Jurkat-Raji PD-1/PD-L2 assay (Bio-IC™)

Antagonist screening assay for PD-1/PD-L2 axis

The PD-1/PD-L2 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 PD-1/PD-L2 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 PD-L2 restore T cell activity and represent promising immunotherapeutic approaches, particularly in cases where monotherapies have limited success.

 More details
 

PD-1/PD-L2 Bio-IC™ assay principle (click to enlarge)

The PD-1/PD-L2 Bio-IC™ assay is comprised of two cell lines:

• Jurkat-Lucia™ TCR-hPD-1

Jurkat-Lucia™ TCR-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 PD-1 immune checkpoint receptor.

• Raji-APC-hPD-L2

Raji-APC-hPD-L2 are engineered human B cells acting as antigen-presenting cells (APCs). They stably express the cognate TCR [HLA::peptide] complex and the PD-L2 immune checkpoint ligand. These cells express endogenous levels of CD80/86, the CD28 ligand, and PD-L1, the canonical PD-1 ligand.
 

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  PD-1/PD-L1 and PD-1/PD-L2. These three interactions prevent the Jurkat-Lucia™ TCR-hPD-1 cells from expressing Lucia®.

In the presence of PD-1, PD-L1, and/or PD-L2 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-L1 and PD-L2 blocking antibodies results in stronger inhibition than when each antibody is used alone (see Figures). 

Jurkat-Lucia™ TCR-hPD-1 key features

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

Raji-APC-hPD-L2 key features

• Stable specific [HLA::peptide] expression
• Stable hPD-L2 overexpression
• Endogenous hPD-L1 and hCD80 expression

Read our review on Immune Checkpoint Blockade

Learn more about Immune Checkpoint Antibodies.

Figures

Validation of human PD-1 expression by Jurkat-Lucia™ TCR-hPD-1 cells.

Jurkat-Lucia™ NFAT and Jurkat-Lucia™ TCR-hPD-1 cells were incubated with APC-conjugated Anti-hPD-1 mAb for 30 minutes.
The binding affinity was then measured using flow cytometry.

Validation of human PD-L1 and PD-L2 expression by Raji-APC-hPD-L2 cells. Raji‑APC-hPD-L2 cells were incubated with APC‑conjugated Anti-hPD-L1 (A) or PE-conjugated Anti-hPD-L2 (B) mAbs for 30 minutes. The binding affinity was then measured using flow cytometry.

Activation of Jurkat-Lucia™ TCR-hPD-1 cells using Anti-hPD-1 biosimilar mAbs. Raji-APC-hPD-L2 and Jurkat-Lucia™ TCR-hPD-1 cells were incubated with gradient concentrations of Anti-hPD-1 hIgG1 mAbs (Ni: Nivolumab variable region; Pem: Pembrolizumab variable region) or an Anti-β-Gal-hIgG1 negative control antibody for 24 hours. NFAT activation, reflecting the disruption of PD-1/PD-L1 or PD-1/PD-L2 inhibitory interactions, was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. The fold increase over non induced cells (no mAbs) is shown.

Activation of Jurkat-Lucia™ TCR-hPD-1 cells using Anti-hPD-L1 or Anti-hPD-L2 mAbs, alone or in combination. Raji-APC-hPD-L2 and Jurkat-Lucia™ TCR-hPD-1 cells were incubated with gradient concentrations of Anti-hPD-L1 biosimilar (Atezo: Atezolizumab variable region), Anti-hPD-L2 mAb (24F: clone 24F.10C12), or an Anti-b-Gal-hIgG1 negative control antibody for 24 hours (A, B). The combination of Anti-hPD-L1 and Anti-hPD-L2 mAbs compared to each mAb alone is shown in panel (B). NFAT activation, reflecting the disruption of PD-1/PD-L1 or/and PD-1/PD-L2 inhibitory interactions, 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 PD-1, PD-L1, and PD-L2 expression have been verified by flow cytometry.
• Reporter activity is validated using InvivoGen's Anti-hPD-1, Anti-hPD-L1, and Anti-hPD-L2 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-hPD-1 cells
• 1 vial of Raji-APC-hPD-L2 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. 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].

IC signal: PD-1 and its ligands PD-L1 and PD-L2

— 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].

— PD-L2 (programmed cell death ligand 2; also known as CD273 or B7-DC) is a transmembrane protein that has more restricted expression in antigen-presenting cells, such as dendritic cells and macrophages [3, 4]. It serves as a second ligand for PD-1, complementing PD-L1 in mediating peripheral immune tolerance and maintaining immune homeostasis during infection [3-5]. Like PD-L1, PD-L2 is also co-opted by cancer cells and tumor-associated immune cells to suppress T cell-dependent anti-tumor immunity [5, 6]. 

Elevated PD-L2 expression has been observed in various malignancies, including lung, colorectal, and breast cancers, and is often associated with poor prognosis [7]. The continuous exploration of the clinical application of PD-L2 has brought exciting prospects for promoting cancer treatment, including small molecules, PD-L2-specific vaccines, and monoclonal antibodies (mAbs) [7]. However, the clinical approval of PD-L2-specific therapeutics for tumor treatment remains elusive, necessitating further investigation and discovery [7].

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. Latchman, Y.  et al. 2001. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2, 261–268.
5. Yearley, J.H. et al. 2017. PD-L2 expression in human tumors: relevance to anti-PD-1 therapy in cancer. Clin Cancer Res 23, 3158–3167.
6. Rozali, E.N. et al. 2012. Programmed death ligand 2 in cancer-induced immune suppression. Clin Dev Immunol 2012, 656340.
7. Yang Y, et al. 2024. Programmed cell death-ligand 2: new insights in cancer. Front Immunol. 2024 Mar 28;15:1359532.

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