Anti-PD-1/PD-L1 Cell-based Assay

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Principle of PD-1/PD-L1 BioIC™ cellular assay

PD-1/PD-L1 immune checkpoint inhibitor screening assay

InvivoGen offers a cell-based assay specifically designed to provide a biologically relevant, sensitive, and well-controlled alternative to the use of primary human T cells for screening antibody-, Fc-fusion protein-, or small molecule-based inhibitors of the PD-1/PD-L1 immune checkpoint (IC) axis.
This assay is comprised of a reporter T cell line and an antigen-presenting cell line (APC). T cells are derived from a human Jurkat reporter T cell line expressing a specific T cell receptor (TCR). APCs are derived from a human Raji cell line expressing a specific [HLA::peptide] complex.

• Jurkat-Lucia™ TCR-hPD-1 Cells – Reporter T Cells
• Raji-APC-hPD-L1 Cells – Antigen-Presenting Cells

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.
The first signal (signal 1) is delivered upon recognition of a specific antigenic peptide complexed with HLA molecules ([HLA::peptide]) on Raji-APC cells by the TCR on Jurkat-Lucia™ cells. The second signal (signal 2), also known as the co-stimulation signal, is operated by the interaction of CD80/86 and CD28 molecules at the surface of the APC and T cells, respectively. An additional regulatory signal inhibits T-cell activation upon T-cell PD-1 and APC PD-L1 engagement (IC signal).

 More details

Assay principle:

Jurkat-Lucia™ TCR-hPD-1 cells derive from the Jurkat-Lucia™ NFAT reporter cell line. They stably express the Lucia luciferase reporter gene under the control of an ISG54 minimal promoter fused to six NFAT response elements. Activation of these cells is measured as a bioluminescent signal produced by the Lucia luciferase using the appropriate detection reagent QUANTI-Luc™ 4 Lucia/Gaussia.

• The Jurkat-Lucia™ TCR-hPD-1 and Raji-APC-hPD-L1 co-culture allows the following signal integration: IC signal inhibits (signal 1 + signal 2) and subsequent NFAT activation in Jurkat-Lucia™ TCR-hPD-1 cells. Lucia luciferase reporter is not expressed.
• Upon addition of either anti-PD-1 or anti-PD-L1 antibodies to the co-culture, the IC signal is not delivered and (signal 1 + signal 2) induces NFAT activation in Jurkat-Lucia™ TCR-hPD-1 cells. Lucia luciferase reporter is expressed.

EC₅₀ values of the bioluminescent signal can be compared to assess the antibody-blocking potency (see Figures).

T-cell key features:

• Stable specific [HLA::peptide]-restricted TCR
• Stable hCD28 overexpression
• Stable hPD-1 overexpression
• NFAT-inducible Lucia luciferase reporter activity

APC key features:

• Stable specific [HLA::peptide] expression
• Endogenous hCD80/86 expression
• Stable hPD-L1 overexpression

Read our review on Immune Checkpoint Blockade

Learn more about Immune Checkpoint Antibodies.

Figures

Validation of human CD28 and PD-1 expression by Jurkat-Lucia™ TCR-hPD-1 cells.

Jurkat-Lucia™ NFAT and Jurkat-Lucia™ TCR-hPD-1 cells were incubated with a PE‑conjugated Anti-hCD28 (A) or APC-conjugated Anti-hPD-1 (B) mAb for 30 minutes.
The binding affinity was then measured using flow cytometry.

Validation of human PD-L1 expression by Raji-APC-hPD-L1 cells.

Raji-APC-Null and Raji‑APC-hPD-L1 cells were incubated with a PE‑conjugated Anti-hPD-L1 mAb for 30 minutes.
The binding affinity was then measured using flow cytometry.

Activation of Jurkat-Lucia™ TCR-hPD-1 cells.
Raji-APC-hPD-L1 and Jurkat-Lucia™ TCR-hPD-1 cells were incubated with gradient concentrations of AntihPD-1 hIgG1 (Ni: Nivolumab variable region; Pem: Pembrolizumab variable region) or Anti-hPD-L1 mAbs for 6 hours. NFAT activation, reflecting the disruption of PD-1/PD-L1 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

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, 100 µg/ml Normocin™

Antibiotic resistance:

• Jurkat-Lucia™ TCR-hPD-1 cells: Blasticidin, Zeocin®, Hygromycin, and G418 (Geneticin)
• Raji-APC-hPD-L1 cells: Blasticidin and G418 (Geneticin)

Quality Control:

• Human CD28, PD-1, and PD-L1 expression have been verified by flow-cytometry.
• Reporter activity has been validated using InvivoGen's anti-hPD-1 and anti-hPD-L1 antibodies.
• The stability for 20 passages following thawing has been verified.
• Both cell lines are guaranteed mycoplasma-free.
   

These products are covered by a Limited Use License (See Terms and Conditions).

Contents

Please note: Both cell lines are sold together and cannot be sold separately.

• 3-7 x 10⁶ Jurkat-Lucia™ TCR-hPD-1 cells in a cryovial or shipping flask
• 3-7 x 10⁶ Raji-APC-hPD-L1 cells in a cryovial or shipping flask
• 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). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

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 PD-L1

— 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].

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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