HEK-Blue™ hTLR8

Human TLR8 / NF-κB / SEAP reporter HEK293 cells

TLR8 activation in HEK-Blue™ hTLR8 cells

HEK-Blue™ hTLR8 cells were derived from the human embryonic kidney HEK293 cell line to monitor the activation of human TLR8. The endosomal location of TLR8 allows it to scan for the presence of microbial RNA in the phagocytic cargo. TLR8 activation leads to NF-κB/AP1- and IRF-mediated production of type I IFNs (IFN-α/β) and pro-inflammatory cytokines [1]. Importantly, findings regarding mouse TLR8 are not transposable to its human counterpart [2,3], and TLR13 has been suggested as a murine TLR8 homolog [4].

Structural analyses have revealed that TLR8 possesses two binding sites with distinct specificities.
- Site 1 binds uridine or synthetic base analogs, such as R848 (Resiquimod).
- Site 2 binds ssRNA with uridine (U) and guanosine (G) motifs. Both uridine and U(G) ssRNA appear to arise from RNA-degradation [5].
Of note, Site 1 occupancy allows the receptor dimerization and signaling with ad hoc ligand concentration. ssRNA-binding to Site 2 is not sufficient for the formation of a signaling competent TLR dimer but it strongly enhances the binding affinity of Site 1 [5].

HEK-Blue™ hTLR8 cells express the human TLR8 gene and an NF-κB/AP-1-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. SEAP levels produced upon TLR8 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.

The parental cell line of HEK-Blue™ hTLR8 cells is HEK-Blue™ Null1 cells.

HEK-Blue™ hTLR8 cells are selectable with Blasticidin and Zeocin™.

References:

1. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31.  
2. Heil F. et al., 2004. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science. 303:1526.
3. Eigenbrod T. & Dalpke A.H., 2015. Bacterial RNA: an underestimated stimulus for innate immune responses. J. Immunol. 195:411  
4. Choo M.K. et al., 2017. TLR sensing of bacterial spore-associated RNA triggers host immune responses with detrimental effects. J. Exp. Med. 214:1297.
5. Tanji H. et al., 2015. Toll-like receptor 8 senses degradation products of single-stranded RNA. Nat. Struct. Mol. Biol. 22:109.

Figures

Dose-response of HEK-Blue™ hTLR8 cells to synthetic base analogs. HEK-Blue™ hTLR8 cells were cultured in HEK-Blue™ Detection medium with increasing concentrations of a TLR8 agonist (TL8-506), various TLR7/8 agonists (R848, CL097, CL075) or TLR7 agonists (CL264, Imiquimod, Gardiquimod). After 24h incubation, TLR8-induced NF-κB/AP1 responses were assessed by measuring SEAP levels in the supernatant by reading the OD at 630 nm. OD fold increase over non-induced cells is shown.

Species-driven TLR8 differential responses. HEK-Blue™ hTLR8 or mTLR8 were cultured in HEK-Blue™ Detection medium with 1 μg/ml R848 (TLR7/8 agonist), 3 μg/ml Imiquimod (TLR7 agonist), 5 μg/ml ssRNA40/LyoVec™ (referred as human TLR8 agonist), or 1 μg/ml TL8-506 (TLR8 agonist, VTX-2337 analog). After 24h incubation, TLR8-induced NF-κB/AP1 responses were assessed by measuring SEAP levels in the supernatant by reading the OD at 630 nm. OD fold increase over non-induced cells is shown.

HEK-Blue™ hTLR8 cells were stimulated with various TLR and NOD agonists: Pam3CSK4 (100 ng/ml), Poly(I:C) (50 ng/ml), LPS-EB ultrapure (100 ng/ml), recombinant flagellin from S. typhimurium (10 ng/ml), CL264 (1 µg/ml), CL097 (1 µg/ml), ssRNA40/LyoVec™ (5 µg/ml), ODN 2006 (10 µg/ml), C12-iE-DAP (100 ng/ml), L18-MDP (100 ng/ml), and TNF-α (100 ng/ml). After 18h incubation (24h incubation for CL264, C12-iE-DAP and L18-MDP ligands), NF-kB-induced SEAP activity was assessed using QUANTI-Blue™ and reading the OD at 655 nm.

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:

• The expression of the human TLR8 gene has been confirmed by RT-PCR.
• The activation of NF-κB/AP1 upon TLR8 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™ hTLR7 cells is HEK-Blue™ Null1 cells.

All of these products are covered by a Limited Use License (See Terms and Conditions).

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml Blasticidin (10 mg/ml)
• 1 ml Zeocin™ (100 mg/ml)
• 1 ml 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)