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human tlr7 dual reporter hek 293 cells  (InvivoGen)


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    InvivoGen human tlr7 dual reporter hek 293 cells
    (A) Representative flow cytometry of Tfh cells, GC B cells, and Spike-specific GC B cells (S+) from wild type (WT) and Myd88 −/− mice. (B) Tfh cell, GC B cell, and S+ GC B cell absolute numbers, as detailed in A . (C) Absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and <t>Tlr7</t> −/− mice. (D) Frequencies and absolute numbers of Tfh cells and GC B cells, as detailed in A , in mice treated with isotype, anti-IL-1R, or anti-IL-18 mAb. (E) IFN-α levels 8 hours after immunization. Mice were treated with isotype or anti-IL-1R mAb. (F) IFN-α levels in WT and Irf3/7−/− mice 8 hours after immunization. (G) Frequency and absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and Irf3/7−/− mice. In ( A-G ), mice received 3 μg of Spikevax; n = 6-10 mice per group from 2-3 independent experiments. An unpaired two-tailed Mann-Whitney U test was conducted.
    Human Tlr7 Dual Reporter Hek 293 Cells, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+tlr7/HEK-Dual+hTLR7+Cells/pmc12878702-155-0-8
    Average 93 stars, based on 26 article reviews
    human tlr7 dual reporter hek 293 cells - by Bioz Stars, 2026-09
    93/100 stars

    Images

    1) Product Images from "Distinct components of mRNA vaccines cooperate to instruct efficient germinal center responses"

    Article Title: Distinct components of mRNA vaccines cooperate to instruct efficient germinal center responses

    Journal: Cell

    doi: 10.1016/j.cell.2025.11.023

    (A) Representative flow cytometry of Tfh cells, GC B cells, and Spike-specific GC B cells (S+) from wild type (WT) and Myd88 −/− mice. (B) Tfh cell, GC B cell, and S+ GC B cell absolute numbers, as detailed in A . (C) Absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and Tlr7 −/− mice. (D) Frequencies and absolute numbers of Tfh cells and GC B cells, as detailed in A , in mice treated with isotype, anti-IL-1R, or anti-IL-18 mAb. (E) IFN-α levels 8 hours after immunization. Mice were treated with isotype or anti-IL-1R mAb. (F) IFN-α levels in WT and Irf3/7−/− mice 8 hours after immunization. (G) Frequency and absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and Irf3/7−/− mice. In ( A-G ), mice received 3 μg of Spikevax; n = 6-10 mice per group from 2-3 independent experiments. An unpaired two-tailed Mann-Whitney U test was conducted.
    Figure Legend Snippet: (A) Representative flow cytometry of Tfh cells, GC B cells, and Spike-specific GC B cells (S+) from wild type (WT) and Myd88 −/− mice. (B) Tfh cell, GC B cell, and S+ GC B cell absolute numbers, as detailed in A . (C) Absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and Tlr7 −/− mice. (D) Frequencies and absolute numbers of Tfh cells and GC B cells, as detailed in A , in mice treated with isotype, anti-IL-1R, or anti-IL-18 mAb. (E) IFN-α levels 8 hours after immunization. Mice were treated with isotype or anti-IL-1R mAb. (F) IFN-α levels in WT and Irf3/7−/− mice 8 hours after immunization. (G) Frequency and absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and Irf3/7−/− mice. In ( A-G ), mice received 3 μg of Spikevax; n = 6-10 mice per group from 2-3 independent experiments. An unpaired two-tailed Mann-Whitney U test was conducted.

    Techniques Used: Flow Cytometry, Two Tailed Test, MANN-WHITNEY

    Related Articles

    Expressing:

    Article Title: Structure-aware machine learning identifies microRNAs operating as Toll-like receptor 7/8 ligands
    Article Snippet: .. HEK-Blue TM cells expressing mouse TLR7, human TLR7, or human TLR8, as well as the respective control cell lines HEK-Blue TM Null2-k, Null1-k and Null-1 (Invivogen, San Diego, CA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen #41,965,062, Carlsbad, CA, USA). .. The DMEM was supplemented with 10% heat-inactivated foetal calf serum (FCS, Gibco #10,082-147, Thermo Fisher Scientific, Waltham, MA, USA) and penicillin (100 U/ml)/streptomycin (100 μg/ml; Gibco #15,140-122, Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Hepatitis C Virus-Induced Exosomal MicroRNAs and Toll-Like Receptor 7 Polymorphism Regulate B-Cell Activating Factor
    Article Snippet: .. Finally, we used HEK293 cells stably expressing human TLR7 and containing an NF-κB reporter gene (InvivoGen, USA) as a specific cell model to validate whether HCV patient-derived exosomes modulate NF-κB and BAFF expression upon TLR7 engagement. ..

    Article Title: Substituted purines as TLR7 agonists
    Article Snippet: .. HEK293 cell lines stably expressing human TLR7 or TLR8 together with an NF-κB-driven-secreted alkaline phosphatase (SEAP) reporter were invented at InvivoGen (San Diego, CA, USA) and used to assess compounds of the present invention for TLR7 and TLR8 agonist activities. ..

    Article Title: The efficacy of a 2,4-diaminoquinazoline compound as an intranasal vaccine adjuvant to protect against influenza A virus infection in vivo.
    Article Snippet: .. Cells and viruses Human embryonic kidney (HEK) Blue cells expressing null (Null1), human TLR7 (hTLR7), human TLR8 (hTLR8), mouse TLR7 (mTLR7), and mouse TLR8 (mTLR8) were purchased from InvivoGen. .. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Atlas Biologicals) and antibiotics, such as blasticidin, Zeocin, and Normocin, according to the manufacturer’s instructions.

    Control:

    Article Title: Structure-aware machine learning identifies microRNAs operating as Toll-like receptor 7/8 ligands
    Article Snippet: .. HEK-Blue TM cells expressing mouse TLR7, human TLR7, or human TLR8, as well as the respective control cell lines HEK-Blue TM Null2-k, Null1-k and Null-1 (Invivogen, San Diego, CA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen #41,965,062, Carlsbad, CA, USA). .. The DMEM was supplemented with 10% heat-inactivated foetal calf serum (FCS, Gibco #10,082-147, Thermo Fisher Scientific, Waltham, MA, USA) and penicillin (100 U/ml)/streptomycin (100 μg/ml; Gibco #15,140-122, Thermo Fisher Scientific, Waltham, MA, USA).

    Cell Culture:

    Article Title: Structure-aware machine learning identifies microRNAs operating as Toll-like receptor 7/8 ligands
    Article Snippet: .. HEK-Blue TM cells expressing mouse TLR7, human TLR7, or human TLR8, as well as the respective control cell lines HEK-Blue TM Null2-k, Null1-k and Null-1 (Invivogen, San Diego, CA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen #41,965,062, Carlsbad, CA, USA). .. The DMEM was supplemented with 10% heat-inactivated foetal calf serum (FCS, Gibco #10,082-147, Thermo Fisher Scientific, Waltham, MA, USA) and penicillin (100 U/ml)/streptomycin (100 μg/ml; Gibco #15,140-122, Thermo Fisher Scientific, Waltham, MA, USA).

    Modification:

    Article Title: Structure-aware machine learning identifies microRNAs operating as Toll-like receptor 7/8 ligands
    Article Snippet: .. HEK-Blue TM cells expressing mouse TLR7, human TLR7, or human TLR8, as well as the respective control cell lines HEK-Blue TM Null2-k, Null1-k and Null-1 (Invivogen, San Diego, CA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen #41,965,062, Carlsbad, CA, USA). .. The DMEM was supplemented with 10% heat-inactivated foetal calf serum (FCS, Gibco #10,082-147, Thermo Fisher Scientific, Waltham, MA, USA) and penicillin (100 U/ml)/streptomycin (100 μg/ml; Gibco #15,140-122, Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: TLR8 is activated by 5'-methylthioinosine, a Plasmodium falciparum-derived intermediate of the purine salvage pathway.
    Article Snippet: Parasite cultures were routinely checked for mycoplasma contamination with a PCR mycoplasma test kit (ATCC, Manassas, US) or with a reporter HEK-293T cell line expressingTLR2-and a NF-kB luciferase (kind gift of Markus Schnare, Philipps University Marburg). .. Human embryonic kidney cells (HEK-293T) stably transfected with human TLR8 (293xl-htlr8) or human TLR7 (293xl-htlr7) were obtained from InvivoGen (Toulouse, France) and cultivated in Dulbecco’s modified Eagle’s medium (Life Technologies, Darmstadt, Germany) supplemented with 10% FCS (Gibco), 2 mM L-glutamine, 100 U/mL penicillin G and 100 mg/mL streptomycin sulphate (PAN Biotech, Aidenbach, Germany), 0.1% 2-mercaptoethanol (Gibco). ..

    Stable Transfection:

    Article Title: Hepatitis C Virus-Induced Exosomal MicroRNAs and Toll-Like Receptor 7 Polymorphism Regulate B-Cell Activating Factor
    Article Snippet: .. Finally, we used HEK293 cells stably expressing human TLR7 and containing an NF-κB reporter gene (InvivoGen, USA) as a specific cell model to validate whether HCV patient-derived exosomes modulate NF-κB and BAFF expression upon TLR7 engagement. ..

    Article Title: Substituted purines as TLR7 agonists
    Article Snippet: .. HEK293 cell lines stably expressing human TLR7 or TLR8 together with an NF-κB-driven-secreted alkaline phosphatase (SEAP) reporter were invented at InvivoGen (San Diego, CA, USA) and used to assess compounds of the present invention for TLR7 and TLR8 agonist activities. ..

    Article Title: TLR8 is activated by 5'-methylthioinosine, a Plasmodium falciparum-derived intermediate of the purine salvage pathway.
    Article Snippet: Parasite cultures were routinely checked for mycoplasma contamination with a PCR mycoplasma test kit (ATCC, Manassas, US) or with a reporter HEK-293T cell line expressingTLR2-and a NF-kB luciferase (kind gift of Markus Schnare, Philipps University Marburg). .. Human embryonic kidney cells (HEK-293T) stably transfected with human TLR8 (293xl-htlr8) or human TLR7 (293xl-htlr7) were obtained from InvivoGen (Toulouse, France) and cultivated in Dulbecco’s modified Eagle’s medium (Life Technologies, Darmstadt, Germany) supplemented with 10% FCS (Gibco), 2 mM L-glutamine, 100 U/mL penicillin G and 100 mg/mL streptomycin sulphate (PAN Biotech, Aidenbach, Germany), 0.1% 2-mercaptoethanol (Gibco). ..

    Article Title: Supporting Information Discovery of Imidazoquinolines with Toll-Like Receptor 7/8 Independent Cytokine Induction
    Article Snippet: .. Human embryonic kidney (HEK) cells that were stably transfected with human TLR7 or TLR8 and an NF-κB – responsive secreted embryonic alkaline phosphatase (SEAP) gene (HEK-TLR7/8) were purchased from InvivoGen (San Diego, CA). .. The procedure used to measure TLR7 or TLR8 agonist activity was conducted as described by Hood et al. 4 HEK-TLR7/8 cells were stimulated with 30 μM of compound in a 96-well plate in DMEM containing 10% FBS and 0.01% Normocin (InvivoGen) for 24 h. 20 μL of the supernatant from each well was incubated with Quanti-blue substrate solution (InvivoGen) at 37 °C for 1 h and absorbance was read at 650 nm using a Synergy plate reader (Biotek, Winooski, VT).

    Activation Assay:

    Article Title: Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response
    Article Snippet: .. For assessing TLR7 or TLR9 activation by CLs, HEK-Blue null, HEK-Blue human TLR7, and HEK-Blue human TLR9 reporter cells (InvivoGen) were stimulated with indicated concentrations of R-DOTAP, PMA (Sigma), R848 (InvivoGen), ODN2006 (InvivoGen), or ODN2395 (InvivoGen) for 24 h. After stimulation, cell supernatants were assayed for TLR and NF-κB–induced SEAP activity using QUANTI-Blue reagent according to the manufacturer’s instruction. ..

    Activity Assay:

    Article Title: Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response
    Article Snippet: .. For assessing TLR7 or TLR9 activation by CLs, HEK-Blue null, HEK-Blue human TLR7, and HEK-Blue human TLR9 reporter cells (InvivoGen) were stimulated with indicated concentrations of R-DOTAP, PMA (Sigma), R848 (InvivoGen), ODN2006 (InvivoGen), or ODN2395 (InvivoGen) for 24 h. After stimulation, cell supernatants were assayed for TLR and NF-κB–induced SEAP activity using QUANTI-Blue reagent according to the manufacturer’s instruction. ..

    Transfection:

    Article Title: TLR8 is activated by 5'-methylthioinosine, a Plasmodium falciparum-derived intermediate of the purine salvage pathway.
    Article Snippet: Parasite cultures were routinely checked for mycoplasma contamination with a PCR mycoplasma test kit (ATCC, Manassas, US) or with a reporter HEK-293T cell line expressingTLR2-and a NF-kB luciferase (kind gift of Markus Schnare, Philipps University Marburg). .. Human embryonic kidney cells (HEK-293T) stably transfected with human TLR8 (293xl-htlr8) or human TLR7 (293xl-htlr7) were obtained from InvivoGen (Toulouse, France) and cultivated in Dulbecco’s modified Eagle’s medium (Life Technologies, Darmstadt, Germany) supplemented with 10% FCS (Gibco), 2 mM L-glutamine, 100 U/mL penicillin G and 100 mg/mL streptomycin sulphate (PAN Biotech, Aidenbach, Germany), 0.1% 2-mercaptoethanol (Gibco). ..

    Article Title: Supporting Information Discovery of Imidazoquinolines with Toll-Like Receptor 7/8 Independent Cytokine Induction
    Article Snippet: .. Human embryonic kidney (HEK) cells that were stably transfected with human TLR7 or TLR8 and an NF-κB – responsive secreted embryonic alkaline phosphatase (SEAP) gene (HEK-TLR7/8) were purchased from InvivoGen (San Diego, CA). .. The procedure used to measure TLR7 or TLR8 agonist activity was conducted as described by Hood et al. 4 HEK-TLR7/8 cells were stimulated with 30 μM of compound in a 96-well plate in DMEM containing 10% FBS and 0.01% Normocin (InvivoGen) for 24 h. 20 μL of the supernatant from each well was incubated with Quanti-blue substrate solution (InvivoGen) at 37 °C for 1 h and absorbance was read at 650 nm using a Synergy plate reader (Biotek, Winooski, VT).



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    93
    OriGene tlr7
    Functional evaluation of the 6 rare <t>TLR7</t> variants. Previous known TLR7 LOF variants N75H, Q710Rfs*18 and V795F (References 12,22), GOF variant: R28G (Reference 71), and common variants Q11L and V222D were included in the functional evaluation as controls. A Immunobloting of TLR7 (140kD) WT and variants using N-terminal and C-terminal primary antibodies. B HEK293 T were or not stimulated with R848 1 μg/mL, CL264 5 μg/mL, R837 5 μg/mL for 24 h. NFκB response was measured using a Dual-Luciferase Reporter, Luciferase/Renilla ratios were normalized against the stimulated WT variant values. Mean ± SEM of n = 3 experiments. Two-way ANOVA with Dunnett’s post hoc test. Variants with less than 25% of the activity of the stimulated WT variant were considered LOF. EV: Empty vector; WT: Wild Type; LOF: Loss of function; GOF: Gain of function; NS: non-stimulated; * p < 0.0332; ** p < 0.0021; *** p < 0.0002; **** p < 0.0001
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    (A) Representative flow cytometry of Tfh cells, GC B cells, and Spike-specific GC B cells (S+) from wild type (WT) and Myd88 −/− mice. (B) Tfh cell, GC B cell, and S+ GC B cell absolute numbers, as detailed in A . (C) Absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and Tlr7 −/− mice. (D) Frequencies and absolute numbers of Tfh cells and GC B cells, as detailed in A , in mice treated with isotype, anti-IL-1R, or anti-IL-18 mAb. (E) IFN-α levels 8 hours after immunization. Mice were treated with isotype or anti-IL-1R mAb. (F) IFN-α levels in WT and Irf3/7−/− mice 8 hours after immunization. (G) Frequency and absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and Irf3/7−/− mice. In ( A-G ), mice received 3 μg of Spikevax; n = 6-10 mice per group from 2-3 independent experiments. An unpaired two-tailed Mann-Whitney U test was conducted.

    Journal: Cell

    Article Title: Distinct components of mRNA vaccines cooperate to instruct efficient germinal center responses

    doi: 10.1016/j.cell.2025.11.023

    Figure Lengend Snippet: (A) Representative flow cytometry of Tfh cells, GC B cells, and Spike-specific GC B cells (S+) from wild type (WT) and Myd88 −/− mice. (B) Tfh cell, GC B cell, and S+ GC B cell absolute numbers, as detailed in A . (C) Absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and Tlr7 −/− mice. (D) Frequencies and absolute numbers of Tfh cells and GC B cells, as detailed in A , in mice treated with isotype, anti-IL-1R, or anti-IL-18 mAb. (E) IFN-α levels 8 hours after immunization. Mice were treated with isotype or anti-IL-1R mAb. (F) IFN-α levels in WT and Irf3/7−/− mice 8 hours after immunization. (G) Frequency and absolute numbers of Tfh cells and GC B cells, as detailed in A , in WT and Irf3/7−/− mice. In ( A-G ), mice received 3 μg of Spikevax; n = 6-10 mice per group from 2-3 independent experiments. An unpaired two-tailed Mann-Whitney U test was conducted.

    Article Snippet: Human TLR7 Dual Reporter HEK 293 Cells , InvivoGen , Cat#hkd-htlr7.

    Techniques: Flow Cytometry, Two Tailed Test, MANN-WHITNEY

    Anti-human TLR7 mAb inhibits the response of TLR7 in human PBMCs and the human TLR7 transgenic mice. (A) Human TLR7 expression in the bone marrow (BM)-derived cDCs from WT or HuTLR7 transgenic (Tg) mice. Cells were fixed and permeabilized for the detection of HuTLR7. (B) Inhibition of TLR7 response in the BM-cDCs derived from HuTLR7-Tg mice. After 4 hours of incubation with Abs, cells were stimulated by TLR7 or TLR9 ligand. Cells were incubated for 24 hours, and the concentrations of IL-12 p40 in the culture medium were measured by ELISA. (C) The inhibitory effect of clone S1 on TLR7 response in HuTLR7-Tg mice. 25 mg/kg of anti-HuTLR7 mAb (clone S1) and control IgG1 (clone TF904) were administrated to WT ( N = 5) and HuTLR7-Tg mice ( N = 6). Seven days after Ab administration, 20 μg of R848 was intraperitoneally administered. Six hours after R848 injection, blood was collected and the serum levels of IL-12 p40 were determined by ELISA. (D) TLR7 and TLR8 expression in human PBMCs. After staining with mAbs to indicated lineage markers, the cells were fixed and intracellular TLR7 or TLR8 was stained by clone S1 or anti-HuTLR8 (clone GH1.7), respectively. (E) Inhibition of TLR7 responses by anti-TLR7 mAb. Human PBMCs were treated with mAbs for 4 hours, and then stimulated with TLR7 ligand (CL264) or TLR9 ligand (CpG-K3). 24 hours after stimulation, culture supernatants were collected and concentrations of IL-6 were measured by ELISA. At least 3 independent experiments were performed (A-E), and representative histograms were shown (A and D). Wells of ELISA were triplicated, and the mean SD was shown (B and E). Dots in the graph indicate the data from individual mice (C). PBMCs were collected from at least 3 healthy donors (D and E). Data were statistically analyzed by one-way ANOVA with multiple comparisons (B and E) or Student’s T-test (C). N.S., not significant; ** P < 0.01; *** P < .001. See also Supplementary Fig. 4.

    Journal: International Immunology

    Article Title: Anti-human TLR7 antibody for therapeutic intervention in systemic lupus erythematosus

    doi: 10.1093/intimm/dxaf046

    Figure Lengend Snippet: Anti-human TLR7 mAb inhibits the response of TLR7 in human PBMCs and the human TLR7 transgenic mice. (A) Human TLR7 expression in the bone marrow (BM)-derived cDCs from WT or HuTLR7 transgenic (Tg) mice. Cells were fixed and permeabilized for the detection of HuTLR7. (B) Inhibition of TLR7 response in the BM-cDCs derived from HuTLR7-Tg mice. After 4 hours of incubation with Abs, cells were stimulated by TLR7 or TLR9 ligand. Cells were incubated for 24 hours, and the concentrations of IL-12 p40 in the culture medium were measured by ELISA. (C) The inhibitory effect of clone S1 on TLR7 response in HuTLR7-Tg mice. 25 mg/kg of anti-HuTLR7 mAb (clone S1) and control IgG1 (clone TF904) were administrated to WT ( N = 5) and HuTLR7-Tg mice ( N = 6). Seven days after Ab administration, 20 μg of R848 was intraperitoneally administered. Six hours after R848 injection, blood was collected and the serum levels of IL-12 p40 were determined by ELISA. (D) TLR7 and TLR8 expression in human PBMCs. After staining with mAbs to indicated lineage markers, the cells were fixed and intracellular TLR7 or TLR8 was stained by clone S1 or anti-HuTLR8 (clone GH1.7), respectively. (E) Inhibition of TLR7 responses by anti-TLR7 mAb. Human PBMCs were treated with mAbs for 4 hours, and then stimulated with TLR7 ligand (CL264) or TLR9 ligand (CpG-K3). 24 hours after stimulation, culture supernatants were collected and concentrations of IL-6 were measured by ELISA. At least 3 independent experiments were performed (A-E), and representative histograms were shown (A and D). Wells of ELISA were triplicated, and the mean SD was shown (B and E). Dots in the graph indicate the data from individual mice (C). PBMCs were collected from at least 3 healthy donors (D and E). Data were statistically analyzed by one-way ANOVA with multiple comparisons (B and E) or Student’s T-test (C). N.S., not significant; ** P < 0.01; *** P < .001. See also Supplementary Fig. 4.

    Article Snippet: Humanized anti-human TLR7 mAb (DS-7011a) and isotype control were prepared by Daiichi Sankyo Co., Ltd. Anti-Mouse IgG, anti-mouse IgG1, streptavidin (StAv), anti-DYKDDDDK, anti-mouse CD3, anti-mouse CD19, anti-mouse B220, anti-mouse CD21, anti-mouse CD23, anti-mouse CD11c, anti-mouse I-A/I-E, anti-mouse Ly6C, anti-mouse Ly6G, anti-mouse Siglec-H, anti-human CD19, anti-human CD4, anti-HLA-DR, anti-human CD11c, anti-human CD1c, and anti-human CD16 were purchased from BioLegend (San Diego, CA, USA).

    Techniques: Transgenic Assay, Expressing, Derivative Assay, Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Control, Injection, Staining

    Humanized anti-TLR7 mAb, DS-7011a, is internalized into cells and inhibits the TLR7 response. (A–C) Intracellular staining of Ba/F3 cells expressing indicated TLRs. Gray histograms and red histograms show staining with the isotype control and DS-7011a, respectively. Alexa fluor 488 (AF488)-labeled antibodies were used for (C). (D) Inhibitory effect of DS-7011a on IL-6 production by CL264 stimulation and IFN-α production by ssRNA9.2s stimulation in human PBMCs. TLR7-dependent cytokine production by DS-7011a. Whole human PBMCs were incubated with DS-7011a for 4 hours before stimulation with CL264 (1 μg/mL) or ssRNA9.2s (1 μg/mL) as a TLR7 ligand, respectively. Twenty hours after stimulation, culture supernatants were collected and concentrations of IL-6 and IFN-α were measured by AlphaLISA. (E) Inhibitory effect of DS-7011a on IFN-α production by ssRNA9.2s stimulation in purified human pDCs. Purified human pDCs were incubated with DS-7011a for 4 hours before stimulation with ssRNA9.2s (1 μg/mL) as a TLR7 ligand. Twenty hours after stimulation, culture supernatants were collected and concentration of IFN-α was measured by AlphaLISA. (F) Inhibition of TLR7-dependent IgG1 production by DS-7011a. Purified human B cells were incubated with DS-7011a for 4 hours before stimulation with CL264. Five days after stimulation, culture supernatants of B cells were collected, and concentrations of IgG1 were measured by cell-based ELISA. (G) Illustration of the method for antibody uptake assay. (H) Internalization of DS-7011a in human PBMCs. AF488-labeled humanized anti-TLR7 mAb (DS-7011a) and isotype control mAb were incubated with PBMCs for 24 hours at 37°C. Antibody on the cell surface was quenched by anti-AF488, and the fluorescence of internalized antibody was detected by flow cytometry. Gray histograms show the data without antibody. Red histograms show the data with AF488-labeled antibodies. At least three independent experiments were performed, and representative histograms were shown (A–C, and H). PBMCs, purified pDCs, and purified B cells were collected from at least 2 healthy donors (A–F, H, and Supplementary Fig. 5). Wells of AlphaLISA or cell-based ELISA were triplicated, and the mean SD was shown (D–F). See also Supplementary Fig. 5.

    Journal: International Immunology

    Article Title: Anti-human TLR7 antibody for therapeutic intervention in systemic lupus erythematosus

    doi: 10.1093/intimm/dxaf046

    Figure Lengend Snippet: Humanized anti-TLR7 mAb, DS-7011a, is internalized into cells and inhibits the TLR7 response. (A–C) Intracellular staining of Ba/F3 cells expressing indicated TLRs. Gray histograms and red histograms show staining with the isotype control and DS-7011a, respectively. Alexa fluor 488 (AF488)-labeled antibodies were used for (C). (D) Inhibitory effect of DS-7011a on IL-6 production by CL264 stimulation and IFN-α production by ssRNA9.2s stimulation in human PBMCs. TLR7-dependent cytokine production by DS-7011a. Whole human PBMCs were incubated with DS-7011a for 4 hours before stimulation with CL264 (1 μg/mL) or ssRNA9.2s (1 μg/mL) as a TLR7 ligand, respectively. Twenty hours after stimulation, culture supernatants were collected and concentrations of IL-6 and IFN-α were measured by AlphaLISA. (E) Inhibitory effect of DS-7011a on IFN-α production by ssRNA9.2s stimulation in purified human pDCs. Purified human pDCs were incubated with DS-7011a for 4 hours before stimulation with ssRNA9.2s (1 μg/mL) as a TLR7 ligand. Twenty hours after stimulation, culture supernatants were collected and concentration of IFN-α was measured by AlphaLISA. (F) Inhibition of TLR7-dependent IgG1 production by DS-7011a. Purified human B cells were incubated with DS-7011a for 4 hours before stimulation with CL264. Five days after stimulation, culture supernatants of B cells were collected, and concentrations of IgG1 were measured by cell-based ELISA. (G) Illustration of the method for antibody uptake assay. (H) Internalization of DS-7011a in human PBMCs. AF488-labeled humanized anti-TLR7 mAb (DS-7011a) and isotype control mAb were incubated with PBMCs for 24 hours at 37°C. Antibody on the cell surface was quenched by anti-AF488, and the fluorescence of internalized antibody was detected by flow cytometry. Gray histograms show the data without antibody. Red histograms show the data with AF488-labeled antibodies. At least three independent experiments were performed, and representative histograms were shown (A–C, and H). PBMCs, purified pDCs, and purified B cells were collected from at least 2 healthy donors (A–F, H, and Supplementary Fig. 5). Wells of AlphaLISA or cell-based ELISA were triplicated, and the mean SD was shown (D–F). See also Supplementary Fig. 5.

    Article Snippet: Humanized anti-human TLR7 mAb (DS-7011a) and isotype control were prepared by Daiichi Sankyo Co., Ltd. Anti-Mouse IgG, anti-mouse IgG1, streptavidin (StAv), anti-DYKDDDDK, anti-mouse CD3, anti-mouse CD19, anti-mouse B220, anti-mouse CD21, anti-mouse CD23, anti-mouse CD11c, anti-mouse I-A/I-E, anti-mouse Ly6C, anti-mouse Ly6G, anti-mouse Siglec-H, anti-human CD19, anti-human CD4, anti-HLA-DR, anti-human CD11c, anti-human CD1c, and anti-human CD16 were purchased from BioLegend (San Diego, CA, USA).

    Techniques: Staining, Expressing, Control, Labeling, Incubation, Purification, Concentration Assay, Inhibition, In-Cell ELISA, Fluorescence, Flow Cytometry

    TLR7 gene signature scores are enhanced in SLE patients and their subgroups. (A) Comparison of SLE patients and healthy subjects for TLR7 and TLR8 gene signature scores in blood samples ( GSE88884 , SLE, N = 1760; healthy, N = 60. GSE65391 , SLE, N = 924; healthy, N = 72. GSE49454 , SLE, N = 157; healthy, N = 20). (B) Ethnic differences of TLR7 gene signature scores (African American, N = 233; Native American, N = 249; Asian, N = 19; Multiple, N = 39; Caucasian, N = 1215) ( GSE88884 ). (C) TLR7 gene signature scores of SLE patients with or without drug treatment (No treatment, N = 40; steroid (iv), N = 86; steroid (po), N = 331; HCQ, N = 190; MMF, N = 358) and healthy subjects ( N = 72) in blood samples ( GSE65391 ). Data were statistically analyzed by Welch’s two-sample t -test (A), Dunnett’s multiple comparison test (B), and Tukey’s multiple comparison test (C). N.S., not significant; **, P < .01; ***, P < .001. See also , , and Supplementary Table 1.

    Journal: International Immunology

    Article Title: Anti-human TLR7 antibody for therapeutic intervention in systemic lupus erythematosus

    doi: 10.1093/intimm/dxaf046

    Figure Lengend Snippet: TLR7 gene signature scores are enhanced in SLE patients and their subgroups. (A) Comparison of SLE patients and healthy subjects for TLR7 and TLR8 gene signature scores in blood samples ( GSE88884 , SLE, N = 1760; healthy, N = 60. GSE65391 , SLE, N = 924; healthy, N = 72. GSE49454 , SLE, N = 157; healthy, N = 20). (B) Ethnic differences of TLR7 gene signature scores (African American, N = 233; Native American, N = 249; Asian, N = 19; Multiple, N = 39; Caucasian, N = 1215) ( GSE88884 ). (C) TLR7 gene signature scores of SLE patients with or without drug treatment (No treatment, N = 40; steroid (iv), N = 86; steroid (po), N = 331; HCQ, N = 190; MMF, N = 358) and healthy subjects ( N = 72) in blood samples ( GSE65391 ). Data were statistically analyzed by Welch’s two-sample t -test (A), Dunnett’s multiple comparison test (B), and Tukey’s multiple comparison test (C). N.S., not significant; **, P < .01; ***, P < .001. See also , , and Supplementary Table 1.

    Article Snippet: Humanized anti-human TLR7 mAb (DS-7011a) and isotype control were prepared by Daiichi Sankyo Co., Ltd. Anti-Mouse IgG, anti-mouse IgG1, streptavidin (StAv), anti-DYKDDDDK, anti-mouse CD3, anti-mouse CD19, anti-mouse B220, anti-mouse CD21, anti-mouse CD23, anti-mouse CD11c, anti-mouse I-A/I-E, anti-mouse Ly6C, anti-mouse Ly6G, anti-mouse Siglec-H, anti-human CD19, anti-human CD4, anti-HLA-DR, anti-human CD11c, anti-human CD1c, and anti-human CD16 were purchased from BioLegend (San Diego, CA, USA).

    Techniques: Comparison

    Development of anti-human TLR7 monoclonal antibodies. (A) Staining pattern of anti-HuTLR7 monoclonal antibodies (mAbs). Ba/F3 cells were fixed and permeabilized for intracellular staining of HuTLR7-FH (FLAG-6xHis) and mouse TLR7 (MsTLR7)-FH. (B) NF-κB-GFP reporter assay using the B-cell lymphoma cell line Ramos. Cells were treated by 10 μg/mL of mAbs for 4 hours and stimulated with the indicated ligands. 22 hours after stimulation, the expression of GFP was measured with flow cytometry to detect the activation of NF-κB. (C) Representative histograms of (B). (D) Ramos cells were treated with the indicated mAbs at the indicated concentrations for 4 hours and stimulated with 250 ng/mL of R848. The expression of NF-κB-GFP was measured by flow cytometry. At least three independent experiments were performed (A-D). Data are shown as individual points and as means for each experimental group (B and D). See also Supplementary Fig. 3.

    Journal: International Immunology

    Article Title: Anti-human TLR7 antibody for therapeutic intervention in systemic lupus erythematosus

    doi: 10.1093/intimm/dxaf046

    Figure Lengend Snippet: Development of anti-human TLR7 monoclonal antibodies. (A) Staining pattern of anti-HuTLR7 monoclonal antibodies (mAbs). Ba/F3 cells were fixed and permeabilized for intracellular staining of HuTLR7-FH (FLAG-6xHis) and mouse TLR7 (MsTLR7)-FH. (B) NF-κB-GFP reporter assay using the B-cell lymphoma cell line Ramos. Cells were treated by 10 μg/mL of mAbs for 4 hours and stimulated with the indicated ligands. 22 hours after stimulation, the expression of GFP was measured with flow cytometry to detect the activation of NF-κB. (C) Representative histograms of (B). (D) Ramos cells were treated with the indicated mAbs at the indicated concentrations for 4 hours and stimulated with 250 ng/mL of R848. The expression of NF-κB-GFP was measured by flow cytometry. At least three independent experiments were performed (A-D). Data are shown as individual points and as means for each experimental group (B and D). See also Supplementary Fig. 3.

    Article Snippet: Humanized anti-human TLR7 mAb (DS-7011a) and isotype control were prepared by Daiichi Sankyo Co., Ltd. Anti-Mouse IgG, anti-mouse IgG1, streptavidin (StAv), anti-DYKDDDDK, anti-mouse CD3, anti-mouse CD19, anti-mouse B220, anti-mouse CD21, anti-mouse CD23, anti-mouse CD11c, anti-mouse I-A/I-E, anti-mouse Ly6C, anti-mouse Ly6G, anti-mouse Siglec-H, anti-human CD19, anti-human CD4, anti-HLA-DR, anti-human CD11c, anti-human CD1c, and anti-human CD16 were purchased from BioLegend (San Diego, CA, USA).

    Techniques: Bioprocessing, Staining, Reporter Assay, Expressing, Flow Cytometry, Activation Assay

    Inhibitory clones of anti-humanTLR7 mAb recognize the N-terminal region of TLR7. (A) Membrane-permeabilized staining of Ba/F3 cells expressing human/mouse chimeric TLR7 proteins with indicated clones. Configurations of chimeric proteins are shown to the right. The transmembrane region and TIR domain were not shown. (B) Schematic representation of the structure of monkey TLR7 (PDB ID: 5GMF). The amino acid sequence of the epitope of clone S1 is the same in human TLR7 and monkey TLR7. Front (left) and top (upper right) views, and molecular surface (lower right) are shown. TLR7 and its dimerization partner are colored green and gray. The red regions from N276 to I313 show the epitope of clone S1. The blue and yellow regions show nucleoside-binding sites and dimerization interfaces, respectively. The C, O, N, and P atoms of the ligands are colored yellow (uridine) or cyan (ssRNA), red, blue, and orange, respectively. At least 3 independent experiments were performed, and representative data were shown (A).

    Journal: International Immunology

    Article Title: Anti-human TLR7 antibody for therapeutic intervention in systemic lupus erythematosus

    doi: 10.1093/intimm/dxaf046

    Figure Lengend Snippet: Inhibitory clones of anti-humanTLR7 mAb recognize the N-terminal region of TLR7. (A) Membrane-permeabilized staining of Ba/F3 cells expressing human/mouse chimeric TLR7 proteins with indicated clones. Configurations of chimeric proteins are shown to the right. The transmembrane region and TIR domain were not shown. (B) Schematic representation of the structure of monkey TLR7 (PDB ID: 5GMF). The amino acid sequence of the epitope of clone S1 is the same in human TLR7 and monkey TLR7. Front (left) and top (upper right) views, and molecular surface (lower right) are shown. TLR7 and its dimerization partner are colored green and gray. The red regions from N276 to I313 show the epitope of clone S1. The blue and yellow regions show nucleoside-binding sites and dimerization interfaces, respectively. The C, O, N, and P atoms of the ligands are colored yellow (uridine) or cyan (ssRNA), red, blue, and orange, respectively. At least 3 independent experiments were performed, and representative data were shown (A).

    Article Snippet: Humanized anti-human TLR7 mAb (DS-7011a) and isotype control were prepared by Daiichi Sankyo Co., Ltd. Anti-Mouse IgG, anti-mouse IgG1, streptavidin (StAv), anti-DYKDDDDK, anti-mouse CD3, anti-mouse CD19, anti-mouse B220, anti-mouse CD21, anti-mouse CD23, anti-mouse CD11c, anti-mouse I-A/I-E, anti-mouse Ly6C, anti-mouse Ly6G, anti-mouse Siglec-H, anti-human CD19, anti-human CD4, anti-HLA-DR, anti-human CD11c, anti-human CD1c, and anti-human CD16 were purchased from BioLegend (San Diego, CA, USA).

    Techniques: Clone Assay, Membrane, Staining, Expressing, Sequencing, Binding Assay

    Functional evaluation of the 6 rare TLR7 variants. Previous known TLR7 LOF variants N75H, Q710Rfs*18 and V795F (References 12,22), GOF variant: R28G (Reference 71), and common variants Q11L and V222D were included in the functional evaluation as controls. A Immunobloting of TLR7 (140kD) WT and variants using N-terminal and C-terminal primary antibodies. B HEK293 T were or not stimulated with R848 1 μg/mL, CL264 5 μg/mL, R837 5 μg/mL for 24 h. NFκB response was measured using a Dual-Luciferase Reporter, Luciferase/Renilla ratios were normalized against the stimulated WT variant values. Mean ± SEM of n = 3 experiments. Two-way ANOVA with Dunnett’s post hoc test. Variants with less than 25% of the activity of the stimulated WT variant were considered LOF. EV: Empty vector; WT: Wild Type; LOF: Loss of function; GOF: Gain of function; NS: non-stimulated; * p < 0.0332; ** p < 0.0021; *** p < 0.0002; **** p < 0.0001

    Journal: Journal of Clinical Immunology

    Article Title: From Rare to Common: Genetic Insights into TLR7 Variants in a Multicentric Spanish Study on COVID-19 Severity

    doi: 10.1007/s10875-025-01892-0

    Figure Lengend Snippet: Functional evaluation of the 6 rare TLR7 variants. Previous known TLR7 LOF variants N75H, Q710Rfs*18 and V795F (References 12,22), GOF variant: R28G (Reference 71), and common variants Q11L and V222D were included in the functional evaluation as controls. A Immunobloting of TLR7 (140kD) WT and variants using N-terminal and C-terminal primary antibodies. B HEK293 T were or not stimulated with R848 1 μg/mL, CL264 5 μg/mL, R837 5 μg/mL for 24 h. NFκB response was measured using a Dual-Luciferase Reporter, Luciferase/Renilla ratios were normalized against the stimulated WT variant values. Mean ± SEM of n = 3 experiments. Two-way ANOVA with Dunnett’s post hoc test. Variants with less than 25% of the activity of the stimulated WT variant were considered LOF. EV: Empty vector; WT: Wild Type; LOF: Loss of function; GOF: Gain of function; NS: non-stimulated; * p < 0.0332; ** p < 0.0021; *** p < 0.0002; **** p < 0.0001

    Article Snippet: A TLR7 vector template (pCMV6-TLR7) was generated by inserting TLR7 (RC207515, OriGene, Rockville, MD, USA) into a pCMV6-AC-Myc-DDK Mammalian Expression Vector (PS100007; OriGene).

    Techniques: Functional Assay, Variant Assay, Western Blot, Luciferase, Activity Assay, Plasmid Preparation