Number of T cells per well

[Guest Sylvain]

Hi,

I'm new to the ELISPOT world, I would like to detect tumor-Ag specific CD8 T cells (human) within T cells sorted from peripheral blood. I plan to stimulate sorted T cells with specific peptides of interest and look at IFNg secretion. I did it by flow cytometry but the background was to high to discriminate specific anti-tumor responses I was looking for. I have a few questions:

1. Ag specific T cells will be of very low frequency within my samples, I was planning to use a large number of cells for all tested peptides that I would plate in several wells to increase the probabilty to find some specific T cells. How many T cells can I plate in one well? 

2. Would you prefer to perform those test in autopresentation or to load peptides on T2 cells for example?

3. How long should I culture the T cells with the peptides?

4. Would the ELISPOT Plus kit increase the sensitivity and decrease the background compared to classic ELISPOT kit? 

Thank you very much for your help,

Best,

Sylvain.

[Jens@mabtech.com]

Hi Sylvain, and welcome to the Mabtech forum!

It sounds like a wise choice to go for ELISpot to detect tumor-antigen specific T cells. Here are my answers to your questions:

1. Given that the antigen-specific T cells are very low-frequent, I think you're doing the right thing sorting the T cells first. Normally in ELISpot, you plate between 50k and 500k cells, and in your case I would plate 300k - 400k T cells per well. That way the cells aren't too crowded but you still have plenty of purified T cells to evaluate in each well.

2. The phenomenon of T-to-T cell presentation do indeed exist in the context of peptide pulsing (although I'm not entirely sure how it works? Is the exogenous peptide added in such an excess that it replaces the endogenous peptide in the MHC groove at the cells surface? Or does it work by another mechanism?): Both other customers and ourselves have successfully pulsed total CD3 as well as pure CD8 T cell cultures with peptides resulting in good IFN-gamma secretion. I cannot tell if a pulsed T2 cell line would present the peptides in a more satisfactory way. You would have to evaluate that in your own setting. But for sure: It is possible to perform the test using only T cell cultures. 

3. When talking IFN-gamma secretion, our experience is that most activity is over by 24 hours. Thus, to be sure you're getting the most sensitivity our of your assay, incubate the ELISpot plate for 24 hours. (If someone else that Sylvain reads this, please note that other analytes than IFN-gamma due to kinetics might benefit from longer or shorter culture times.)

4. The pre-coated ELISpotPLUS or ELISpotPRO kits won't give a higher sensitivity vs the classic ELISpot, but the pre-coated plates generally give lower background and less variability. Since high background was one of your concerns, I would go for either of those kits.

Good luck with your experiment, and please come back with any further questions you might have!

Kind regards,

Jens

[Guest Sylvain]

Hi Jens,

Thank you very much for your reply! I'll have a try with your suggestions then!

Best,

Sylvain.