T-cell cytotoxicity assays
A T-cell cytotoxicity assay, also known as a CD8+ T-cell cytotoxicity assay, measures the ability of cytotoxic T lymphocytes to kill target cells. In drug discovery, this assay helps us understand the immune-modulating actions of new therapeutics and optimize their potency.
T-cell cytotoxicity refers to the normal function of cytotoxic T cells, also known as CD8+ T cells, to find and kill target cells as part of the body’s defense mechanisms. This includes killing cells infected by viruses, cancer cells, or foreign cells. This process is a part of the adaptive immune response.
T-cell cytotoxicity assays provide essential information about T-cell activation during the development of various drug modalities and the data needed to optimize their potency.
Autoimmune disorders: Disturbed T-cell activation leads to reactions against healthy tissues in autoimmune disorders. Sensitive assays monitor the efficacy of drugs in these conditions.
Vaccine development: T-cell cytotoxicity assays are used in vaccine development to ensure that the immunogen activates T-cells produced by the vaccine.
Gene therapies: In gene therapies using viral vectors such as AAV or lentiviruses, assays monitor immune responses toward the vector and the capsid proteins. This ensures the drug does not activate cytotoxic T-cells, which could degrade the drug, affect biodistribution, shedding, and pharmacokinetic data preclinically, and challenge the drug’s efficacy clinically.
CAR-T therapies: T-cell cytotoxicity assays measure the potency and efficacy of CAR-T therapies, where T-cells are genetically modified to recognize and attack tumor cells.
High throughput T-cell cytotoxicity assays
Various cellular assays, including cell culturing and flow cytometry-based methods, are commonly used to assess T cell cytotoxicity. However, there are faster and more sensitive alternatives available.
Validated qPCR/dPCR assays
qPCR and dPCR offer higher sensitivity than methods such as flow cytometry, allowing for faster processing of multiple samples in parallel. Validated assays ensure reliable and robust results with short lead times and high throughput capacity. These techniques are particularly advantageous for detecting low-abundance targets and distinguishing between highly similar sequences. qPCR and dPCR quantify transgene expression and copy numbers in various tissues and biofluids and are commonly used to measure the expression of transgenic chimeric antigen receptors (CARs) at the mRNA level and the presence of transgene DNA. This analysis provides insights into the kinetics and expansion of CAR-T cells in vivo, offering valuable information about therapeutic efficacy and biological response.
To understand cytotoxic T-cell activation, qPCR and dPCR assays are used to measure the expression of key molecules at the mRNA level:
- Cytotoxic molecules: These include perforin and granzymes. Once activated, cytotoxic T cells release granules containing perforin, which forms pores in the target cell membrane, allowing granzymes to enter and induce apoptosis.
- Activation markers include markers such as CD69, which indicates early activation, and CD25, which signifies later stages of the immune response.
- Cytokines like IFN-γ, TNF-α, and IL-2 critical for the immune response.
Gene expression analysis can also measure genes related to the apoptosis of target tumor cells. This includes the expression of pro-apoptotic genes like BAX and anti-apoptotic genes like BCL-2, which helps assess whether CAR-T cells successfully induce cell death.
TATAA Pro Perspectives
Our approach
We design and validate qPCR and dPCR assays for your gene of interest, with custom extractions from any sample. Your samples are analyzed using high-throughput instrumentation in a GLP-accredited laboratory.
Proteomics your way – Quantify all essential proteins simultaneously with just microliters of sample
For those who think of mass spectrometry or ELISA when proteomics comes up, think again.
We use Olink’s Proximity Extension Assay (PEA) technology, which combines the specificity of antibodies with the sensitivity of qPCR or NGS readouts.
Olink offers pre-designed and technically validated immunology panels that quantify critical proteins involved in T-cell cytotoxicity activation. These panels require only microliters of blood or plasma and generate data as Normalized Protein eXpression (NPX) values, allowing for easy comparison across different samples and conditions.
The immunological panels include Olink® Target 48 Cytokine, which quantifies key cytokines in human or mouse plasma or blood. Most Olink panels also work for non-human primates (NHP) – contact us for more information.
Additionally, Olink provides two Target 96 panels, each quantifying 92 immunoproteins. These panels require only microliters of blood or plasma from human (or potentially NHP) samples.
Just before summer, Olink launched the new Olink® Target 48 Immune surveillance panel, an extension of these panels.
Protein lists for the relevant panels:
TATAA Pro Perspectives
Our Olink team and lab
We are a certified Olink provider with a dedicated lab team running the assays, utilizing all the necessary instruments and robots to automate steps from dilution to analysis. Our services also include data analysis using Olink software. We offer the complete range of Olink’s platform services.
The Olink Target 48 panels provide both absolute and relative quantification. These panels include cytokines and other immune-related proteins, making them ideal for monitoring immune responses. They are perfect for assessing desired immune reactions in vaccines and CAR-T therapies, as
Immunosequencing entails the high-throughput sequencing of T-cell (TCR) and B-cell receptors (BCR), offering invaluable insights into the immune response within CAR-T therapies. Download the blog as PDF. T-cell malignancies following CAR-T therapy There is a potential risk of secondary cancers
