ABOUT
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.
The CTLA4/CD80 Bio-IC™ assay is comprised of two cell lines:
– 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 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.
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.
InvivoGen also offers
SPECIFICATIONS
Specifications
CTLA4/CD80
Human
Screening of antibody-based inhibitors of the CTLA-4/CD80 axis, Flow cytometry
Complete IMDM (see TDS)
Verified using Plasmotest™
Each lot is functionally tested and validated.
CONTENTS
Contents
-
Product:CTLA-4/CD80 Bio-IC™
-
Cat code:rajkt-ctla4
-
Quantity:3-7 x 10^6 cells (x2)
- 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
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. 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].
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.
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
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.
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
Cell culture media
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.
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.
Cell line culture
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.
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)
Assays
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.
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).
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.