IL-3 Reporter HEK 293 Cells

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IL-3 responsive STAT5-SEAP reporter assay

Signaling pathway in HEK-Blue™ IL-3 cells

HEK-Blue™ IL-3 cells are designed to monitor human IL-3-induced STAT5 stimulation or inhibition. This bioassay can be used for screening activatory molecules, such as engineered cytokines, or inhibitory molecules, such as neutralizing antibodies. The reliable and consistent performance of HEK-Blue™ IL-3 cells makes them suitable for release assays of therapeutic molecules that inhibit IL-3 signaling, such as Talacotuzumab, a monoclonal antibody targeting the IL-3Rα chain of IL-3 receptor.

Key features

• Readily assessable STAT5-SEAP reporter activity
• Convenient readout using  QUANTI-Blue™ Solution
• High sensitivity to human (h) IL-3 activity
• Stability guaranteed for 20 passages

Applications

• Therapeutic development
• Drug screening
• Release assay

Interleukin-3 (IL-3) is a cytokine that plays an important role in the recruitment, differentiation, and survival of a wide variety of hematopietic cells, expecially during the course of inflammation. It is currently regarded as a regulator of inflammation with either protective or detrimental effects in the response to infections, immune mediated diseases, and hematologic cancers.

 More details

Figures

Validation of the expression of human IL-3Rα and b-chain subunits by HEK-Blue™ IL-3 cells. 5 x 10⁵ cells were incubated with either a (A) APC-conjugated isotype control (blue) or a APC-conjugated Anti-hIL-3Rα mAb (red), or (B) PE-conjugated isotype control (blue) or a PE-conjugated Anti-β-chain mAb (red) for 30 minutes. The binding affinity was then measured using flow cytometry.

Dose-response of HEK-Blue™ IL-3 cells to recombinant IL-3. Cells were stimulated with increasing concentrations of recombinant human IL-3 (hIL-3) and mouse IL-3 (mIL-3). After overnight incubation, the STAT5-induced SEAP activity was determined using QUANTI-Blue™, a SEAP detection reagent. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Dose-dependent inhibition of HEK-Blue™ IL-3 cell response using Talacotuzumab biosimilar. Increasing concentrations of Anti-hIL-3Rα Talacotuzumab biosimilar (60 ng/ml - 30 µg/ml) were incubated with HEK-Blue™ IL-3 cells for 1 h before the addition of recombinant human IL-3 (100 pg/ml). 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).

Response of HEK-Blue™ IL-3 cells to a panel of cytokines. Cells were stimulated with various human and mouse recombinant cytokines: 1 ng/ml of hIL-3, 100 ng/ml of mIL-3, hIL-5, hGM-CSF, hIL-6, hIL-27, IFN-γ, and 1000 U/ml hIFN-α. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ The OD at 650 nm is shown as mean ± SEM.

Specifications

Cell type: Epithelial

Tissue origin: Human Embryonic Kidney

Target: IL-3

Specificity: Human

Reporter gene: SEAP

Antibiotic resistance: Blasticidin, Hygromycin, Zeocin®

Detection range: Human IL-3: 0.1 -100 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 of 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

Cell line description

HEK-Blue™ IL-3 cells were generated by stable transfection of the human embryonic kidney HEK293 cell line with the gene encoding the human IL-3 receptor (IL-3Rα chain and common β-chain (CD131)) and STAT5 to obtain a fully active IL-3 signaling pathway. In addition, a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene was introduced. The binding of IL-3 to its receptor triggers a signaling cascade leading to STAT5 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™  IL-3 cells detect human (h) IL-3, but not mouse (m) IL-3. These cells also respond, to a weaker extent, to human IFN-γ. However, they do not respond to other STAT5-signaling cytokines of the common β-chain family: IL-5 and GMCSF (see figures).
 

IL-3 background

Interleukin-3 (IL-3) belongs to the common β chain (βc) cytokine family, originally identified as a multi-colony stimulation factor (CSF). It is now regarded as a regulator of inflammation [1, 2].

IL-3 expression is induced in response to inflammation, and is highly restricted to T cells. Under some circumstances, IL-3 may also be produced by macrophages, basophils, mast cells, NK cells and stromal cells [1, 3]. IL-3 binds a heterodimeric receptor comprising the βc (CD131) and IL-3Rα (CD123) subunits. It signals through tyrosine kinases of the Janus family (JAK2) and signal transducer and transcription activators (STATs), notably STAT5 [1, 3]. IL-3 supports the survival, proliferation, differentiation, polarization, or recruitment of immune and non-immune cells [2, 3]. Notably, as a multi-CSF, IL-3 targets a wide spectrum of hematopoietic cells, including eosinophils, basophils, plasmacytoid dendritic cells, neutrophils, and progenitor cells [3].

Relevance for therapeutics development

Depending on the clinical context, strategies have been investigated to either boost or disrupt IL-3 signaling [2, 3]. The administration of IL-3 has been evaluated as a treatment for patients with cytopenia (e.g. after chemotherapy). On the contrary, monoclonal antibodies or antibody-drug conjugates against IL-3Rα have been used in clinical trials to treat hematologic cancers. Inded, the IL-3Rα subunit of the IL-3 receptor is overexpressed in acute myeloid lymphoma (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), B-cell acute lymphoblastic leukemia, or Hodgkin lymphoma  [2, 3].

Talacotuzumab (CSL362) is a monoclonal antibody targeting the IL-3Rα subunit, and exhibiting a modified Fc region for enhanced ADCC functions. Thus, it disrupts the IL-3 signaling and kills the malignant cells that overexpress IL-3Rα [5]. This antibody has reached phase 2/3 in a clinical trial to treat AML patients (NCT02472145).

​References:

1. Dougan, M. et al., 2019. GM-CSF, IL-3, and IL-5 family of cytokines: regulators of inflammation. Immunity. 50(4):796-811.
2. Podolska, M.J. et al., 2024. IL-3: key orchestrator of inflammation. Front Immunol. 15:1411047.
3. Pant, H. et al., 2023. Translating the biology of β common receptor-engaging cytokines into clinical medicine. J. Allergy & Clin Immunol. 151(2):324-344.
4. Busfield, S.J. et al., 2014. Targeting of acute myeloid leukemia in vitro and in vivo with an anti-CD123 mAb engineered for optimal ADCC. Leukemia. 28(11):2213-2221.
5. Xie, L.H. et al., 2017. CD123 target validation and preclinical evaluation of ADCC activity of anti-CD123 antibody CSL362 in combination with NKs from AML patients in remission. Blood Cancer J 7(6):e567.

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