Jurkat-Raji CTLA4/CD80 assay (Bio-IC™)

Antagonist screening assay for CTLA4/CD80 axis

The CTLA4/CD80 Bio-IC™ assay is a bioluminescent, cell-based system designed for the screening of antibody-, Fc-fusion protein-, or small-molecule antagonists of the CTLA4/CD80 immune checkpoint (IC) axis.
This IC interaction delivers inhibitory signals that prevent T cells from eliciting an immune response. Inhibitors of CTLA-4 restore T cell activity and are among the most promising immunotherapeutic approaches.

 More details

CTLA4/CD80 Bio-IC™ assay principle (click to enlarge)

The CTLA4/CD80 Bio-IC™ assay is comprised of two cell lines:

• Jurkat-Lucia™ TCR-hCTLA4

Jurkat-Lucia™ TCR-hCTLA4 are engineered human T cells that stably express a specific TCR and an NFAT-Lucia luciferase reporter. They also overexpress the CD28 costimulatory receptor and the CTLA4 immune checkpoint receptor.

• Raji-APC-Null

Raji-APC-Null are engineered human B cells acting as antigen-presenting cells (APCs). They stably express the cognate TCR [HLA::peptide] complex. These cells express endogenous levels of CD80/86, the ligand shared by CD28 and CTLA4.
 

Assay principle

The co-culture of these two cell lines mimics the immune synapse between T cells and APCs, leading to the inactivation of the reporter T cells.

The immune synapse results from the activatory interactions of the TCR/[HLA::peptide] complex and CD28/CD80, and the concomitant inhibitory interactions of  CTLA4/CD80. These three interactions prevent the Jurkat-Lucia™ TCR-hCTLA4 cells from expressing Lucia®.

In the presence of CTLA4 antagonists, the IC-mediated inhibition is removed, leading to T cell activation and Lucia® production. The potency of these antagonists can be evaluated by assessing Lucia® activity using QUANTI-Luc™ 4 Lucia/Gaussia detection reagent (see Figures). 

Jurkat-Lucia™ TCR-hCTLA4 key features

• Stable specific [HLA::peptide]-restricted TCR
• Stable hCTLA4 and hCD28 overexpression
• NFAT-inducible Lucia luciferase reporter activity
• No Lucia® expression in the absence of IC inhibitor(s)
• Lucia® expression in the presence of IC inhibitor(s)

APC key features:

• Stable specific [HLA::peptide] expression
• Endogenous hCD80 expression

Read our review on Immune Checkpoint Blockade

Learn more about Immune Checkpoint Antibodies.

Figures

Validation of human CD28 and CTLA4 overexpression by Jurkat-Lucia™ TCR-hCTLA4 cells. Jurkat-Lucia™ TCR-hCTLA4 cells were incubated with a PE-conjugated Anti-hCD28 (A) or APC-conjugated Anti-hCTLA4 (B) mAb for 30 minutes. The binding affinity was then measured using flow cytometry.

Validation of endogenous human CD80 expression by Raji-APC-Null cells. Raji-APC-Null cells were incubated with a PE‑conjugated Anti-hCD80 mAb for 30 minutes. The binding affinity was then measured using flow cytometry.

 

Activation of Jurkat-Lucia™ TCR-hCTLA4 cells using Anti-hCTLA4 biosimilar mAb. Raji-APC-Null and Jurkat-Lucia™ TCR-hCTLA4 cells were incubated with gradient concentrations of the biosimilar Anti-hCTLA4-hIgG1 Ipilimumab or Anti-β-Gal-hIgG1, as a negative control, for 24 hours. NFAT activation, reflecting the disruption of CTLA4/CD80 inhibitory interaction, was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. The fold increase over non induced cells (no mAbs) is shown.

 

Specifications

Cell type: Lymphoblastic

Tissue origin: Human T lymphocytes & Human B cell lymphoma

Specificity: Human

Reporter gene: Lucia®

Antibiotic resistance: Blasticidin, Zeocin®, Hygromycin, G418 (Geneticin)

Growth medium: Complete IMDM (see TDS)

Growth properties: Suspension

Quality Control:

• Human CTLA4, CD28, and CD80 expression have been verified by flow cytometry.
• Reporter activity is validated using InvivoGen's Anti-hCTLA4-hIgG1 antibody.
• The stability for 20 passages following thawing is confirmed.
• Both cell lines are guaranteed mycoplasma-free.

Contents

Please note: Both cell lines are sold together.

• 1 vial of Jurkat-Lucia™ TCR-hCTLA4 cells
• 1 vial of Raji-APC-Null cells
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Hygromycin (100 mg/ml)
• 1 ml of G418 (Geneticin) (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml).
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent

Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)

Details

The activation of T lymphocytes initiates their proliferation and yields a variety of effector functions that allow combating microbial infections, as well as developing tumors. The current paradigm is that full activation of T cells requires at least 2 signals upon contact with antigen-presenting cells (APCs) [1, 2].

Signal 1 is delivered through the interaction of the T cell receptor (TCR) and a specific antigenic peptide associated with an MHC (major histocompatibility complex) molecule on APCs. Signal 2 is delivered through the interaction of CD28, the prototypical T cell co-stimulatory molecule, and its ligands, CD80 or CD86, expressed by the APC. However, a number of other molecules, named immune checkpoints (IC), have been reported to regulate the onset and the limitations of T cell activities.  Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) receptor and its ligand, CD80/86, are among the best-characterized suppressive immune checkpoints [3].

Signal 1: TCR and [HLA::peptide]

The 'classical' and most represented TCR is an 80 to 90 kDa heterodimer composed of one α chain and one β chain. The αβTCR is a transmembrane protein expressed by developing and mature T cells. It features an extracellular ligand-binding pocket and a short cytoplasmic tail. Each αβTCR is restricted to a specific complex made of an antigenic peptide and a class I or class II MHC molecule. Human MHC molecules are also known as HLA (human leukocyte antigen). Because of its short cytoplasmic tail, the TCR, once engaged,  lacks the ability to signal and requires non-covalent association with the CD3 to trigger downstream intracellular signaling and T cell activation [1, 2]. Importantly, signal 1 without co-stimulation results in T cell unresponsiveness or 'anergy', a tolerance mechanism that guards against premature activation.

Signal 2: CD28 and CD80/86

CD28 is a homodimeric and transmembrane protein expressed by T cells. Nearly all human CD4+ T cells and 50% of human CD8+ T cells express CD28. The CD28 interaction with CD80 (aka B7-1) or CD86 (aka B7-2) on APCs, in conjunction with TCR engagement, triggers a co-stimulation signal (signal 2). It results in T cell proliferation, cytokine production, cell survival, and cellular metabolism [1, 2].

Suppressive CTLA4/CD80 IC signal 

The cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) is an inhibitory receptor and immune checkpoint expressed by activated and regulatory T cells [3]. CTLA-4 outcompetes CD28 for binding to CD80 or CD86 expressed by antigen-presenting cells. Thereby, CTLA-4 upregulation by T cells prevents overstimulation by arresting both proliferation and activation [3].

References:

1. Budd R.C. & Fortner K.A., 2017. Chapter 12 - T Lymphocytes. Kelley and Firestein's Textbook of Rheumatology (Tenth Edition). pages 189-206.
2. Smith-Garvin J.E. et al., 2009. T Cell Activation. Ann. Rev. Immunol. 27:591-619.
3. Ribas A. and Wolchock J.D., 2018. Cancer immunotherapy using checkpoint blockade. Science. 359:1350-55.

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