Performance of Image-Based Label-Free Tracking and Quantification of T-Cell Activation for Use in Cancer Immunotherapy Applications

Interest in cancer immunotherapy research has dramatically increased in recent years, due to the success of a variety of therapies now available, including:

• cancer vaccines
• antibodies or other proteins used to either stimulate or block immune checkpoint pathways
• cells from a patient's own immune system (natural killer or T-cells)

One particular area of interest is the use of activated T-cells to target particular cancers. This has led to unprecedented results where 94% of patients with acute lymphoblastic leukemia (ALL) saw symptoms completely disappear in one study and > 80% of blood cancer patients experienced remission in a second study¹. The process involves removing a portion of the patient’s peripheral blood, isolating the T-cells, activating and expanding the isolated cells, and finally reintroducing them back into the patient².

A critical part of the process is the directed activation and expansion of isolated T-cells. Following antigen binding, as the cells become activated, increased proliferative rates are exhibited and are visualized as aggregates in a clustering effect within the well³. To properly track and quantify these phenotypic changes, cell imaging is essential. The brightfield channel of BioTek’s Cytation Cell Imaging Multi-Mode Reader allows for label-free monitoring of T-cell activation. When integratedas part of the BioSpa Live Cell Imaging System, the results from multi-day activation experiments can be easily quantified.

In the experiment below isolated T-cells were placed into wells of a 24-well plate at a concentration of 100,000 cells per well in the presence of anti-CD3 and anti-CD28 antibodies, plus varying concentrations of an IL-2 Superkine (AdipoGen Life Sciences).

250 ng/mL anti-CD3/250 ng/mL anti-CD28/ 100 ng/mL IL-2 Superkine

3MB - 250 ng/mL anti-CD3/250 ng/mL anti-CD28/ 0 ng/mL IL-2 Superkine

Higher levels of cell proliferation, as indicated by an increase in dark, highly confluent areas within the image, are observed when the cells are in the presence of the antibodies and IL-2 superkine, compared to antibodies alone.

Quantification of activation can also be achieved through determination of the level of confluency and area covered by cell aggregates.

The results validate the ability of BioTek’s instrumentation to be used for this critical step of the T-cell immunotherapy process.

1. Yuhas, A. Cancer researchers claim 'extraordinary results' using T-cell therapy. The Guardian, [Online] Feb 15, 2016. https://www.theguardian.com/science/2016/feb/15/cancer-extraordinary-results-t-cell-therapy-research-clinical-trials (accessed Jul 14, 2017). 
2. Topalian, S.L.; Weiner, G.J.; Pardoll, D.M. Cancer immunotherapy comes of age. J Clin Oncol. 2011, 29, 4828–4836. 
3. Purtic, B.; Pitcher, L.A.; van Oers, N.S.C.; Wülfing, C. T cell receptor (TCR) clustering in the immunological synapse integrates TCR and costimulatory signaling in selected T cells. PNAS. 2005, 102(8), 2904-2909. 

By: BioTek Instruments, Brad Larson, Principal Scientist