T cell killing assessment

Explicyte is offering a specialized in vitro T cell cytotoxicity-mediated killing assay to evaluate the ability of test compounds to modulate the immune response of effector cell subsets. Our sensitive and quantitative assay is based on a 96-w plate co-culture of human tumor cells* and primary immune cells and consists in the kinetic monitoring of tumor cell proliferation and death thereby enabling to measure the killing immune response towards tumor cells by analyzing cell death specifically within the tumor cell population.
 
* Contact us to know the tumor cell lines available in this assay
 

Assay principle

  • Co-culture of target tumor cells with immune cells (PBMCs, purified T cells…) in the presence of an activator cocktail to promote killing activity.
  • Real-time monitoring of tumor cell count (tumor cells stably expressing a nuclear fluorescent probe) as a surrogate of cell proliferation, and of tumor cell death using a mix-and-read caspase 3/7 fluorescent reagent, over several days in the continuous presence of immune cells.
  • Automated image analysis & segmentation enables apoptotic death quantitation specifically within tumor cells (caspase 3/7 fluorescent probe within the nuclear probe-expressing cells).
  • Ability to multiplex with additional functional measures such as effector cytokine release.

Illustrative data 

Immune cell killing of H1299 lung cancer cells
Immune cell-mediated killing of H1299 lung cancer cells in the presence of increasing concentrations of αCD3. H1299 tumor cells stably expressing a nuclear red fluorescent protein are cultured, at an appropriate effector:target ratio, with inactivated or αCD3-activated hPBMC.

 

αCD3 induces hPBMC-mediated apoptotic killing of target tumor cells (a). Live cell real time detection images of apoptotic H1299 tumor cells at 72h post-culture with inactivated and αCD3-activated hPBMC. Tumor cell count (nuclear red probe-expressing cells, (a, b, d, e)) and apoptosis (caspase 3/7 green fluorescent probe, (a, b, d, e)) are kinetically monitored by live cell imaging. Segmentation masks & image analysis are performed (c, f) to quantify apoptosis specifically within the tumor cell population (i.e. caspase 3/7 green fluorescent probe within the nuclear red probe-expressing cells – yellow objects). Arrows depicted on the image indicate apoptotic tumor cells and arrowheads apoptotic non-tumor cells. Kinetic and dose-dependent effect of αCD3 in inducing hPBMC-mediated apoptotic killing of H1299 cancer cells (b). Tumor cell apoptosis is quantified and results are then normalized to tumor cell count, and plotted either overtime on a 120h post-treatment period or as integrated AUC values over this time period.

Immune cell-mediated killing of H1299 lung cancer cells in the presence of increasing concentrations ...

Immune cell killing of A549 lung cancer cells
Promotion of hPBMC-mediated killing of A549 lung cancer cells by increasing concentrations of αCD3.

A549 tumor cells stably expressing a nuclear fluorescent probe are cultured, at an appropriate E:T ratio, with inactivated or αCD3-activated hPBMC. Tumor cell count and apoptosis are kinetically monitored by live cell imaging. Apoptosis is quantified specifically within the tumor cell population and results are then normalized to tumor cell count and can be plotted overtime on a 120h post-treatment period (a) or expressed as integrated AUC values over this time period (b). 

Promotion of hPBMC-mediated killing of A549 lung cancer cells by increasing concentrations ...

Immune cell killing of SKOV-3 ovarian cancer cells
Kinetic monitoring of SKOV-3 ovarian cancer cell count (optional, as a surrogate of tumor cell proliferation) over a 120h period. Real-time counting of live tumor cells (red nuclear probe) reveals time-dependent and dose-dependent effect of hPBMC-activating αCD3 thereby resulting in a decrease of tumor cells number in the co-culture.  

Kinetic monitoring of SKOV-3 ovarian cancer cell count (optional, as a surrogate of tumor ...