Agonists of Toll-like Receptor 9 Containing Synthetic Dinucleotide Motifs (original) (raw)
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Veterinary Immunology and Immunopathology, 2008
Synthetic agonists of TLR9 containing novel DNA structures and R 0 pG (wherein R = 1-(2 0-deoxy-b-D-ribofuranosyl)-2-oxo-7deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T H-1type-immune responses and potently reverse allergen-induced T H-2 responses to T H-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5 + and CD21 +) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-g, IL-10, TGF-b, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5 + T cells nor CD21 + B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-g on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-b and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T H-1 immune response including T-and B-cell proliferation.
Immunology, 2007
Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, plays an important role in the differentiation and activation of B cells. Mutations affecting Btk cause immunodeficiency in both humans and mice. In this study we set out to investigate the potential role of Btk in Toll-like receptor 9 (TLR9) activation and the production of pro-inflammatory cytokines such as interleukin (IL)-6, tumour necrosis factor (TNF)-α and IL-12p40. Our data show that Btk-deficient B cells respond more efficiently to CpG-DNA stimulation, producing significantly higher levels of pro-inflammatory cytokines but lower levels of the inhibitory cytokine IL-10. The quantitative reverse transcription–polymerase chain reaction (RT-PCR) analysis presented in this work shows that mRNA production of one of the important new members of the IL-12 family, IL-27, was significantly increased in Btk-deficient B cells after CpG-DNA stimulation. In this study, we demonstrate significant differences in CpG responsiveness between transitional 1 (T1) and T2 B cells for survival and maturation. Furthermore, TLR9 expression, measured both as protein and as mRNA, was increased in Btk-defective cells, especially after TLR9 stimulation. Collectively, these data provide evidence in support of the theory that Btk regulates both TLR9 activation and expression in mouse splenic B cells.
Cancer Genetics and Cytogenetics, 2009
Chronic infection and resulting inflammation promote tumor development and progression, and Toll-like receptors (TLRs) may play an important role in this process. The aim of this study was to determine whether CpG oligonucleotides (CpG-ODN), which are Toll-like receptor 9 (TLR9) agonists, can promote inflammatory cytokines release from the prostate cancer PC-3 cells through activation of nuclear factor-kB (NF-kB). Flow cytometry, semiquantitative real-time reverse transcriptaseepolymerase chain reaction, enzyme-linked immunosorbent assay, and immunofluorescence analysis were used to detect the transforming growth factor-b1 (TGF-b1) and interleukin-8 (IL-8) release and NF-kB activation in PC-3 cells after CpG-ODN stimulation. CpG-ODN promoted the expression and secretion of immunosuppressive cytokines TGF-b1 and IL-8 from PC-3 cells. In addition, after CpG-ODN stimulation, NF-kB nuclear translocation was also observed in PC-3 cells, contributing to CpG-induced upregulation of IL-8 and TGF-b1. Thus, TLR9 agonists may promote IL-8 and TGF-b1 production in human prostate cancer cells through NF-kB activation. Ó
Cellular Immunology, 2020
Toll-like receptor (TLR)4 and TLR9 agonists, MPL and CpG, are used as adjuvants in vaccines and have been investigated for their combined potential. However, how these two combined agonists regulate transcriptional changes in innate immune cells and cells at the site of vaccination has not been thoroughly investigated. Here, we utilized transcriptomics to investigate how CpG, MPL, and CpG+MPL impact gene expression in dendritic cells (DC) in vitro. Principal component analysis of transcriptional changes after single and combined treatment indicated that CpG, MPL, and CpG+MPL caused distinct gene signatures. CpG+MPL induced antiviral gene expression and activated the interferon regulatory factor pathway. In vitro changes were associated with lower in vivo morbidity upon viral challenge, elevated systemic cytokine protein production, local cytokine mRNA expression, and increased migratory monocyte derived DC populations in the draining lymph node following vaccination with CpG+MPL. This report suggests that CpG+MPL enhances transcription of antiviral and inflammatory genes and increases DC migration.
Synthesis and immunological activities of novel agonists of toll-like receptor 9
Cellular Immunology, 2010
Novel agonists of TLR9 with two 5 0 -ends and synthetic immune stimulatory motifs, referred to as immune modulatory oligonucleotides (IMOs) are potent agonists of TLR9. In the present study, we have designed and synthesized 15 novel IMOs by incorporating specific chemical modifications and studied their immune response profiles both in vitro and in vivo. Analysis of the immunostimulatory profiles of these IMOs in human and NHP cell-based assays suggest that changes in the number of synthetic immunostimulatory motifs gave only a subtle change in immune stimulation of pDCs as indicated by IFN-a production and pDC maturation while the addition of self-complementary sequences produced more dramatic changes in both pDC and B cell stimulation. All IMOs induced cytokine production in vivo immediately after administration in mice. Representative compounds were also compared for the ability to stimulate cytokine production in vivo (IFN-a and IP-10) in rhesus macaques after intra-muscular administration.
Immunology, 2010
The in vitro Peripheral Tissue Equivalent (PTE) module is a three-dimensional tissue-engineered endothelial cell/collagen matrix culture system, which has been reported to reproduce in vivo physiological conditions and which generates dendritic cells (DC) autonomously. In the present study, we used the PTE module to investigate the immunopotency of Toll-like receptor (TLR) agonists, including polyinosine-polycytidylic acid, Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR agonists in the PTE module induced a wide range of cytokines, including interleukins 1a/b, 6, 8 and 10 and tumour necrosis factor-a. Compared with traditional peripheral blood mononuclear cell (PBMC) cultures, the PTE module produced twofold to 100-fold higher levels of cytokine secretion, indicating that it can be a highly sensitive assay system. This increased sensitivity is the result of the natural synergy between the leucocytes and the endothelium. Furthermore, the application of TLR agonists, such as lipopolysaccharide and Gardiquimod, to the PTE module enhanced DC differentiation and promoted DC maturation, as indicated by up-regulated expression of CD83, CD86 and CCR7(CD197). In addition, functional assays indicated PTE-derived DC treated with Gardiquimod, a TLR-7 agonist, significantly augmented anti-tetanus toxoid antibody production. Interestingly, replacing PBMC with purified myeloid cells (CD33 + ) significantly reduced the responsiveness of the PTE module to TLR stimulation. The reduced sensitivity was partly the result of the removal of plasmacytoid DC that participated in the response to TLR stimulation and sensitization of the PTE module. Overall, the in vitro PTE module clearly demonstrated the effects of TLR agonists on DC generation, maturation and antigen-presenting capacity, and may serve as a sensitive and predictive test bed for the evaluation of adjuvant candidates.
Development of a Clinical Assay To Evaluate Toll-Like Receptor Function
Clinical and Vaccine Immunology, 2006
Toll-like receptors (TLRS) recognize pathogen-associated molecular patterns to enable innate immune responses. A number of genetic defects influencing the function of these receptors have been identified and are associated with recurrent and/or severe infection. Our goal was to develop a reproducible assay of TLR function in order to evaluate patients with recurrent infection who would be suspected of having a genetic defect affecting TLR signaling. We chose to study peripheral blood mononuclear cells (PBMCS) to avoid potential influences of soluble factors contained in whole blood, and we utilized ligands for TLRS 1/2, 2/6, 3, 4, 5, 6, 7, and 9. Tumor necrosis factor (TNF) production in PBMC supernatants was measured by an enzyme-linked immunosorbent assay after TLR ligand stimulation and was dependent on gene transcription and NF-B activation. Some variables affecting the assay were assessed, including the effects of: blood anticoagulant, serum-containing media, incubation time, ligand storage, blood storage time, and cell cryopreservation. By using optimized assay conditions, effective concentrations of individual ligands and mean responses to those ligands were established for healthy control donors. Finally, three patients with a mutation in the IKBKG gene, encoding the NF-B essential modulator (NEMO) protein, were evaluated as disease controls and were almost uniformly below the standard deviation of healthy donors for all ligands tested. Although a number of variables influence TLR ligand-induced TNF responses, this assay can be optimized for potential clinical use to screen patients with primary immunodeficiencies affecting TLR function.