ABOUT
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.
The PD-1/PD-L2 Bio-IC™ assay is comprised of two cell lines:
– 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 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
- 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.
Disclaimer: These cells are for internal research use only and are covered by a Limited Use License (See Terms and Conditions). Additional rights may be available.
SPECIFICATIONS
Specifications
PD-1/PD-L1, PD-1/PDL-2
Human
Screening of antibody-based inhibitors of the PD-1/PD-L1 & PD-1/PD-L2 axes, Flow cytometry
Complete IMDM (see TDS)
Verified using Plasmotest™
Each lot is functionally tested and validated.
CONTENTS
Contents
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Product:PD-1/PD-L2 Bio-IC™
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Cat code:rajkt-pdl2
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Quantity:3-7 x 10^6 cells (x2)
- 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
Please note: Both cell lines are sold together and cannot be sold separately.
Shipping & Storage
- Shipping method: Dry ice
- Liquid nitrogen vapor
- Upon receipt, store immediately in liquid nitrogen vapor. Do not store cell vials at -80°C.
Storage:
Caution:
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].
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.
DOCUMENTS
Documents
Technical Data Sheet
Validation Data Sheet
Safety Data Sheet
Certificate of analysis
Need a CoA ?
You may also need
Citations
Powered by BiozFAQ
Handling of cells upon arrival
In general, InvivoGen’s cells are shipped on dry-ice and since dry-ice is not nearly as cold as liquid nitrogen, thawing of the cells technically begins during transport. Thus, we recommend a full thaw upon receipt to ensure the best recovery results.
NOTE: this is not applicable in some countries in Asia where cell lines are sent at room temperature using our specially designed flask
The most important step is the thawing procedure upon receipt of the cells.
Do not put your cells at -80°C or in liquid nitrogen as this may damage the cells.
You must propagate the cells immediately upon receipt.
Below are a few tips we recommend if you encounter any issues during initial culture:
• For the first 2-3 passages, grow cells in media containing 20% FBS and no antibiotics.
• Do not allow cells to reach 100% confluency. Please check the cells as regularly as possible.
• The cells should not be grown in 20% FBS for too long. Use media with 10% FBS after 2 or 3 passages.
• When making frozen stocks, continue growing additional cultures in case there is a problem with the frozen stock.
If you are working with THP1 cells in particular, here are a few additional tips:
• THP1 cells need to be passaged between 3 x 105 and 7 x 105 cells per mL (we recommend 5 x 105 cells per ml). Below this, your cells will take a long time to grow and above 2 x 106 cells per ml you may have toxicity.
• Between each passage, do not centrifuge the cells. THP1 cells grow better in conditioned media, therefore when passaging cells, leave 1 – 2 ml of the old media and add fresh media on top. Do not leave more than 50% of conditioned media as the cells will not have enough nutrients to properly grow.
After thawing, the cells can be more sensitive to selective antibiotics due to the initial low levels of resistance markers. Therefore, it is recommended to wait for 2 - 3 passages before adding the selective antibiotics in order to avoid further stress to the cells during the first couple of passages.
Cell culture media
Many of InvivoGen’s cell lines are engineered with a SEAP (Secreted Embryonic Alkaline Phosphatase) reporter system, and FBS, in many cases, contains traces of alkaline phosphatase (AP) that interferes with the test results. Therefore, to avoid background noise we recommend working with inactivated serum with all of our cells.
In house we inactivate serum by heating at 56°C for 30 minutes. It is important to wait for the water bath to reach the 56°C before placing the tube of serum in to ensure the serum is fully inactivated.
In house we always use heat-inactivated serum for the freezing medium, growth medium and test medium. However, please note that it is not a problem if you wish to use non-heat-inactivated in the freezing medium.
It is important to choose an endotoxin-free serum for the culture of our reporter cell lines. InvivoGen systematically asks for the Certificate of Analysis of different batches of serum to ensure a low level of endotoxin as to not activate the NFkB pathway.
100 U/ml Penicillin and 100 µg/ml Streptomycin
No, this shouldn’t be a problem as long as there is at least 2 mM L-Glutamine in the medium. Most DMEM formulations already contain 4mM of L-Glutamine. However, if you need to add L-Glutamine to your medium you will only need to add 2 mM for the cells to grow properly.
Both Glutamine and GlutaMax have been tested in-house and can be used, with no difference in the cells noted.
Cell line culture
When the HEK-Blue™ cells are non-adherent, either they were diluted too harshly at the start or they have grown over-confluent in a small flask and suffocated.
To avoid this in the future:
• Change the medium and seed the cells at a density of approximately 1.5 x 106 cells/mL in a T25 flask.
• Wash the cells before putting them into a new flask. Sometimes when the cells are non-adherent, it is due to the clustering of both live and dead cells. Therefore, this will get rid of any remaining DMSO which could affect the adhesion of the cells to the flask.
• Use medium with 20% FBS.
• The use of CellBIND flasks can sometimes help to increase attachment and growth of the cells (however CellBIND flasks are not required in the normal protocol)
It isn’t normal for your cells to divide only once a week. Depending on the clone, they usually have a doubling time of 24 – 72 hours. THP1 cells must be cultured at quite a high concentration (at least 5 x 105 cells/ml). Please note that our R&D teams have noticed that THP1 cells often die when they are diluted too harshly. Also, it may help to not add Normocin™ while waiting for improvement to their growth.
It is not a problem if the cells are clumping, as long as they are growing fine and have normal morphology. You may want to centrifuge the cells to get rid of the dead cells as sometimes this is the reason for clumping. Please note that we recommend homogenizing the cells before performing your assays.
We recommend to centrifuge at 120 G for 10 minutes.
Assays
They are semi-quantitative tests. However, they can be used for quantitative measurements by making a standard curve using a positive control.
If you cannot run the detection assays immediately, you can store supernatant samples for up to a week at 4°C. Supernatant samples are active for a longer time when kept at -20°C. However, in this case we would recommend supplementing the test medium with 20% FBS to protect the SEAP as much as possible during the freezing process. Do not repeat freeze/thaw cycles.
It depends on the inhibitor. If the inhibitor blocks a receptor or an element in the signaling pathway, we recommend pre-incubating the inhibitor with the cells for at least 30 minutes. If the inhibitor is binding or blocking the ligand (i.e. an antibody against a specific cytokine) then it is best to pre-incubate the inhibitor with the ligand prior to adding to the cells.
In house the cells are seeded at the same time as adding the ligand (ligand first).
To limit the activation of NFκB before stimulation (lower background):
• Use healthy cells that have been passaged at least 24 hours before the assay
• Do not allow cells to be greater than 80 % confluent
• Use pre-warmed PBS to rinse your cells
• Use heat-inactivated FBS (to eliminate residual alkaline phosphatase in the serum)
• Do not centrifuge the cells prior to stimulation
• Rather than trypsin, use PBS for 2 - 3 min @ 37°C and gently pipet up and down to detach cells
• Do not use an excessive number of cells per well
(approximately 50 000 cells/well as recommended on the TDS).
We recommend to not use Normocin™ in the test medium with all of our cell lines as it is better to reduce the number of potential interfering agents in the medium when performing the assay. The same goes for the use of selective antibiotics
It is hard to estimate the deviation factor regarding cell passage number. However, we note very little difference in our experimental results, with no more than the slight variations you expect due to handling errors.
Yes, InvivoGen’s reporter cell lines can be used in ELISA and Western Blots. However, please note that we do not sell them for this purpose.
Frozen stock preparation
For adherent cells, we recommend DMEM, 20% (v/v) fetal bovine serum (FBS), and 10% (v/v) DMSO.
For suspension cells, we recommend fetal bovine serum (FBS) and 5-10% (v/v) DMSO
We highly recommend keeping a flask of cells running until you begin thawing your frozen stock in case something has gone wrong during the freezing step.
Reporter system
Our reporter cells require the activation of just one transcription factor (NFκB and/or AP-1) to produce a signal.
InvivoGen’s Lucia® luciferase is completely synthetic and is not related to any natural luciferase.