TCR-T and CAR-T/NK Cell Exhaustion Assay

One of the mechanisms that prevent the destruction of the immune system is functional silencing T cells, known as T cell exhaustion, which is also utilized by viruses and cancers to induce immune escape. T cell exhaustion is a state of T cell dysfunction that occurs in many cancers. In the case of tumors and chronic infections, NK cells exhibit an exhausted state similar to T cells, showing poor effector function and altered phenotype. Exhaustion of T/NK cells in the tumor microenvironment (TME) results in a decrease in the ability to produce effective immune responses to eliminate tumors. Reversing or preventing CAR-T/NK cells exhaustion is the main research field of cancer immunotherapy.

Currently, multiple phenotypes and functional parameters need to be analyzed to identify CAR-T/NK cell exhaustion. Better understanding of the CAR-T/NK cell exhaustion mechanism and the identification of unique phenotypic markers of T/NK cells will greatly promote the development of adoptive cell transfer strategies.

Creative Biolabs's proprietary assays effectively mimic the tumor microenvironment and determine the exhaustion status of T/NK cells, allowing you to simultaneously measure expression levels of up to hundreds of cytokines using standard Western blotting equipment. Flow cytometry is used to detect CAR T cell–infiltrated tumor cells, followed by detection of multiple markers in one sample.

Hallmarks of T Cell Exhaustion

T Cell ExhaustionFig1 T Cell Exhaustion Markers (Okoye I S, 2017)

CD8 + T cells

  • Co-expressing multiple inhibitory receptors: PD-1, CTLA-4, LAG-3, TIM-3, 2B4 / CD244 / SLAMF4, CD160, TIGIT
  • Loss of IL-2 production, proliferative capacity, in vitro cytolysis activity
  • Impaired production of TNF-α, IFN-γ and cc(β) chemokines
  • Degranulation; expression of high levels of granzyme B

CD4 + T cells

  • Co-expressing multiple inhibitory receptors: PD-1, CTLA-4, LAG-3, TIM-3, 2B4 / CD244 / SLAMF4, CD160, TIGIT
  • Loss of IL-2 production, proliferative capacity, in vitro cytolysis activity
  • Impaired production of TNF-α, IFN-γ and cc(β) chemokines

NK Cell ExhaustionFig2 NK Cell Exhaustion (Bi J and Tian Z, 2017)

Hallmarks of NK Cell Exhaustion

  • Co-expressing multiple inhibitory receptors: NKG2D, CD16, NCRs, CD226, 2B4, IFN-γ
  • Impaired cytolytic activity
  • Immune checkpoint blockade can potentially reverse NK cell exhaustion

T/NK Cell Exhaustion Assay Workflow

In this assay, fresh isolated human PBMCs are stimulated with a fixed concentration of compounds for three days. The media is harvested for hallmark measurements.

Workflow

For more details, please feel free to contact us for project quotations and more detailed information.

References

  1. Okoye I S.; et al. Coinhibitory Receptor Expression and Immune Checkpoint Blockade: Maintaining a Balance in CD8+ T Cell Responses to Chronic Viral Infections and Cancer. Frontiers in Immunology. 2017, 8:1215.
  2. Bi J and Tian Z. NK Cell Exhaustion. Frontiers in Immunology. 2017, 8:760.

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