IFN-γ Reporter HEK 293 Cells

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IFN-γ responsive STAT1-SEAP reporter cells

Signaling pathway in HEK-Blue™ IFN-γ cells

HEK-Blue™ IFN-γ cells are designed to monitor human type II interferon IFN-γ-induced STAT1 stimulation or inhibition. This colorimetric bioassay can be used for screening activatory molecules, such as engineered cytokines, or inhibitory molecules, such as neutralizing antibodies.

HEK-Blue™ IFN-γ cells respond specifically to recombinant human IFN-γ. The reliable and consistent performance of HEK-Blue™ IFN-γ cells makes them suitable for release assays of therapeutic molecules that inhibit IFN-γ signaling, such as Fontolizumab, a monoclonal antibody that targets IFN-γ and prevents its binding to its receptor (see figures).
 

Key Features

• Readily assessable STAT1-SEAP reporter activity
• Convenient readout using  QUANTI-Blue™ Solution
• High sensitivity to human (h) IFN-γ activity
• Stability guaranteed for 20 passages

Applications

• Therapeutic development
• Drug screening
• Release assay

Interferon-gamma (IFN-γ) is a pro-inflammatory cytokine that plays a key role in innate and adaptive immune responses to intracellular pathogens and tumor immunosurveillance.

 More details

InvivoGen’s products are for research use only, and not for clinical or veterinary use.

Figures

Dose-response of HEK-Blue™ IFN-γ cells to human and mouse IFN-γ. Cells were stimulated with increasing concentrations of recombinant human (h) and mouse (m) IFN-γ. After overnight incubation, the SEAP activity was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. The optical density (OD) at 650 nm is shown as mean ± SEM.

Response of HEK-Blue™ IFN-γ cells to a panel of cytokines. Cells were stimulated with various human recombinant cytokines: 100 U/ml hIFN-α2b or hIFN-β-1a, 1 ng/ml hIFN-γ, 10 ng/ml mIFN-γ, 100 ng/ml IL-28a, or 10 ng/ml hTNF-α After overnight incubation, SEAP activity was assessed using QUANTI-Blue™ Solution. The OD at 630 nm is shown as mean ± SEM.

Dose-dependent inhibition of HEK-Blue™ IFN-γ cells response using Fontolizumab biosimilar. Increasing concentrations of Anti-hIFN-γ-hIgG1 (0.1 ng/ml - 10 µg/ml) were incubated with recombinant human (h)IFN-γ (3 ng/ml) for 1 h before the addition of HEK-Blue™ IFN-γ cells. After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution. Data are shown in percentage of activity (%) (mean ± SEM).

Specifications

Cell type: Epithelial

Tissue origin: Human Embryonic Kidney

Target: IFN-γ

Specificity: Human

Reporter gene: SEAP

Antibiotic resistance: Blasticidin, Zeocin®

Detection range: Human IFN-γ: 0.1 -10 ng/ml

Growth medium: Complete DMEM (see TDS)

Growth properties: Adherent

Mycoplasma-free: Verified using Plasmotest™

Quality control: Each lot is functionally tested and validated.

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 and some areas in Asia)

Details

Cell line description

HEK-Blue™ IFN-γ cells were generated by stable transfection of the human embryonic kidney HEK293 cell line with the gene encoding the human STAT1 to obtain a fully active type II IFN signaling pathway. The other genes of the pathway (IFNGR1, IFNGR2, JAK1, and JAK2) are naturally expressed by these cells. In addition, a STAT1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene was introduced. The binding of IFN-γ to its receptor triggers a signaling cascade leading to STAT1 activation and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue™ IFN-γ cells detect human (h) IFN-γ, but not mouse (m) IFN-γ. Of note, these cells do not respond to either type I IFNs (IFN-α/β) or type III IFNs (IFN-λ) (see figures).
 

IFN-γ background

IFN-γ, also known as Type II IFN or immune interferon, is predominantly produced by innate immune cells, such as Natural Killer (NK) cells and innate lymphoid type 1 cells (ILC1), and activated adaptive immune cells, such as Th1 CD4+ T cells and cytotoxic CD8+ T cells [1]. This cytokine is produced as a secreted homodimeric molecule in response to infections and growing tumors [1, 2]. IFN-γ engages a receptor composed of two IFN-γR1 chains and two IFNγ-R2a, thus forming a hexameric complex [2]. While IFN-γR1 is constitutively expressed on all nucleated cells, the expression of IFNγ-R2 is tightly regulated. The binding of IFN-γ to its receptors triggers a JAK1/JAK2 signal transduction leading to the activation of STAT1. Activated STAT1 forms homodimers that are translocated to the nucleus where they bind interferon-gamma-activated sites (GAS) in the promoter of interferon-stimulated genes (ISGs). ISGs encode many products with direct effector or regulatory immune functions [1]. Thus IFN-γ plays a versatile role in immune responses and tissue homeostasis [1].

Relevance for therapeutics development

IFN-γ contributes to the pathogenesis of inflammatory diseases, such as Crohn's disease, psoriasis, haemophagocytic lymphohistiocytosis (HLH), and macrophage activation syndrome (MAS) [3-5].
Fontolizumab (aka HuZAF™) is a therapeutic, humanized monoclonal antibody (mAb) that targets IFN-γ. By binding to IFN-γ, Fontolizumab prevents it from interacting with its receptor (IFN-γR) on the surface of immune cells, thus inhibiting downstream inflammatory response. Fontolizumab was used as an immunosuppressive drug to treat inflammatory diseases, including Crohn's disease and psoriasis. While the mode of action was promising, clinical trials did not demonstrate sufficient clinical benefit [4, 5].
Emapalumab is a fully human monoclonal antibody (mAb) that targets both free and receptor-bound IFN-γ, preventing downstream signaling. This therapeutic antibody was FDA-approved in 2018 for treating pediatric and adult patients with HLH [3]. 

The administration of recombinant IFN-γ is a strategy for enhancing anti-infectious and anti-tumoral innate and adaptive immune responses.
Interferon gamma-1b (Actimmune®) is a form of recombinant human IFN-γ approved by the FDA to treat infections associated with chronic granulomatous disease and to slow the progression of severe malignant osteopetrosis [6]. IFN-γ monotherapy to treat cancer has been of limited success. This is partly explained by IFN-γ's short half-life and dual anti- and pro-tumor activities [7, 8]. Multiple clinical trials are ongoing to explore the combination of IFN-γ with other cancer therapeutics [8].
Another strategy relies on the engineering of IFN-γ partial agonists to tune IFN-γ receptor signaling output. Interestingly, such recombinant IFN-γ variants can exhibit biased gene-expression profiles, such as the retention of upregulation of class I molecules and impaired induction of inhibitory checkpoint molecules by cancer cells [2]. These results demonstrate that the two opposing functions of IFN-γ in the tumor microenvironment can be decoupled, offering a route for therapeutic applications.

References:

1. Ivashkiv L.B., 2018. IFNγ: signalling, epigenetics and roles in immunity, metabolism, disease and cancer immunotherapy. Nat Rev Immunol. 18(9):545-558
2. Mendoza, J.L., et al., 2019. Structure of the IFNγ receptor complex guides design of biased agonists. Nature. 567(7746):56-60.
3. Vallurupalli M. & Berliner N., 2019. Emapalumab for the treatment of relapsed/refractory hemophagocytic lymphohistiocytosis. Blood. 134(21):1783-1786.
4. Reinisch W et al., 2009. Fontolizumab in moderate to severe Crohn's disease: A phase 2, randomized, double-blind, placebo-controlled, multiple-dose study. Infl Bowel Dis., 16(2):233-242.
5. Harden J.L., et al., 2015. Humanized anti-IFNg (HuZAF) in the treatment of psoriasis. J. Aller Clin Immunol. 135(2):553.
6. Silva, A.C. & Lobo, J.M. Sousa., 2020. Cytokines and growth factors. Current Applications of Pharmaceutical Biotechnology. 87-113.
7. Castro, F., et al., 2018. Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion. Front Immunol. 9:847.
8. Yi, M., et al., 2024. Targeting cytokine and chemokine signaling pathways for cancer therapy. Signal Transduction and Targeted Therapy. 9(1):176.

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