Project description:We report the gene expression differences between OT-1 T cells activated alone or in the presence of TLR1/2 (Pam3CSK4) or TLR7 (Gardiquimod). By activating OT-1 splenocytes in the presence or absence of the TLR agonists, isolating RNA from purified CD8+ T cells we show that cells activated in the presence of either TLR1/2 or TLR7 agonists had similar transcriptional profiles demonstrating an increase in Th1 cytokine expression over cells that were activated alone. This study provides a key piece of information for those designing T cell mediated therapies.
Project description:Analysis of monocyte-derived dendritic cells (MoDC) gene expression signature induced by TLR agonists the in presence or in the absence of PP2.
Project description:Toll like receptors (TLRs) sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). Since pathogens may contain several agonists we asked whether different TLRs may synergize in DC activation. We report that in human and mouse DC TLR3 or TLR4 potently synergize with TLR7, TLR8 or TLR9 in the induction of selected cytokine genes. Upon synergistic stimulation, IL-12, IL-23 and Delta-4 are induced at levels 50-100 fold higher than those induced by optimal concentrations of single agonists, leading to enhanced and sustained TH1 polarizing capacity. Using microarray analysis we show that only 1.5% of the transcripts induced by single TLR agonists are synergistically regulated by combinations of TLR4 and TLR8 agonists.. These results identify a combinatorial code by which DCs discriminate pathogens and provide (suggest) a rationale to design adjuvants for TH1 responses. Series_overall_design: 3 untreated, 3 treated with LPS at 2h, 3 treated with LPS at 8h, 3 treated with R848 at 2h, 3 treated with R848 at 8h, 3 treated with LPS + R848 at 2h, 3 treated with LPS + R848 at 8h Keywords: other
Project description:Goal of this study was to compare transcriptional changes of in vivo (MOG/CFA) activated 2D2 T cells in the absence or presence of NIK
Project description:Apilimod is a reported TLR pathway antagonist in clinical trial. To understand the mode of action of apilimod, we applied global microarray analysis to explore the effect of apilimod on gene expression of RAW cell in the absence of presence of R848 (TLR7 ligand).
Project description:Apilimod is a reported TLR pathway antagonist in clinical trial. To understand the mode of action of apilimod, we applied global microarray analysis to explore the effect of apilimod on gene expression of RAW cell in the absence of presence of R848 (TLR7 ligand). RAW cells were stimulated with R848 in the absence or presence of apilimod (1μM). mRNAs were extracted at 0h, 1h,3h, 7h, and 22h for affymetrix analysis. The inactive analog of apilimod (API09) was included as a control.
Project description:Immunosuppressive microenvironments block the activity of tumoricidal T and NK cells, allowing cancers to avoid immune elimination. Monocytic myeloid-derived suppressor cells (mMDSC) are an important component of these immunosuppressive milieus. Human mMDSC respond to stimulation via their Toll-like receptors by differentiating into macrophage. Agonists targeting TLRs 1/2 (such as PAM3) induce mMDSC to mature into immumosuppressive M2-like macrophage through a process that involves TNF and IL6. Agonists targeting TLRs 7/8 (such as R848) cause the same precursors to mature into tumoricidal M1-like macrophages, a process in which IL12 plays a central role. The immune suppression mediated by mMDSC is reversed by exposure to TLR 7/8 agonists which thus might be useful adjuncts for tumor immunotherapy. In this study we looked at several time points. hMDSC from four different donors were sorted from PBMC then cultured in the presence of R848, Pam 3 or left unstimulated for 30, 75 and 225 minutes. Late time points included samples from seven donors after 3 days in culture with the same TLR ligands. For microarray experiments total RNA was extracted, amplified into aRNA and coupled to Cy5. Similarly amplified RNA from universal RNA was coupled to Cy3 and used as a reference for each array.
Project description:Among both healthy and immunocompromised patient populations, pneumonia is a leading cause of death worldwide. Yet, despite structural vulnerability resulting in recurrent exposure to pathogens, the lungs’ mucosal immunity successfully suppresses most infections. We recently reported that these innate defenses can be substantially augmented by inhalational exposure to a crude bacterial lysate, protecting broadly against respiratory pathogens, including lethal pneumonia caused by bacteria, fungi or viruses. The phenomenon of inducible resistance is associated with rapid pathogen killing in the lungs and persists in the absence of the typical leukocytes of innate immunity. Rather, the respiratory epithelium appears to be the predominant effector. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors crucial to host defense through the sensing of pathogen associated molecular patterns. Given the importance of TLRs to mucosal immunity, the presence of numerous pathogen associated molecular patterns in the bacterial lysate, and the induction of many TLR-dependent genes following lysate treatment, we hypothesized that induced resistance follows simultaneous stimulation of multiple TLRs. To test this, we challenged mice deficient in TLR/IL1R adaptor proteins and found that resistance could not be induced in mice lacking MyD88. Having identified this phenomenon to be MyD88-dependent, we sought to determine whether the protective phenomenon could be recapitulated by treatment with synthetic TLR agonists. Mice were treated with aerosolized TLR ligands, alone and in combination, prior to infection with virulent pathogens. While limited protection against pneumonia was afforded by the individual TLR ligands, we discovered that the synergistic combination of diacylated lipopetide TLR2/6 agonist Pam2CSK4 and CpG oligodeoxynucleotide TLR9 agonist ODN2395 induced profound resistance against all tested pathogens. This combination also induced greater than additive pathogen killing in the lungs of challenged mice, and we found that the combination could effectively induce pathogen killing by respiratory epithelial cells in vitro. In order to better understand the mechanisms underlying the inducible pathogen killing by this unique combination of TLR agonists, we performed microarray analysis of murine MLE-15 respiratory epithelial cells following 4 h treatment with PBS (sham treatment), Pam2CSK4 alone, ODN2395 alone, or the combination of both agonists, using Illumina Sentrix MouseRef-8 v2 BeadChips. This allows for assessment of differential gene expression, not only between treated and untreated, but between single and combination treated. The intent of the experiment is to gain insight into the transcriptionally-regulated means by which TLR2/6 and TLR9 signaling pathways synergistically interact. Keywords: Differential expression, epithelium, in vitro