Jurkat-Raji ICOS/ICOS-L assay (Bio-IC™)

Antagonist screening assay for ICOS/ICOS-L axis

InvivoGen offers a cellular assay specifically designed for screening antibody-, Fc-fusion protein-, or small-molecule antagonists of the ICOS/ICOS-L immune checkpoint (IC) axis. This assay is comprised of:

Principle of ICOS/ICOS-L cellular assay
(click to enlarge)

• Jurkat-Lucia™ hICOS: Reporter T cells
• Raji-hICOS-L: Antigen-presenting cells (APCs)

​These paired cell lines allow the mimicking of the immune synapse between T cells and APCs through the interaction of cell surface molecules delivering T-cell activation signals.

Inducible Co-stimulator (ICOS, CD278) is an immunostimulatory IC and a member of the CD28 superfamily. Expression of ICOS is rapidly induced in  CD4 and CD8 T cells upon their activation, whereas its ligand ICOS-L (CD275), is mostly expressed by APCs [1]. 

 More details

Assay principle:

This assay relies on the co-culture of Jurkat-Lucia™ hICOS and Raji-hICOS-L cells:
– Jurkat-Lucia™-ICOS cells express a hICOS-CD3ζ fusion protein along with the Lucia luciferase reporter gene under the control of an ISG54 minimal promoter fused to six NFAT response elements. CD3ζ is a key component of the T-cell receptor (TCR) and CD3 complex that triggers TCR downstream signaling.
– Raji-hICOS-L cells express the ICOS-L activating IC ligand.
In the presence of a potent ICOS or ICOS-L antagonist, the ICOS-CD3ζ-mediated activation is blocked and there is no Lucia production. Activation of the reporter T cells can be readily measured using QUANTI-Luc™ 4 Lucia/Gaussia detection reagent (see Figures).

T-cell key features:

• Stable hICOS-CD3ζ expression
• NFAT-inducible Lucia luciferase reporter activity

APC key features:

• Stable hICOS-L overexpression

InvivoGen also offers Jurkat-Lucia™ hICOS cells without Raji-hICOS-L cells to measure the potency of agonists of the ICOS/ICOS-L axis.

Read our review on Immune Checkpoint Blockade

 Learn more about Immune Checkpoint Antibodies.

​Reference:

1. Amatore, F. et al. 2020. Role of ICOS in cancer immunotherapy. Expert Opin Biol Ther 20, 141-150.

Figures

Validation of human ICOS overexpression by Jurkat-Lucia™hICOS cells. Jurkat-Lucia™ hICOS and Jurkat-Lucia™ NFAT cells were incubated with a PE‑conjugated Anti-hICOS mAb for 30 minutes. The binding affinity was measured using flow cytometry. Unstained Jurkat-Lucia™ hICOS cells are shown in grey.

Validation of human ICOS-L overexpression by Raji-hICOS-L cells. Raji-hICOS-L and Raji-Null cells were incubated with a PE‑conjugated Anti-hICOS-L mAb for 30 minutes. The binding affinity was measured using flow cytometry. Unstained Raji-hICOS-L cells are shown in grey.

Activation of Jurkat-Lucia™-hICOS cells. Raji-hICOS-L and Jurkat-Lucia™-hICOS cells were incubated with gradient concentrations of antagonist Anti-hICOS-L hIgG2, antagonist Anti-h/mICOS mIgG2a, or control Anti-β-Gal hIgG1 monoclonal antibodies for 6 hours. NFAT activation in the Jurkat-Lucia™-hICOS cells, reflecting the interaction of ICOS-L with the ICOS-CD3ζ fusion protein was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™ 4 Reagent. Percentages of the maximal responses are shown (mean + SEM).

Specifications

Growth medium: IMDM, 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin

Antibiotic resistance:

• Jurkat-Lucia™ hICOS cells: Blasticidin and Zeocin®
• Raji-hICOS-L cells: Blasticidin

Quality Control:

• Human ICOS and ICOS-L expression have been verified by flow-cytometry.
• Reporter activity has been validated following the co-culture of the two cell lines.
• The stability for 20 passages following thawing has been verified.
• Both cell lines are guaranteed mycoplasma-free.

Contents

Please note: Both cell lines are sold together.

• 3-7 x 10⁶ Jurkat-Lucia™ hICOS cells
• 3-7 x 10⁶ Raji-hICOS-L cells 
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

 Shipped on dry ice (Europe, USA, Canada)

Details

Inducible Co-stimulator (ICOS, CD278) is an immunostimulatory IC and a member of the CD28 superfamily. Expression of ICOS is rapidly induced in  CD4+ and CD8+ T cells upon their activation, whereas its ligand ICOS-L (also known as CD275), is mostly expressed by antigen-presenting cells [1]. The interaction between ICOS and ICOS-L delivers a secondary co-stimulatory signal through the activation of the transcription factor AKT, which promotes T cell proliferation and differentiation as well as the production of cytokines  [1]. In tumor immunity, ICOS is involved in the amplification of the anti-tumor cytotoxic CD8+ T cell response, as well as the 'pro-tumor' function and maintenance of regulatory T cells (Tregs). Therefore, both agonistic and antagonistic monoclonal antibodies (mAbs) targeting this pathway are being investigated in combinational cancer immunotherapy [2]. Notably, ICOS agonistic mAbs have been shown to potentiate the effects of anti-CTLA-4 mAbs [3]. 

​References:

1. Amatore, F. et al. 2020. Role of ICOS in cancer immunotherapy. Expert Opin Biol Ther 20, 141-150.
2. Solinas, C. et al. 2020. The rationale behind targeting the ICOS-ICOS ligand costimulatory pathway in cancer immunotherapy. ESMO Open 5.
3. Soldevilla, M.M. et al. 2019. ICOS Costimulation at the Tumor Site in Combination with CTLA-4 Blockade Therapy Elicits Strong Tumor Immunity. Mol Ther 27, 1878-1891.

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