Jurkat-Raji (PD-1/CTLA4)/PD-L1 assay (Bio-IC™)

Antagonist screening assay for PD-1/PD-L1 and CTLA4/CD80 axes

The (PD-1/CTLA4)/PD-L1 Bio-IC™ assay is a bioluminescent, cell-based system designed for the exploration of combination therapies that block the PD-1/PD-L1 and/or CTLA4/CD80 immune checkpoint (IC) axes.
These IC interactions deliver inhibitory signals that prevent T cells from eliciting an immune response. Combined inhibitors of PD-1, PD-L1, and/or CTLA4 restore T cell activity and represent promising immunotherapeutic approaches, particularly in cases where monotherapies have limited success.

 More details
 

(PD-1/CTLA4)/PD-L1 assay principle (click to enlarge)

The (PD-1/CTLA4)/PD-L1 Bio-IC™ assay is comprised of two cell lines:

• Jurkat-Lucia™ TCR-hCTLA4-PD-1

Jurkat-Lucia™ TCR-hCTLA4-hPD-1 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 and PD-1 immune checkpoint receptors.

• Raji-APC-hPD-L1

Raji-APC-hPD-L1 are engineered human B cells acting as antigen-presenting cells (APCs). They stably express the cognate TCR [HLA::peptide] complex and the PD-L1 immune checkpoint ligand. 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 and PD-1/PD-L1. These three types of interaction prevent the Jurkat-Lucia™ TCR-hCTLA4-hPD-1 cells from expressing Lucia®.

In the presence of  PD-1, PD-L1, and/or CTLA4 antagonists, the IC-mediated inhibition is removed, leading to T cell activation and Lucia® production. The potency of these antagonists, used alone or in combination, can be evaluated by assessing Lucia® activity using QUANTI-Luc™ 4 Lucia/Gaussia detection reagent.

The combination of both PD-1 and CTLA4 blocking antibodies results in stronger inhibition than when each antibody is used alone (see Figures). 

Jurkat-Lucia™ TCR-hCTLA4-hPD-1 key features:

• Stable specific [HLA::peptide]-restricted TCR
• Stable hCD28, hCTLA4, and hPD-1 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)

Rahi-APC-hPD-L1 key features:

• Stable specific [HLA::peptide] expression
• Stable hPD-L1 overexpression
• Endogenous hCD80 expression

Read our review on Immune Checkpoint Blockade

Learn more about Immune Checkpoint Antibodies.

Figures

Validation of human CD28, CTLA4, and PD-1 expression by Jurkat-Lucia™ TCR-hCTLA4-hPD-1 cells. Jurkat-Lucia™ TCR-hCTLA4-hPD-1 cells were incubated with a PE‑conjugated Anti-hCD28 (A), APC-conjugated Anti-hPD-1 (B), or APC-conjugated Anti-hCTLA4 (C) mAbs for 30 minutes. The binding affinity was then measured using flow cytometry.

Validation of human CD80 and PD-L1 expression by Raji-APC-hPDL1-derived cells. Raji‑APC-hPD-L1 cells were incubated with a PE‑conjugated Anti-hCD80 (A) or APC-conjugated Anti-hPD-L1 (B) mAbs for 30 minutes. The binding affinity was then measured using flow cytometry.

Activation of Jurkat-Lucia™ TCR-hCTLA4-hPD-1 cells using Anti-hPD-1 and Anti-hCTLA4 biosimilar mAbs, alone or in combination. Raji-APC-hPD-L1 and Jurkat-Lucia™ TCR-hCTLA4-hPD-1 cells were incubated - either alone or in combination - with gradient concentrations of Anti-hPD-1 mAb (Nivolumab variable region), Anti-hCLTA4 biosimilar (Ipilimumab), or an Anti-b-Gal-hIgG1 negative control antibody for 24 hours. NFAT activation, reflecting the disruption of the PD-1/PD-L1 or 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), Puromycin

Growth medium: Complete IMDM (see TDS)

Growth properties: Suspension

Quality Control:

• Human PD-1, CTLA4, CD28, PD-L1, and CD80 expression have been verified by flow cytometry.
• Reporter activity is validated using InvivoGen's Anti-hPD-1 and Anti-hCTLA4 antibodies.
• 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 and cannot be sold separately.

• 1 vial of Jurkat-Lucia™ TCR-hCTLA4-hPD-1 cells
• 1 vial of Raji-APC-hPD-L1 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 Puromycin (10 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. PD-1 (programmed cell death 1) receptor and its ligand, PD-L1, 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 IC signals: PD-1/PD-L1 and CTLA4/CD80

— PD-1 (programmed cell death 1; also known as CD279) is a type I transmembrane protein expressed at the cell surface of activated and exhausted conventional T cells. PD-1 is an inhibitory immune checkpoint that prevents T-cell overstimulation and host damage. PD-1 interaction with its ligands PD-L1 and PD-L2 induces inhibition of TCR signaling [3]. PD-L1 (programmed cell death ligand 1; also known as CD274 or B7-H1) is a transmembrane protein expressed at the cell surface of hematopoietic and nonhematopoietic cells and is induced by pro-inflammatory cytokines, such as in the tumor microenvironment  [3].  PD-L1 is one ligand for PD-1, an inhibitory immune checkpoint receptor that is expressed by activated and exhausted T cells. PD-1:PD-L1 interaction induces inhibition of TCR signaling, thereby preventing T-cell overstimulation and host damage [3].

— 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 expressed by antigen-presenting cells. Thereby, CTLA-4 upregulation by T cells prevents overstimulation by arresting both proliferation and activation [3].

Combination therapies targeting PD-1/PD-L1 and CTLA4/CD80

Approaches combining the use of two IC blocking monoclonal antibodies may offer enhanced therapeutic efficacy, particularly in cases where monotherapies have limited success, as demonstrated by scientific reports [4, 5] and clinical trials [6, 7]. 
The combination has shown improved outcomes in several settings, though it is also associated with increased immune-related adverse events and cumulative toxicity. Ongoing research continues to refine patient selection and optimize treatment protocols to maximize benefits while managing risks.

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.
4. Lisi L. et al., 2022. Clinical experience with CTLA-4 blockade for cancer immunotherapy: From the monospecific monoclonal antibody ipilimumab to probodies and bispecific molecules targeting the tumor microenvironment. Pharmacol Research 175:105997.
5. Blanchard L. et al., 2025. Fc-optimized anti-CTLA-4 antibodies increase tumor-associated high endothelial venules and sensitize refractory tumors to PD-1 blockade. Cell Reports Médicine. DOI 10.1016/j.xcrm.2025.102141.
6. NCT03215706 : https://clinicaltrials.gov/study/NCT03215706?term=NCT03215706&rank=1&tab=results (accessed June 2025)
7. Long, GV et al., 2025. Ipilimumab plus nivolumab versus nivolumab alone in patients with melanoma brain metastases (ABC): 7-year follow-up of a multicentre, open-label, randomised, phase 2 study. The Lancet Oncology, Volume 26, Issue 3, 320 – 330 .

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