Comparison of human B cell activation by TLR7 and TLR9 agonists.

BACKGROUND: Human B cells and plasmacytoid dendritic cells (pDC) are the only cells known to express both TLR7 and TLR9. Plasmacytoid dendritic cells are the primary IFN-alpha producing cells in response to TLR7 and TLR9 agonists. The direct effects of TLR7 stimulation on human B cells is less understood. The objective of this study was to compare the effects of TLR7 and TLR9 stimulation on human B cell function. RESULTS: Gene expression and protein production of cytokines, chemokines, various B cell activation markers, and immunoglobulins were evaluated. Purified human CD19+ B cells (99.9%, containing both naïve and memory populations) from peripheral blood were stimulated with a TLR7-selective agonist (852A), TLR7/8 agonist (3M-003), or TLR9 selective agonist CpG ODN (CpG2006). TLR7 and TLR9 agonists similarly modulated the expression of cytokine and chemokine genes (IL-6, MIP1 alpha, MIP1 beta, TNF alpha and LTA), co-stimulatory molecules (CD80, CD40 and CD58), Fc receptors (CD23, CD32), anti-apoptotic genes (BCL2L1), certain transcription factors (MYC, TCFL5), and genes critical for B cell proliferation and differentiation (CD72, IL21R). Both agonists also induced protein expression of the above cytokines and chemokines. Additionally, TLR7 and TLR9 agonists induced the production of IgM and IgG. A TLR8-selective agonist was comparatively ineffective at stimulating purified human B cells. CONCLUSION: These results demonstrate that despite their molecular differences, the TLR7 and TLR9 agonists induce similar genes and proteins in purified human B cells.

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

BACKGROUND: 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. RESULTS: Global gene expression was evaluated using the Affymetrix U133A GeneChip(R) and selected genes were confirmed using real time TaqMan(R) 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 IFNalpha subtypes, IFNalpha2, alpha5, alpha6, alpha8, alpha1/13, alpha10, alpha14, alpha16, alpha17, alpha21. 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. CONCLUSION: 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.

Distinct indirect pathways govern human NK-cell activation by TLR-7 and TLR-8 agonists.

NK cells limit the emergence of cancers and viral infections by surveillance of 'missing-self' and 'induced-self' ligands, and by direct recognition of pathogen-associated molecules. We examined individual roles for Toll-like receptors (TLRs)-7 and -8 in human NK-cell activation using synthetic, small molecule agonists of either TLR-7 (imiquimod and 3M-001), TLR-8 (3M-002) or both TLR-7/8 (3M-003 and R-848) for comparison with known ligands of TLR-2 to -9. Tracking cytokine production in PBMC initially revealed that a subset of TLR agonists including polyinosinic-polycytidylic acid (poly I:C), 3M-002, 3M-003, R-848 and single-stranded RNA trigger relatively high levels of IFN-gamma expression by NK cells. Isolated NK cells did not express TLR-7 or TLR-8. Unlike MALP-2 and poly I:C, 3M-001-3 did not induce expression of either CD69 or IFN-gamma by purified NK cells suggesting indirect activation. IL-18 and IL-12p70 were primarily required for induction of IFN-gamma by both synthetic and natural TLR-8 ligands, while type I IFN was required for induction of CD69 on NK cells by the TLR-7 agonist 3M-001. In addition to expression of IFN-gamma and CD69, relative induction of NK-cell cytotoxicity by TLR-7 and TLR-8 agonists was compared. Immune response modifiers (IRMs) with a TLR-8 agonist component (3M-002 and 3M-003) stimulated greater levels of K562 cytolysis than achieved with 3M-001 or IL-2 (1000 units ml(-1)). In vivo NK-cell cytotoxicity was also enhanced by R-848, but not in type I IFNR-deficient mice. We conclude that IRMs can modulate NK-cell function both in vitro and in vivo and that distinct indirect pathways control human NK-cell activation by TLR-7 and TLR-8 agonists.