tlr3 agonist poly i c  (Tocris)

Journal: Cancer biology & therapy

Article Title: β-amino acid substitution in the SIINFEKL antigen alters immunological recognition.

doi: 10.1080/15384047.2025.2486141

Figure Lengend Snippet: Figure 4. Immunization with native peptides, but not with β-modified peptides, along with TLR3 and TLR9 agonists improved survival in tumor-bearing mice. C57BL/6 mice (n = 5 mice per group, 25 mice total) were implanted with E.G7-OVA cells (1 × 106 cells per mouse) subcutaneously. Nine days post implantation 2 × 106 OT-I splenocytes (from one OT-I mouse) were adoptively transferred intraperitoneally. The following day, mice were immunized with either TLR3 (100 μg) and TLR9 (50 μg) agonists only or with native or β-modified peptide (100 μg) combined with TLR3 and TLR9 agonists subcutaneously. (a). Schematic of the study design is represented on a timeline. (b). Tumor volumes were measured every other day and plotted. Statistical analysis performed using two-way ANOVA (mixed-effects model) for measurements from day 20. (c). Survival curve was plotted, and statistical analysis was performed using Log-rank (Mantel-Cox) test. Mice were euthanized when the tumor size reached >2 cm3. The experiment was repeated twice, and each group consisted of five mice. Tumors were collected on day 16 (additional 25 mice) and processed into single-cell suspension to be analyzed by flow cytometry. (d).Percentage of tumor-infiltrating tetramer positive CD8 T cells were plotted as a bar graph, statistical analysis was performed using one-way ANOVA. *p < .05, **p < .01, and ****p < .0001.

Article Snippet: TLR3 agonist (Poly I:C) (Cat.# vacpic) and TLR9 agonist, CpG (Cat.# trlr-2395) were purchased from InvivoGen (San Diego, CA).

Techniques: Modification, Suspension, Flow Cytometry

Journal: Cancer biology & therapy

Article Title: β-amino acid substitution in the SIINFEKL antigen alters immunological recognition.

doi: 10.1080/15384047.2025.2486141

Figure Lengend Snippet: Figure 5. β-modified peptides are immunogenic but result in antigen-specific CD8 T cells do not cross react with the native epitope. Dendritic cells were isolated from C57BL/6 splenocytes using negative selection and incubated with 1 µg/ml of native or β-modified peptides for 4 h. DCs were then washed and stained for analysis by flow cytometry. (a). Percentage of DCs that are stained for SIINFEKL bound to H2-kb antibody. (b). Naïve C57BL/6 mice (n = 4 per group, 36 mice total) were immunized twice with either TLR3 (100 μg) and TLR9 (50 μg) agonists only (untreated) or with native or β-modified peptide (100 μg) combined with TLR3 and TLR9 agonist subcutaneously. The immunizations were 1 week apart, and the mice were euthanized 1 week after the second immunization. Spleens were collected and processed for analysis by flow cytometry. Gating was performed to identify live CD3+ CD8+ SIINFEKL tetramer+ single cells. (c). ELISpot plate was coated with anti-mouse IFN-γ antibody and 0.2 × 106 splenocytes from each mouse spleen were plated. Cells were stimulated for 48 h with either media alone (no antigen), native peptide (2 µg/ml) or the β-specific peptide (2 µg/ml) that was used to immunize the mice. IFN-γ-secreting cells were counted, with the data for each mouse presented as bar graphs. Statistical analysis was performed using one-way ANOVA, *p < .05, ***p < .001 and ****p < .0001.

Article Snippet: TLR3 agonist (Poly I:C) (Cat.# vacpic) and TLR9 agonist, CpG (Cat.# trlr-2395) were purchased from InvivoGen (San Diego, CA).

Techniques: Modification, Isolation, Selection, Incubation, Staining, Flow Cytometry, Enzyme-linked Immunospot

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A Differentially expressed genes (DEGs) in full-thickness bladder tissues from HIC patients and controls identified via RNA sequencing (|log₂FC|≥2; adjusted p < 0.05, two-sided Wald test using DESeq2). Gene Ontology (GO) ( B ) and KEGG ( C ) enrichment analyses of DEGs (two-sided hypergeometric test with FDR correction). D UMAP visualization of single-cell transcriptomic landscape of urothelium, with urothelial cells (UCs) identified by KRT19 and UPK1A expression. GO ( E ) and KEGG ( F ) analyses of upregulated DEGs in UCs (two-sided hypergeometric test with FDR correction). GO ( G ) and KEGG ( H ) analyses of downregulated DEGs in UCs (two-sided hypergeometric test with FDR correction). I , J Gene Set Enrichment Analysis (GSEA) of altered pathways in UCs (two-sided permutation test with 1000 permutations, with multiple testing corrected using the FDR). The exact p value for TLR signaling of UCs in panel I is 0.0009. K Expression of Toll-like receptor (TLR) subtypes in UCs from HIC bladders. mRNA ( L ) and protein ( M ) expression levels of TLR3 in isolated urothelium from control and HIC patients ( n = 7 control vs. 10 HIC; data show median (IQR); two-sided Mann–Whitney U test; ns not significant; Bar for panel M: 1 cm). N Immunostaining showing the distribution of TLR3 protein in the urothelium of patients with HIC ( n = 7 control vs. 10 HIC; one section and field per patient; Bar: 100 μm). IHC immunohistochemistry, HL Hunner lesions, U urothelium, LP lamina propria (blue line), M muscularis. Source data are provided as a Source Data file.

Article Snippet: To explore the role of Toll-like receptor 3 (TLR3), TCDCA-pretreated HUCs were treated with either a TLR3 agonist (Poly(I:C) Sodium Salt, CST, Cat. 61401; 5 μg/mL) or TLR3 siRNA (Toll-like Receptor 3 siRNA I, CST, Cat. 6236; 100 nM).

Techniques: RNA Sequencing, Expressing, Isolation, Control, MANN-WHITNEY, Immunostaining, Immunohistochemistry

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A Experimental workflow of E. avium colonization in antibiotic-pretreated mice (Abx-mice, n = 6 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . B Increased abundance of E. avium confirmed by metagenomic sequencing ( n = 6 per group). C Evaluation of voiding behavior following E. avium transplantation ( n = 6 per group; one measurement per mouse). D Measurement of mechanical pain threshold post-transplantation ( n = 6 per group). E Representative histological analysis of bladder tissues after E. avium transplantation ( n = 6 per group; one section and field per mouse). F , G Quantification of blood and urinary bile acids using comprehensive targeted bile acid profiling ( n = 6 per group). H Cell viability of human urothelial cells (HUCs) following TCDCA or TUDCA treatment ( n = 3 independent experiments). I Expression of ZO-1, TNF-α, and TLR3 following TCDCA (200 µM) exposure ( n = 3 independent experiments). J Experimental workflow of intravesical instillation of TCDCA in rats ( n = 5 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . K Evaluation of voiding function after TCDCA instillation ( n = 5 per group; each rate measured once). L Assessment of mechanical pain threshold following TCDCA exposure ( n = 5 per group). M Representative histology of bladder tissues post-instillation ( n = 5 per group; one section per rat, one field quantified per section). For panels ( B – H , K , L ): data are presented as median (IQR). Statistical analysis was performed using the two-sided Mann–Whitney U test. IHC immunohistochemistry, Red arrow: urothelial thinning, detachment, and exposure, Blue line: LP lamina propria, U urothelium, M muscularis. Source data are provided as a Source Data file.

Article Snippet: To explore the role of Toll-like receptor 3 (TLR3), TCDCA-pretreated HUCs were treated with either a TLR3 agonist (Poly(I:C) Sodium Salt, CST, Cat. 61401; 5 μg/mL) or TLR3 siRNA (Toll-like Receptor 3 siRNA I, CST, Cat. 6236; 100 nM).

Techniques: Sequencing, Transplantation Assay, Expressing, MANN-WHITNEY, Immunohistochemistry

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A , B Expression of tight junction protein ZO-1 and inflammatory marker TNF-α after TLR3 intervention in TCDCA-pretreated human urothelial cells (HUCs) ( n = 3 independent experiments). C Transepithelial resistance (TER) changes following TLR3 intervention in TCDCA-pretreated HUCs ( n = 3 independent experiments). D , E Expression of ZO-1 and TNF-α after pentosan polysulfate sodium (PPS) administration in TCDCA-pretreated HUCs ( n = 3 independent experiments). F TER changes following PPS intervention in TCDCA-pretreated HUCs ( n = 3 independent experiments). G Experimental workflow showing TLR3 inhibitor and PPS administration to rats following intravesical TCDCA instillation ( n = 5 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . H Assessment of voiding function (one measurement per rat) and pain threshold after TLR3 inhibition ( n = 5 per group). I Representative histological images of bladder tissues post-TLR3 inhibition ( n = 5 per group; one section and field per rat). For panels ( B , C , E , F , H ): data are presented as median (IQR). Statistical analysis was performed using the Kruskal–Wallis test. ns not significant, IHC immunohistochemistry, Red arrow: urothelial thinning, detachment, and exposure, Blue line: LP lamina propria, U urothelium, M muscularis. Source data are provided as a Source Data file.

Article Snippet: To explore the role of Toll-like receptor 3 (TLR3), TCDCA-pretreated HUCs were treated with either a TLR3 agonist (Poly(I:C) Sodium Salt, CST, Cat. 61401; 5 μg/mL) or TLR3 siRNA (Toll-like Receptor 3 siRNA I, CST, Cat. 6236; 100 nM).

Techniques: Expressing, Marker, Inhibition, Immunohistochemistry

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Levels of TMEFF1 mRNA, as determined by RT–qPCR, in various human cell lines or primary cells. b , TMEFF1 mRNA levels were determined by RT–qPCR in cortical neurons from control and TMEFF1 -KO hPSCs. c , TMEFF1 protein expression was studied by confocal microscopy on cortical neurons derived from healthy control and TMEFF1 -KO hPSCs. Cells were fixed and stained for TMEFF1 (anti-TMEFF1 antibody, green), cell membrane (wheat germ agglutinin (WGA), white) and chromosomes (DAPI, blue). Scale bar, 10 μm. d , Cortical neurons derived from hPSCs from healthy controls, TMEFF1 -KO hPSCs and TLR3 −/− hPSCs were infected with HSV-1 (MOI 0.001) and assessed for HSV-1 titres at the timepoints indicated. TCID 50 , 50% tissue culture infectious dose. e , TMEFF1 mRNA levels were determined by RT–qPCR on hPSC-derived cortical neurons for healthy controls and the two patients with TMEFF1 mutations (P1 and P2). f , Relative abundance of TMEFF1 cDNA isoforms generated from mRNA extracted from hPSC-derived cortical neurons for healthy controls and P2, as assessed by TOPO-TA cloning. g , h , hPSC-derived cortical neurons from a healthy control (H9), the patients with TMEFF1 mutations (P1 and P2) and other TLR3 −/− and IFNAR1 −/− HSE patients were infected with HSV-1 (MOI 0.001) and assessed for HSV-1 titres at the timepoints indicated, without ( g ) or with ( h ) IFNβ pretreatment for 18 h. The data shown in a , b , d , e , g and h are mean ± s.e.m. of three independent experiments. Statistical analysis: for b and e , two-tailed Mann-Whitney U -tests; for d , g and h , mean log-transformed relative values were compared between control cells and TMEFF1 -mutated cells in one-way analysis of variance (ANOVA) with Tukey tests for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For the assessment of TLR3-associated responses, the cells were stimulated with the TLR3 agonist poly(I:C) (Tocris, 4287; a mixture of low-molecular-weight (250–1,000 bp) and high-molecular-weight (more than 1,000 bp)) at a concentration of 25 μg ml −1 and collected for assessment by RT–qPCR at various time points (2 h, 4 h and 6 h).

Techniques: Quantitative RT-PCR, Control, Expressing, Confocal Microscopy, Derivative Assay, Staining, Membrane, Infection, Generated, TA Cloning, Two Tailed Test, MANN-WHITNEY, Transformation Assay

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Electropherogram representation (left panels) of the CRISPR-Cas9-introduced compound-heterozygous TMEFF1 mutations confirmed by Sanger sequencing on genomic DNA from a gene-edited TMEFF1 KO hPSC line (TMEFF1 KO #1). Sequencing results for the parental line (Ctrl parental, BJ1) are also shown. The relative abundance of WT or mutated TMEFF1 cDNA generated from mRNA extracted from the control parental and TMEFF1 KO hPSCs was assessed by TOPO-TA cloning and is shown in the right panels. b , Representative images of cortical neurons from controls (Ctrl 1-H9, Ctrl 2 parental-BJ1) and TMEFF1 KO hPSCs. Cells were fixed and stained with DAPI (blue) and for a neuron-specific marker, microtubule-associated protein 2 (MAP2, green). c , FOXG1 and PAX6 mRNA levels, as measured by RT-qPCR, in cortical neurons from control and TMEFF1 KO hPSCs. SV40-transformed fibroblasts from healthy controls (Fibros ctrl 1, Fibros ctrl 2) were used as a negative control in this assay. d , Representative images of cortical neurons from control (H9) and various patient-specific hPSC lines (P1, P2, IFNAR1 −/− , TLR3 −/− ). Cells were fixed and stained with DAPI (blue), and for a neuron-specific marker MAP2 (green). e , FOXG1 and PAX6 mRNA levels were measured by RT-qPCR in cortical neurons derived from control and patient-specific hPSC lines. SV40-transformed fibroblasts from healthy controls (Fibros ctrl 3, Fibros ctrl 4) were used as a negative control in this assay. f , Electropherogram representation of the three TMEFF1 mutant isoforms, as detected by TOPO-cloning of cDNA from P2’s hPSC-derived cortical neurons.

Article Snippet: For the assessment of TLR3-associated responses, the cells were stimulated with the TLR3 agonist poly(I:C) (Tocris, 4287; a mixture of low-molecular-weight (250–1,000 bp) and high-molecular-weight (more than 1,000 bp)) at a concentration of 25 μg ml −1 and collected for assessment by RT–qPCR at various time points (2 h, 4 h and 6 h).

Techniques: CRISPR, Sequencing, Generated, Control, TA Cloning, Staining, Marker, Quantitative RT-PCR, Transformation Assay, Negative Control, Derivative Assay, Mutagenesis, Cloning

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , TMEFF1 mRNA levels were determined by RT-qPCR, in SV40-transformed fibroblasts from the patients with TMEFF1 mutations, a TLR3 −/− HSE patient, and healthy controls treated with poly(I:C) for 2 or 4 h or left untreated (NS). b , TMEFF1 mRNA levels were measured by RT-qPCR, in SV40-transformed fibroblasts from patients with TMEFF1 mutations, an IFNAR1 −/− HSE patient, and healthy controls treated with IFN-α2b for 8 h or left untreated. c , TMEFF1 mRNA levels were measured by RT-qPCR in cortical neurons derived from control parental or TMEFF1 KO hPSCs, and hPSCs from a TLR3 −/− HSE patient, after treatment with poly(I:C) for 6 h, or without treatment. d , TMEFF1 mRNA levels were measured by RT-qPCR, in cortical neurons derived from control parental or TMEFF1 KO hPSCs, and hPSCs from an IFNAR1 −/− HSE patient treated with IFN-β for 8 h or left untreated. In a - d , two probes, targeting exons 1-2 (upper panels) and exons 9-10 (lower panels) of TMEFF1 were used. The data shown are the means ± SEM from three ( a , b ) or two ( c , d ) independent experiments. e , Abundance of TMEFF1 mRNA, as assessed by RNAseq, in healthy control neurons (Ctrls, n = 6), SNORA31 -mutated ( SNORA31 -MT, n = 8), TLR3 −/− ( n = 2) or STAT1 −/− ( n = 2) hPSC-derived cortical neurons treated with poly(I:C) or IFN-α2b, or left unstimulated (NS). Data are presented as mean ± SD. f , Abundance of TMEFF1 mRNA, as assessed by RNAseq, in healthy controls (Ctrls, n = 6), SNORA31 -mutated ( SNORA31 -MT, n = 8) or STAT1 −/− ( n = 2) hPSC-derived cortical neurons infected with HSV-1 for 24 h, or left unstimulated (NS). g , IFNB1 (upper panel) or IFNL1 (lower panel) mRNA levels were measured by RT-qPCR, in SV40-transformed fibroblasts from the patients with TMEFF1 mutations, a TLR3 −/− HSE patient, and healthy controls, after treatment with poly(I:C) for 2 or 4 h or without treatment. h , MX1 (upper panel) or IFIT1 (lower panel) mRNA levels were measured by RT-qPCR, in SV40-transformed fibroblasts from the patients with TMEFF1 mutations, an IFNAR1 −/− HSE patient, and healthy controls, after treatment with IFN-α2b for 8 h, or without treatment. The data shown in g , and h are the means ± SEM from three independent experiments. i , Basal levels of IFNAR1 (top panel), IFNAR2 (middle panel), and TLR3 (lower panel) mRNA were measured by RT-qPCR, in hPSC-derived cortical neurons from healthy controls (Ctrl 1-H9, Ctrl 2-Parental BJ1), TMEFF1 KO hPSCs, or hPSCs from TMEFF1-mutated patients. j , Levels of MX1 (upper panels) or IFIT1 (lower panels) mRNA were measured by RT-qPCR, in cortical neurons derived from control parental or TMEFF1 KO hPSCs, hPSCs from a TLR3 −/− HSE patient, and an IFNAR1 −/− HSE patient, with and without treatment with poly(I:C) for 6 h (left panels), or with IFN-β for 8 h (right panels). Statistical analysis was performed with two-tailed Mann-Whitney U tests. ns: not significant. k , Scatterplots of the mean log 2 fold-changes in RNAseq-quantified gene induction following stimulation with 100 IU/ml IFN-β for 8 h (upper panel) or HSV-1 (MOI 1) for 24 h (lower panel) in hPSC-derived CNS cortical neurons from two healthy controls (Ctrl1-H9, Ctrl2 parental-BJ1), TMEFF1-mutated patients (TMEFF1 Pts) or TMEFF1 KO hPSCS, or hPSCs from an IFNAR1 −/− HSE patient. Each point represents a single gene. Genes with an absolute fold-change in expression > 2 in response to IFN-β or HSV-1 treatment relative to NS samples in the Ctrl group are plotted. l , Heatmaps of RNA-Seq-quantified gene expression (z-score-scaled DESeq2 vst-normalization) in hPSC-derived CNS cortical neurons from healthy controls (Ctrl 1-H9, Ctrl 2-Parental BJ1) or TMEFF1 KO hPSCS, or hPSCs from an IFNAR1 −/− HSE patient, a TLR3 −/− HSE patient and TMEFF1-mutated P1 and P2 (TMEFF1 Pts), not stimulated (NS), stimulated with HSV-1 for 24 h, or stimulated with IFN-β for 8 h. Duplicates were studied for each set of conditions and mean gene expression levels were used for subsequent analyses. The heatmap includes genes with a relative fold-change in expression > 2 in response to HSV-1 or IFN-β treatment relative to NS samples in the Ctrl group.

Article Snippet: For the assessment of TLR3-associated responses, the cells were stimulated with the TLR3 agonist poly(I:C) (Tocris, 4287; a mixture of low-molecular-weight (250–1,000 bp) and high-molecular-weight (more than 1,000 bp)) at a concentration of 25 μg ml −1 and collected for assessment by RT–qPCR at various time points (2 h, 4 h and 6 h).

Techniques: Quantitative RT-PCR, Transformation Assay, Derivative Assay, Control, Infection, Two Tailed Test, MANN-WHITNEY, Expressing, RNA Sequencing, Gene Expression