Human TLR9 Reporter HEK293 Cells (NF-κB)

NF-κB–SEAP reporter HEK293 cells expressing human TLR9

Signaling pathways in HEK-Blue™ hTLR9 cells

HEK-Blue™ hTLR9 cells were engineered from the human embryonic kidney HEK293 cell line to study the Toll-like receptor 9 (TLR9)-dependent NF-κB pathway. This important pattern recognition receptor (PRR) recognizes unmethylated CpG dinucleotides, a hallmark of microbial or host-derived self DNA and subsequently triggers NF-κB and IRF immune responses [1].

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Description

HEK-Blue™ hTLR9 cells feature the stable expression of the human TLR9 gene as well as an inducible reporter gene for SEAP (secreted embryonic alkaline phosphatase). SEAP levels produced upon TLR9 stimulation can be readily determined by performing the assay in HEK-Blue™ Detection, a cell culture medium that allows for real-time detection of SEAP. Alternatively, SEAP activity may be monitored using QUANTI-Blue™ , a SEAP detection reagent.

HEK-Blue™ hTLR9 cells are highly responsive to TLR9 agonists, such as oligonucleotides containing CpG motifs (CpG ODNs). They show potent NF-κB responses upon incubation of CpG-ODNs of class A (e.g. ODN 2216), class B (e.g. ODN 2006), and class C (e.g. ODN 2395), when compared to their parental cell line HEK-Blue™ Null1 (see figures). 

As TLR9 activates both the NF-κB and IRF signaling pathways, InvivoGen also offers HEK-Dual™ hTLR9 cells; a cell line expressing a dual reporter system comprising an NF-κB-inducible SEAP and an IRF-inducible-Lucia luciferase ( learn more here).

Of note, HEK293 cells express endogenous levels of various PRRs, including TLR3, TLR5, and RIG-I-like receptors, and therefore might respond to their cognate ligands (see figures). 

Key features

• Stable expression of human TLR9
• Strong response to synthetic DNA containing CpG motifs
• Distinct monitoring of TLR9-dependent NF-κB activation by assessing the SEAP activities

Applications

• Defining the role of TLR9-dependent NF-κB signaling pathway
• Screening for novel TLR9 agonists and inhibitors in comparison with their parental cell line HEK-Blue™ Null1
• Studying the differences between human and mouse TLR9 when used with HEK-Blue™ mTLR9 cells

1. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.

Figures

Response of HEK-Blue™-derived cells to TLR9 agonists. HEK-Blue™ Null1 and HEK-Blue™ hTLR9 cells were cultured in HEK-Blue™ Detection reagent and stimulated for 24 hours with 10 µM of the following TLR9 agonists: ODN 2216, ODN 1826, ODN 2006, and ODN 2395. Human TNF-α (1 ng/ml) served as an NF-κB-positive control. After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Dose-response of HEK-Blue™ hTLR9 cells to TLR9 agonists. Cells were cultured in HEK-Blue™ Detection reagent and stimulated with increasing concentrations of ODN 2216, ODN 1826, ODN 2006, and ODN 2395. After 24 hour incubation, the NF‑κB‑induced SEAP activity was determined by reading the OD at 650 nm (mean ± SEM).

Response of HEK-Blue™ hTLR9 cells to various PRR agonists and cytokines. Cells were cultured in HEK-Blue™ Detection reagent and stimulated for 24 hours with cytokines and various TLR agonists: Human TNF-α (NF-κB-positive control, 1 ng/ml), Pam3CSK4 (TLR2 ligand, 100 ng/ml), Poly(I:C) HMW (TLR3 ligand, 100 ng/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 100 ng/ml), FLA-ST UP (TLR5 ligand, 100 ng/ml), R848 (TLR7/8 ligand, 10 µg/ml), ODN 2006 (TLR9 ligand, 10 µg/ml), Tri-DAP (NOD1 ligand, 100 ng/ml), and MDP (NOD2 ligand, 100 ng/ml). After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as OD at 650 nm (mean ± SEM).

NF-κB responses of HEK-Blue™ hTLR9 vs. HEK-Dual™ hTLR9​. HEK-Blue™ hTLR9 and HEK-Dual™ hTLR9 cells were stimulated with various TLR9 agonists and cytokines: TNF-α (10 ng/ml), ODN 2216 (class A, human TLR9-preferred, 10 µM), ODN 1826 (class B, mouse TLR9-preferred, 10 µM), ODN 2006 (class B, human TLR9-preferred, 1 µM) or ODN 2395 (class C, human/mouse TLR9-preferred, 10 µM). 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).

Specifications

Antibiotic resistance: Blasticidin, 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 (h)TLR9 has been verified by RT-qPCR and functional assays.
• The activation of NF-κB/AP1 upon TLR9 stimulation has been verified using functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free.

Note: HEK293 cells express endogenous levels of TLR3, TLR5, and NOD1.
The appropriate parental cell line for HEK-Blue™ hTLR9 cells is HEK-Blue™ Null1. 

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 pouch of HEK-Blue™ Detection (cell culture medium for real-time detection of SEAP)

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

Details

Toll-Like Receptor 9 (TLR9)

The Toll-Like Receptor 9 (TLR9) is an endosomal receptor that triggers NF-κB- and IRF-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [1-3]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA, as well as mitochondrial self-DNA [3,4]. These TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs), which have been extensively studied to improve adaptive immune responses in the context of vaccination [1,3].

TLR9 is mainly expressed in subsets of Dendritic Cells and in B cells of all mammals. In rodents, but not in humans, TLR9 is also expressed in monocytes and macrophages [3]. The structure of the receptor varies by 24% between human TLR9 (hTLR9) and mouse TLR9 (mTLR9) [3]. They recognize different CpG motifs, the optimal sequences being GTCGTT and GACGTT for hTLR9 and mTLR9, respectively [5].
 

CpG ODNs

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs), such as ODN 1018, have been extensively studied as adjuvants [6]. These CpG motifs are present at a 20-fold greater frequency in bacterial DNA compared to mammalian DNA [7]. CpG ODNs are recognized by the Toll-like receptor 9 (TLR9), which is expressed on human B cells and plasmacytoid dendritic cells (pDCs), thereby inducing Th1-dominated immune responses [8]. Pre-clinical studies, conducted in rodents and non-human primates, as well as human clinical trials, have demonstrated that CpG ODNs can significantly improve vaccine-specific antibody responses [6]. Three types of stimulatory CpG ODNs have been identified, types A, B, and C, which differ in their immune-stimulatory activities [9]. 

Get more information about CpG-ODNs Classes.

References

1. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
2. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
3. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
4. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
5. Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA, 98(16):9237-42.
6. Steinhagen F. et al., 2011. TLR-based immune adjuvants. Vaccine 29(17):3341-55.
7. Hemmi H. et al., 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-5.
8. Coffman RL. et al., 2010. Vaccine adjuvants: Putting innate immunity to work. Immunity 33(4):492-503.
9. Krug A. et al., 2001. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol, 31(7): 2154-63.

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