T Cell Killing Assays Available at Reaction Biology
Reaction Biology offers a versatile suite of immuno-oncology assay setups. Since every project is different, we enable a customized study layout with various options for immune cells, target cells, or readouts for our customers to choose from.
Please inquire about further customization requirements to tailor an assay specific to your research needs.
Options for immune cells
- PBMCs
- isolated T cells
Options for target cells
- human or murine tumor cells
- tumor cells stably expressing tumor-associated antigens or luciferase
- 2D or 3D cell culture
Options for readout
- quantification of luciferase-expressing tumor cells
- flow cytometry
- live-cell imaging and high-content imaging
- Bliss factor determination of drug combination therapies
Therapy options
- T cell killing enhanced by T cell activation
- T cell killing mediated by T cell engagement
- further therapy options are available upon request
Killing of luciferase-expressing tumor cells via PBMCs stimulated with anti-CD3 treatment enhanced by immune-checkpoint inhibition
Assay principle
Engaging the T-cell receptor complex leads to activation of resting T cells and induction of cytokine release. In this study example, we determined the potency of drugs enhancing T cell activation by co-cultivating PBMCs with tumor cells in the presence of anti-CD3. Further, we modulated the T cell's killing capacity with the immune checkpoint inhibitor anti-PD1.
Setup
Tumor cells stably expressing luciferase were co-cultured with PBMCs at an E:T (effector to target cell ratio) of 6:1 or a range of E:T ratios, in the presence of anti-CD3 at low concentrations to induce killing at a suboptimal level. In a second experiment, the checkpoint inhibitor anti-PD1 was added at different concentrations (7x semi-log). The co-culture was incubated for 4 days after which the viability of the tumor cells was measured by luciferase activity.
Study results
T cell stimulation via anti-CD3 antibody resulted in activation of T cells and killing of tumor cells (A). This effect was enhanced by drug combination testing with anti-PD1 antibodies (B).
PBMCs from a healthy donor were co-cultured with cancer cell lines HT29 and COLO205 stably transduced with Firefly luciferase (FLuc).
A. An E:T ratio of 6:1 was used with the addition of semi-log dilutions of anti-human CD3 (OKT3).
B. Anti-human CD3 (OKT3) at a concentration of 0,68 ng/ml was used to stimulate T cells in a killing assay with different E:T ratios. Anti-PD1 antibody Nivolumab treatment slightly enhanced tumor cell killing.
The % viability was calculated using tumor cells alone as maximal viability (100%) and Staurosporine treatment as maximal killing (0%).
Combination therapy to enhance BiTE-induced T cell killing of luciferase-labeled B-cell lymphoma cell line OCI-LY1
Assay principle
Bi-specific antibodies contain two binding sites to engage T cells and tumor cells resulting in tumor cell killing by (i) bringing the cells in close proximity, and (ii) stimulating cytokine release in T cells.
In this study example, we determine the capacity of a bi-specific engaging molecule to facilitate tumor cell killing by T cells and the combinatorial potential with other clinically relevant agents.
Setup
CD19-expressing B cell lymphoma cells stably expressing firefly luciferase are co-cultured with PBMCs at an E:T (effector to target cell ratio) of 2:1. We tested the bi-specific T cell engager Blinatumomab recognizing CD19 and CD3 at different concentrations (7x semi-log), with or without additional compounds. The co-culture was incubated for 4 days, after which the viability of the tumor cells was measured by luciferase activity.
Study results
BiTE Blinatumomab and the small molecule Lenalidomide act synergistically to kill CD19-positive B cell lymphoma cells, whereas the combination with S63845 acts antagonistically.
A. PBMCs from a healthy donor were incubated with the B cell lymphoma cell line OCI-LY1 at a 2:1 E:T ratio and treated with a range of 7 concentrations of Blinatumomab. Luciferase expression of live tumor cells was measured after 4 days, and viability was calculated using Staurosporine treated cells as maximal cell death control and untreated target cells as maximal viability control. An IC50 of 0,19 ng/ml was found for Blinatumomab. The combinatorial effects of Blinatumomab with Lenalidomide and MCL1-inhibitor S63845 are shown as examples.
B. Using the bliss factor scoring, we determined which drug combinations produce additive, synergistic, or antagonistic effects for killing tumor cells.