Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists

<p>Abstract</p> <p>Background</p> <p>Plasmacytoid Dendritic Cells (pDC) comprise approximately 0.2 to 0.8% of the blood mononuclear cells and are the primary type 1 interferon (IFN), producing cells, secreting high levels of IFN in response to viral infections. Plasmacytoid dendritic cells express predominantly TLRs 7 & 9, making them responsive to ssRNA and CpG DNA. The objective of this study was to evaluate the molecular and cellular processes altered upon stimulation of pDC with synthetic TLR 7 and TLR 7/8 agonists. To this end, we evaluated changes in global gene expression upon stimulation of 99.9% pure human pDC with the TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011.</p> <p>Results</p> <p>Global gene expression was evaluated using the Affymetrix U133A GeneChip^®and selected genes were confirmed using real time TaqMan^®RTPCR. The gene expression profiles of the two agonists were similar indicating that changes in gene expression were solely due to stimulation through TLR7. Type 1 interferons were among the highest induced genes and included IFNB and multiple IFNα subtypes, IFNα2, α5, α6, α8, α1/13, α10, α14, α16, α17, α21. A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC. Induction of an antiviral state was shown by the expression of several IFN-inducible genes with known anti-viral activity. Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted. The analysis revealed transcription networks that show increased expression of signaling components in TLR7 and TLR3 pathways, and the cytosolic anti-viral pathway regulated by RIG1 and MDA5, suggestive of optimization of an antiviral state targeted towards RNA viruses. The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro-survival gene expression program.</p> <p>Conclusion</p> <p>Thus this study demonstrates that as early as 4 hr post stimulation, synthetic TLR7 agonists induce a complex transcription network responsible for activating pDC for innate anti-viral immune responses with optimized responses towards RNA viruses, increased co-stimulatory capacity, and increased survival.</p

Comparison of human B cell activation by TLR7 and TLR9 agonists

Research articl

Combined TLR and CD40 Triggering Induces Potent CD8+ T Cell Expansion with Variable Dependence on Type I IFN

Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10–20-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)γ production and an enhanced secondary response to antigenic challenge. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNγ, and IL-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity

Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists-7

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists"</p><p>http://www.biomedcentral.com/1471-2172/8/26</p><p>BMC Immunology 2007;8():26-26.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2175514.</p><p></p> 3M-852A and 3M-011. The network was generated from a list of genes with GO process classification of anti-apoptosis, using the shortest path algorithm. The network highlights the large number of anti-apoptotic genes that are increased in expression with a concomitant decrease in expression of key pro-apoptotic genes including several that are transscriptionally regulated by p53. Remaining network details are as described in Figure 6

Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists-2

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists"</p><p>http://www.biomedcentral.com/1471-2172/8/26</p><p>BMC Immunology 2007;8():26-26.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2175514.</p><p></p>on using a Luminex 25-Plex assay. (A) Secreted TNFα, IL8, IFNα, MIP-1α, MIP-1β and IL6. (B) secreted IL1β, IL2R, IL12P70, Rantes, GM-CSF, MCP-1, and IP-10. Dotted bars, vehicle stimulated samples; hatched bars, 3M-852A-stimulated samples and solid bars, 3M-011-stimulated samples. The results are expressed as mean + SD, n = 2 donors

Purified B cells were stimulated with the indicated IRM or with CpG2006

Gene expression changes were assessed by quantitative real time RT-PCR at 2, 8 or 24 hours after stimulation. The log2 of the maximum fold change over the time course from 2 to 24 hours for 1 representative donor is shown. Hierarchical clustering was performed as described in methods.<p><b>Copyright information:</b></p><p>Taken from "Comparison of human B cell activation by TLR7 and TLR9 agonists"</p><p>http://www.biomedcentral.com/1471-2172/9/39</p><p>BMC Immunology 2008;9():39-39.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2503978.</p><p></p