T cell Cytotoxicity and Cell Killing Assays

T cell Cytotoxicity and Cell Killing Assays

ImmuMap offers T cell cytotoxicity assay and cell killing assays by the xCELLigence® RTCA DP instrument. This instrument takes advantage of noninvasive electrical impedance monitoring to quantify cell proliferation, killing, morphology change, and attachment quality in a label-free, real-time manner.

The impedance is measured continually inside the incubator and reported and controlled in real-time by a laptop computer placed outside the incubator. Experiments can run for several days and this cytotoxicity assay is thus much more sensitive than common methods such as the chromium release assay. Also a great advantage of using the xCELLigence platform for cytotoxicity test is that a specific evaluation time point for optimal signal-to-noise ratio does not need to be chosen on beforehand, and the ideal time point for downstream applications can thus easily be identified.

Our services include long-term cytotoxicity assays for revealing T cell killing in cell cultures or compound-mediated cytotoxic effects. We can make use of a broad range of both cancer and healthy cell lines, the latter e.g. taking advantage of induced pluripotent stem cells (iPSCs) of various sorts such as heart, liver and brain. As a supplement to the xCELLigence analysis we perform intra- and extracellular staining for flow cytometry, enabling us to investigate in parallel the direct T cell or other cellular cytotoxicity and in single-cell resolution the functional activation induced after co-culturing of cells or compound of interest and target cells.

Our package of safety analyses is recommended for in vitro safety studies and T cell epitope discovery. We offer evaluation of cytotoxicity/safety against a panel of relevant healthy cell lines of a broad range of cells and compounds, including, but not limited to, TCRs, CARs, potential cytotoxic reagents and medical drugs.

The analysis package is likewise suited for determining the T cell killing of specific targets in T cell epitope discovery studies of e.g. cancer-associated potential T cell epitopes, as we can investigate the processing and presentation of the peptide-MHC complex in question on relevant cancer cell lines through these T cell cytotoxicity assays.

Additionally our xCELLigence® RTCA DP instrument offers the ability for making kinetic measurements of cell invasion and migration using an electronically integrated Boyden chamber. It is thus suited for e.g. analysis of the migration of chemokine-engineered or other cells and a wide range of other applications:

  • Cell adhesion and cell spreading
  • Cell proliferation and cell differentiation
  • Receptor-mediated signaling
  • Virus-mediated cytopathogenicity
  • Continuous quality control of cells

All projects will be tailored to the specific need of individual customers.

The development of TCRs and CARs for the treatment of a broad range of both hematological and solid cancers has reached the clinic in recent years, pioneered by Carl June and coworkers. Clinical results span from complete responses with no or only manageable side effects to severe and sometimes fatal toxicities even though proper pre-clinical safety evaluation was done on beforehand. Especially the study of an affinity-optimized TCR performed by Adaptimmune/Immunocore (Linette et al., Blood, 2013; Cameron et al., Sci Transl Med, 2013) shed light on the need for thorough testing of possible on- or off-target cross-reactivity by cytotoxicity test assays before entering the clinic. Two patients were treated with a TCR recognizing MAGE-A3 and developed fatal cardiac toxicity within five days after T cell administration. This cytotoxicity was later elucidated to be due to cross-recognition by the TCR (off-target) of a peptide-MHC complex from the heart-associated protein titin. Prior to entering the clinic the possible recognition by the TCR in question of cell lines of different origin had been tested intensively, including 10 cardiac-derived primary cells expressing the relevant HLA, and no recognition was found. Post-treatment in vitro investigation revealed the cross reaction against a beating culture of iCell cardiomyocytes generated from iPSCs, while for common myocytes recognition could only be detected after culture at confluence for 32 days. This finding suggests that iPSCs represents a better reflection of the in vivo conditions than heavily cultured cell lines from primary tissue. Therefore we offer testing of iPSCs at ImmuMap.

ImmuMap - DTU Science Park, Diplomvej 377, 2800 Kgs. Lyngby, Denmark - Tel: +45 6178 1882

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