Cellero showcases the latest techniques for measuring immune cell cytotoxicity in a new educational webinar.
Cancer immunotherapy is a rapidly growing area of cancer treatment in which the body’s immune response to cancer cells is artificially stimulated. As the number of cancer immunotherapy treatments in clinical trials continues to increase, it is important that immune cell cytotoxicity is measured accurately so that treatment efficacy can be meaningfully evaluated.
Dr. Anne Lodge, Scientific Consultant at Cellero, has decades of immunology expertise behind her. Dr. Lodge has worked on T cell biology since she was a young graduate student; she made numerous contributions to the development of various immune cell therapeutics even before founding her own immune cell product company in 2004. In hosting this latest webinar, Dr. Lodge hopes to give fellow researchers an overview of the currently available methods for evaluating immune cell cytotoxicity, so they can make an informed decision on which method or methods work best for their lab or program.
Many researchers still consider the chromium51 (51Cr) release assay the gold standard for measuring the effectiveness of cytotoxic immune cells against pathogens. Targeted cancer cells take up 51Cr, which is then released upon cell lysis. However, one obvious drawback to this method is the reliance on radioactivity; spontaneous background release can be quite high, often up to 20%. This increases handling hazards for the researcher, and the short half-life of the radioactive isotope makes the method expensive.
There are, of course, alternatives available. One of the most popular of these is calcein labeling. This method works along the same lines as the 51Cr release assay, but in this case fluorescence rather than radioactivity is measured. While there is no danger from radioactivity, calcein labeling, like radioactive labeling, suffers from high spontaneous release of the fluorescent dye, as well as low intensity of the fluorescent signal, both of which decrease the overall sensitivity of the test.
Luciferase labeling is another alternative cytotoxicity test. It combines high sensitivity with ease of use. Target cells are labeled with luciferase using transduction, a method by which the luciferase gene is transferred into target cells using a viral vector. Target cell death can be calculated by measuring either decreasing luminescence as cancer cells die or increasing luminescence in the cell culture media as cancer cells are lysed. The assay does have some drawbacks, however; it relies on accurate measurement of the percentage of transduced cells and is time consuming compared to other assays.
Dr. Lodge admits that there is one immune cell cytotoxicity assay that she favors over the others – live cell imaging.
Unlike other methods, live cell imaging can be used with adherent cell cultures as well as suspension cell cultures, making it a powerful technique. For the purposes of their lab’s study, Dr. Lodge and her colleagues chose Incucyte® for their live cell analysis.
One reason Dr. Lodge favors live cell imaging is the level of automation involved. Once imaging is set up, researchers can literally walk away and lend their attention to other tasks, coming back at their convenience to analyze the data. And that data is impressive!
Like many techniques, live cell imaging works through the judicious application of cell labeling. Red and green-fluorescent dye can be used to label immune cells versus target cells, or to label cell death indicators such as proteins Annexin V and Caspase 317, both of which are released during cell lysing and apoptosis. Researchers can label and track T cells designed to bind specific target cell antigens. For the purposes of the webinar, Dr. Lodge showed live cell imaging data collected using Cellero’s anti-HPV E7 antigen specific T cells.
Once targets of interest are labeled, computer analysis of images taken over time (generally 18-24 hours) are converted into plots. Scientists can define exactly what they wish to measure; for example, overlapping objects, red objects, or green objects. They can also set up complex analyses to discover which cells and treatments are most effective
Dr. Lodge cautions that live cell imaging works best when T cell specificity is established beforehand using other methods, rather than immediately looking at cytotoxicity against cancer cells. Specificity controls should always be included since factors such as poor antigen presentation by the target cell can confuse results. Nevertheless, she feels that there is no substitute for looking at living immune cells in action-some of the images the group captures are definitely worth a thousand words!
Cellero’s mission is to provide the resources scientists need to develop and commercialize life-saving immunotherapies. That includes not just biological supplies, but expertise. Dr. Lodge says her primary advice to other immuno-oncologists is to keep abreast of new developments and remember that there are always alternative ways to look at data. Please visit our website to discover how Cellero can help advance your research.