How to measure immune cell killing of tumor cells effectively

In this on-demand SelectScience webinar, Dr. Anne Lodge, Chief Science and Innovation Officer at Cellero, discusses how the Incucyte^® Live-Cell Analysis offers walkaway monitoring of tumor cell lysis using fluorescent labels of cytotoxicity. Lodge presents data using tumor antigen-specific T cells from Cellero and widely available tumor cell lines to demonstrate specific lysis of tumor cells. Important technical factors are also discussed.

Read on for highlights of the live Q&A session or register to watch the webinar at any time that suits you.

Q: Do you use preactivated effectors or naive?

AL: The data shown in the presentation is with cells that we have thawed and immediately placed in the assay. NK cells are completely naive and for the T cells, I would consider preactivated.

Q: If the data is different depending on the way they are expressed (area vs count), then how can you rely on it?

AL: They are not necessarily inconsistent, but they reveal something about what is going on in the culture. For example, there could be more cells, if there’s a comparison between different target cell suspensions, or there is a difference in the cell size area.

Q: Can you elaborate a bit on specificity controls?

AL: The use of non-specific effectors is important because we do see non-specific killing. The addition of the appropriate antigenic peptide can also reveal specific killing, especially when there isn't killing in the absence of peptide.

Q: We are trying to do autologous tumor cell killing and don't have a peptide to add. What other controls do you recommend?

AL: You will be trying to see cell killing and be sure it is specific. That is difficult but I would run some allogeneic cells as controls. Those might give you an idea of non-specific killing.

Q: Would you recommend the Annexin V dye for cell death of adherent cells and would you add that dye at the beginning of the incubation with the effector cells or after the incubation?

AL: Yes, you would add the Annexin V at the start of the assay. Sartorius recommends the caspase reagent for adherent cells though.

Q: Does Cellero have any information on which of their T cell lines exhibit killing against specific targets on their site?

AL: No, we're still demonstrating specificity with appropriate targets, but I can tell you that our Her2 specific T cells are cytotoxic against appropriate targets, as are the NY-ESO T cells.

Q: What is the advantage of the Incucyte^® over a flow cytometric assay?

AL: The assay using the Incucyte^® uses fewer cells and needs less hands-on time.

Q: Can you use other reagents besides those from Sartorius?

AL: Yes, there are other reagents that can be used for labeling, particularly for Annexin V. We haven’t tried alternatives because we wanted to limit the variables as we worked out the assay.

Q: With regards to the specific T cell products from Cellero, can you expand them to some extent after buying them?

AL: They can be expanded but we do not support that because it has been difficult for people to reproduce our expansion protocol.

Q: What system have you used to isolate T cells for these assays? What is the minimum percentage of purity of T cells that you prefer in those assays?

AL: There is no minimum purity, we see good cytotoxicity when there is just 10% of the specific effectors present.

Q: In the coculture condition, have you observed the adsorption of T cells? How do you interpret ATP test results?

AL: That's the problem with that method. If T cells are adsorbed (and they are) then they contribute to signal and sort of drown out the loss of signal from target cells.

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