients can be reversed by treatment with TLR7/8 agonists, which induce human mMDSC to differentiate into tumoricidal M1-like macrophages. In contrast, agonists targeting TLR1/2 cause mMDSC to mature into immunosuppressive M2-like macrophages. These two populations of macrophage are phenotypically and functionally discrete and differ in gene expression profile. The ability of TLR7/8 agonists to reverse mMDSC-mediated immune suppression suggests that they might be useful adjuncts for tumor immunotherapy.

Activation of type I interferon‐dependent genes characterizes the “core response” induced by CpG DNA

Synthetic ODNs expressing CpG motifs trigger an innate immune response via TLR9. pDCs are major effectors of this response. Two structurally distinct classes of CpG ODNs have been identified that differentially activate pDCs. “K” ODNs trigger the production of TNF‐α and IL‐6, whereas “D” ODNs preferentially induce the secretion of IFN‐α. As K and D ODNs have distinct therapeutic effects, knowledge of their shared and sequence‐specific activity is of considerable importance. This work uses the CAL‐1 human pDC line to analyze the effect of CpG stimulation on gene expression. Genes up‐regulated by both K and D ODNs (n=92) were largely dependent on type I IFN signaling and characterized functionally by antiviral activity. K ODNs induced a short‐term increase in IFN‐α/β production and uniquely up‐regulated genes that supported antibacterial responses. In contrast, D ODNs triggered a persistent increase in IFN‐α/β production and uniquely up‐regulated genes associated with metabolic functions. Thus, the core functionality of human pDCs mediated by TLR9 ligation rests on a type I IFN response that differs from the response induced by the structural elements unique to specific classes of ODNs.

IRF5 and IRF8 modulate the CAL-1 human plasmacytoid dendritic cell line response following TLR9 ligation.

Synthetic oligonucleotides (ODNs) containing CpG motifs stimulate human plasmacytoid dendritic cells (pDCs) to produce type‐1 interferons (IFNs) and proinflammatory cytokines. Previous studies demonstrated that interferon regulatory factors (IRFs) play a central role in mediating CpG‐induced pDC activation. This work explores the inverse effects of IRF5 and IRF8 (also known as IFN consensus sequence‐binding protein) on CpG‐dependent gene expression in the human CAL‐1 pDC cell line. This cell line shares many of the phenotypic and functional properties of freshly isolated human pDCs. Results from RNA interference and microarray studies indicate that IRF5 upregulates TLR9‐driven gene expression whereas IRF8 downregulates the same genes. Several findings support the conclusion that IRF8 inhibits TLR9‐dependent gene expression by directly blocking the activity of IRF5. First, the inhibitory activity of IRF8 is only observed when IRF5 is present. Second, proximity ligation analysis shows that IRF8 and IRF5 colocalize within the cytoplasm of resting human pDCs and cotranslocate to the nucleus after CpG stimulation. Taken together, these findings suggest that IRF5 and IRF8, two transcription factors with opposing functions, control TLR9 signaling in human pDCs.