TL1A Reporter HEK 293 Cells

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Tumor Necrosis Factor-Like Cytokine 1A Reporter Cells

Signaling pathway in HEK-Blue™ TL1A cells

HEK-Blue™ TL1A cells were engineered from the human embryonic kidney HEK 293 cell line to detect bioactive tumor necrosis factor-like cytokine 1A (TL1A) by monitoring the activation of the AP-1/NF-κB pathway. They can also be used for screening antibodies or small molecule inhibitors targeting the TL1A pathway.

TL1A is a member of the TNF (Tumor Necrosis Factor) superfamily. This cytokine exists as a soluble or transmembrane protein produced by numerous cells including monocytes, macrophages, and dendritic cells [1]. TL1A binding to its receptor DR3 (death receptor 3) and the subsequent signaling events drive the production of pro-inflammatory cytokines and differentiation of T helper subsets [1].

 More details

Cell line description

HEK-Blue™ TL1A cells were generated by stable transfection with the gene encoding for the human TL1A receptor, DR3 (Death Receptor 3, aka TNR25 or Apo-3) and an NF-κB/AP1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of TL1A to its receptor triggers a signaling cascade leading to the activation of NF-κB/AP1, and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue™ TL1A cells detect human and murine TL1A (see figures). Of note, these cells also respond to hIL-1β and hTNF-α which share a common signaling pathway with TL1A (see figures). However, they do not respond to other members of the TFN-α superfamlily: hAPRIL, hBAFF, CD40L, and hRANKL (see figures).

Key features

• Fully functional TL1A signaling pathway
• Readily assessable NF-κB/AP1-inducible SEAP reporter activity
• Strong response to human (h) and murine (m) TL1A
• Response to hIL-1β and hTNF-α
• No response to hAPRIL, hBAFF, CD40L, and hRANKL

Applications

• Detection and quantification of human and murine TL1A activity
• Screening of anti-TL1A and anti-DR3 antibodies
• Screening of small molecule inhibitors of the TL1A pathway

Reference:

1. Xu, W.-D, et al., 2021. Role of TL1A in Inflammatory Autoimmune Diseases: A Comprehensive Review. Frontiers in Immunology, 2022. 13:891328.

Figures

Dose-response of HEK-Blue™ TL1A cells to recombinant TL1A. Cells were stimulated with increasing concentrations of recombinant human TL1A (hTL1A) and murine TL1A (mTL1A). After overnight incubation, the AP-1/NF-κB-induced response was determined using QUANTI‑Blue™ Solution, a SEAP detection reagent. The optical density (OD) at 630 nm is shown as mean ± SEM.

Dose-dependent inhibition of HEK‑Blue™ TL1A cellular response using a neutralizing antibody against human TL1A. An increasing concentration of a neutralizing anti-hTL1A antibody was incubated with recombinant human TL1A (3 ng/ml) for 2 hours before the addition of HEK-Blue™ TL1A cells. After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™. Data are shown as a percentage (%) of activity, mean ± SEM.

Response of HEK-Blue™ TL1A cells to a panel of cytokines. Cells were stimulated with various human and murine recombinant cytokines: 10 ng/ml of hTL1A, mTL1A, hAPRIL, hBAFF, hCD40L, hRANKL, hTNF-α, hIFN-γ, hIL-1β, hIL-6, hIL-27 and 1000 U/ml hIFN-α. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ The OD at 630 nm is shown as mean ± SEM.

Specifications

Antibiotic resistance: Blasticidin and Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2-4 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin®

Specificity: Detects human and mouse TL1A

Detection range:

• Detection range for human TL1A: 300 pg/ml - 30 ng/ml
• Detection range for murine TL1A: 300 pg/ml - 100 ng/ml

Quality Control:

• SEAP reporter activity in response to TL1A is validated using functional assays.
• The stability for 20 passages following thawing is confirmed. 
• These cells are tested for mycoplasma contamination. 

This product is covered by a Limited Use License (See Terms and Conditions).

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 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).

 Shipped on dry ice (Europe, USA, Canada)

Details

Tumor necrosis factor-like cytokine 1A (TL1A), also known as vascular endothelial growth inhibitor (VEGI) or TNFSF15, is part of the tumor necrosis factor (TNF) superfamily.

TL1A is mainly produced by endothelial cells, as well as activated dendritic cells and macrophages. It is synthesized as a membrane-bound trimeric molecule. Alternative splicing or cleavage by the tumor necrosis factor-alpha converting enzyme (TACE) results in soluble TL1A [1]. Both forms bind the homotrimeric transmembrane death receptor 3 (DR3), triggering TRADD/TRAF2/RIP signaling, and ultimately leading to NF-kB and MAPK activation. Subsequent gene expression in target cells drives the production of pro-inflammatory cytokines and differentiation of T helper subsets [1]. Alternatively, as DR3 is a dual signaling death receptor, TRADD also associates with FADD/RIP3/caspase 8 to induce apoptosis or necroptosis [1].

In resting T cells, DR3 is expressed as a soluble decoy receptor, DcR3, that protects the cells from apoptosis. Activated T cells express transmembrane DR3. Their activity can thus be modulated upon TL1A sensing, from proliferation and differentiation to response termination.

Two decades after its discovery, TL1A has become a hot therapeutic target. Abnormal TL1A expression is linked to multiple enteric autoimmune diseases (e.g. Crohn’s disease, ulcerative colitis) and extends to other diseases, including rheumatoid arthritis, psoriasis, and allergic airway inflammation [1, 2]. Genome-wide association studies have also linked TL1A polymorphisms with disease susceptibility, conferring a biomarker relevance on TL1A [1].

Anti-TNFa monoclonal antibodies (mAbs) such as Adalimumab are among industry’s top-selling drugs to treat autoimmune diseases [3]. However, many patients either do not respond to their treatment or suffer from side-effects that are inherent to long-lasting TNF blockage [3]. Targeting TL1A could break the efficacy ceiling that we see with the best drugs out there. Pharmaceutical biotechs now have three neutralizing mAbs in clinical development. Inhibition of TL1A signaling using anti-TL1A Tulisokibart (MK-7240, from Merck), RVT-3101 (from Roche), or Duvakitub (TEV-48574, from Sanofi & Teva Pharmaceuticals) has shown clinical evidence of “best-in-class” potential for treating inflammation and scarring in inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease [4]. The TL1A /DR3 axis could also be targeted to enhance or restore immune responses, such as in exhausted T cells in a tumor environment. Further research should guarantee the clinical usage of DR3 agonists, or inhibitors of the soluble decoy receptor DcR3, either alone or in combination with immune checkpoint blockade.

​References:

1. Xu, W.-D, et al., 2021. Role of TL1A in Inflammatory Autoimmune Diseases: A Comprehensive Review. Frontiers in Immunology, 2022. 13:891328.
2. Schmitt, P., et al., 2024. TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation. J Exp Med, 221(6). doi: 10.1084/jem.20231236.
3. Steeland, S., C. et al., 2018. A New Venue of TNF Targeting. International Journal of Molecular Sciences. 19(5):1442.
4. Neimark, J., 2024. TL1A inhibitors could usher in new era of IBD treatment. www.biospace.com. 

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