Human TLR7 Reporter HEK293 Cells (NF-κB and IRF)

NF-κB–SEAP and IRF–Lucia reporter HEK293 cells expressing human TLR7

Signaling pathways in HEK-Dual™ hTLR7 cells
(click to enlarge and see legend)

HEK-Dual™ hTLR7 cells were engineered from HEK-Dual™ cells, a human embryonic kidney HEK293-derived cell line, to study the human Toll-like receptor 7 (TLR7)-dependent NF-κB and IRF pathways. This important pattern recognition receptor (PRR) recognizes - together with TLR8 - single-stranded (ss)RNA structures, a hallmark of viral replication, and triggers antiviral NF-κB and IRF immune responses [1].

 More details

Description

HEK-Dual™ hTLR7 cells feature the stable expression of the TLR7 gene and a mutated (mut) version of the chaperone protein UNC93B1. They feature two inducible reporter genes for SEAP (secreted embryonic alkaline phosphatase) and Lucia luciferase. As a result, these cells allow the simultaneous study of the NF-κB pathway, by monitoring the activity of SEAP, and the IRF pathway, by assessing the activity of the secreted Lucia luciferase. Upon TLR7 activation, both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Blue™ Solution, a SEAP detection reagent, and QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia luciferase detection reagent. 

HEK-Dual™ hTLR7 cells show potent NF-κB and IRF responses upon incubation with TLR7-specific ligands, such as imiquimod when compared to HEK-Blue™ hTLR7 cells and their parental cell line HEK-Dual™ (see figures). They do not respond to TLR8-specific ligands. However, as they express endogenous levels of various PRRs, such as TLR3 and TLR5, HEK-Blue™ hTLR7 cells may respond to their cognate ligands (see figures).

Key features

• Stable expression of human TLR7 and UNC93B1^mut
• Strong response to TLR7/8- and TLR7-specific ligands
• Distinct monitoring of TLR7-dependent NF-κB or IRF activation by assessing the SEAP and Lucia luciferase activities

Applications

• Defining the role of TLR7-dependent IRF and NF-κB signaling pathways
• Screening for novel TLR7 agonists and inhibitors

References

1. Heil F. et al., 2004. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science. 303:1526.

Figures

NF-κB responses of HEK-Blue™ hTLR7 vs. HEK-Dual™ hTLR7​. HEK-Blue™ hTLR7 and HEK-Dual™ hTLR7 cells were stimulated with various TLR agonists and cytokines: TNF-α (10 ng/ml), Imiquimod (TLR7 agonist; 1 µg/ml), CL264 (TLR7 ligand; 300 ng/ml), R848 (TLR7/8 agonist; 100 ng/ml), and TL8-506 (TLR8 agonist; 10 µg/ml). After overnight incubation, the activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

NF-κB responses in HEK-Dual™ -derived cells. HEK-Dual™ and HEK-Dual™ hTLR7 cells were incubated for 24 hours with cytokines and various TLR agonists: Human TNF-α (NF-κB-positive control, 1 ng/ml), hIFN-β (IRF-positive control, 1000 U/ml), Pam3CSK4 (TLR2 ligand, 100 ng/ml), Poly(I:C) HMW (TLR3 ligand, 10 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 100 ng/ml), FLA-ST UP (TLR5 ligand, 100 ng/ml), Imiquimod (TLR7 ligand, 1 µg/ml), CL264 (TLR7 ligand, 1 µg/ml) R848 (TLR7/8 ligand, 1 µg/ml), CL075 (TLR7/8 ligand, 1 µg/ml), TL8-506 (TLR8 ligand, 1 µg/ml), and ODN 2006 (TLR9 ligand, 10 µg/ml). After 24h incubation, the NF-κB-induced SEAP activity was assessed using QUANTI-Blue™. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

IRF responses in HEK-Dual™ -derived cells. HEK-Dual™ and HEK-Dual™ hTLR7 cells were incubated for 24 hours with cytokines and various TLR agonists: Human TNF-α (NF-κB-positive control, 1 ng/ml), hIFN-β (IRF-positive control, 10 U/ml), Pam3CSK4 (TLR2 ligand, 100 ng/ml), Poly(I:C) HMW (TLR3 ligand, 1 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 100 ng/ml), FLA-ST UP (TLR5 ligand, 100 ng/ml), Imiquimod (TLR7 ligand, 10 µg/ml), CL264 (TLR7 ligand, 10 µg/ml), R848 (TLR7/8 ligand, 1 µg/ml), CL075 (TLR7/8 ligand, 10 µg/ml), TL8-506 (TLR8 ligand, 1 µg/ml), and ODN 2006 (TLR9 ligand, 10 µg/ml). After 24h incubation, the IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown in fold response over non-induced cells (mean ± SEM).

Specifications

Antibiotic resistance: Blasticidin, Hygromycin B, Puromycin, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™

Quality Control:

• Stable expression of human TLR7 and UNC93B1^mut has been verified by RT-qPCR and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free.

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml Blasticidin (10 mg/ml)
• 1 ml Hygromycin B Gold (100 mg/ml)
• 1 ml Puromycin (10 mg/ml)
• 1 ml Zeocin® (100 mg/ml)
• 1 ml Normocin™ (50 mg/ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Details

Toll-Like Receptor 7

In humans, four Toll-Like Receptor (TLR) family members TLR3, TLR7, TLR8, and TLR9, mainly found in the endosome, are specialized in sensing viral-derived components. TLR7 and TLR8 recognize single-stranded (ss)RNA structures, such as viral ssRNA, miRNA, and various synthetic agonists [1]. Despite their similarities in PAMP (pathogen-associated molecular pattern) recognition, structure, and signaling partners, they highly differ in expression profiles and signaling responses, with TLR7 being more involved in the antiviral immune response and TLR8 mastering the production of proinflammatory cytokines [2]. TLR7 is mainly found in plasmacytoid dendritic cells (pDCs) and B cells, whereas TLR8 is highly expressed in monocytes, monocyte-derived DCs (mDCs), and macrophages [3].

TLR7 trafficking and signaling

Upon viral infection, the chaperone protein UNC93B1 interacts with TLR7, thereby facilitating its trafficking from the endoplasmic reticulum via the Golgi into the endosomes [4]. Subsequently, TLR7 undergoes proteolytic cleavage and dimerization [1,3]. Once activated, it recruits the adaptor protein MyD88 to trigger IRF, AP-1, and NF-kB responses via TRAF6 (TNF receptor-associated factor 6) [1,3]. Depending on the stimulus and cell type, TLR7-mediated signaling induces IFN-α and IFN-regulated cytokines or T helper 17 (Th17) polarizing cytokines, such as interleukin (IL)-1β and IL-23 [5].

TLR7 therapeutic targeting

The involvement of nucleic acid-sensing mechanisms in the immune response against infections and other diseases makes them interesting targets for drug design [5]. TLR7 agonists are currently been tested as vaccine adjuvants and immunomodulatory therapeutics. They are extensively studied in the context of viral infection (e.g. SARS-CoV-2, Influenza, HIV), autoimmune (e.g. asthma, Lupus), and autoinflammatory diseases (e.g. cancer) [1-5]. Understanding the fundamental differences between these two related receptors could potentially be harnessed to discover novel drugs and improve vaccine efficacy/safety [5].

​References:

1. Martínez-Espinoza I & Guerrero-Plata A. 2022. The Relevance of TLR8 in Viral Infections. Pathogens. 11(2):134.
2. Salvi V, et al., 2021. SARS-CoV-2-associated ssRNAs activate inflammation and immunity via TLR7/8. JCI Insight.;6(18):e150542.
3. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31.
4. Majer, O., et al. 2019. UNC93B1 recruits syntenin-1 to dampen TLR7 signalling and prevent autoimmunity. Nature 575, 366–370.
5. de Marcken M, et al., 2019. TLR7 and TLR8 activate distinct pathways in monocytes during RNA virus infection. Sci Signal.;12(605):eaaw1347.  

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