Hi everyone!
We're hoping to run an ELISPOT using splenocytes animals that have been cured of tumors with an antibody drug. We are fine with isolating the splenocytes, but are worried about the immunogenicity of our AB12 tumor cells. Therefore, we'd like to prime the splenocytes before adding the T-cells to our plates using either whole tumor cells (since we don't know the exact antigen) or tumor lysates. Does anyone have a protocol for this, especially in any mesothelioma cell line?
Thanks in advance,
Richard Davis, Ph.D.
Radiation Oncology
University of Pennsylvania
Hi,
The problem with using a tumor lysate as antigen is that you will have plenty of antigens that are not your primary T cell antigens, which may mask your signal response. If possible, reduce it to a set of candidate proteins (the exact peptide antigen is not required). I do not have experience in using a full cell lysate as antigen in these assays, and I would be quite skeptical in using them.
Instead, using the tumor cells as antigen presenting cells may be a better solution. The pMHC complexes that have triggered your T cells in vivo will be same in vitro. You may need to optimize timings for detection of your ELISPOT readouts, but this might work fine.
Good luck.
Pulsing APCs with tumor lysates is doable, but it takes a lot of optimization to get it to work for the issues mentioned above. Similarly, tumor antigen presentation my not provide enough co-stimulation to trigger a strong recall response (depends what stage of memory they're in).
One option for consideration is to use gp70/AH1 as a tumor-associated antigen. This antigen is present in most murine tumor cell lines and can be pulsed onto a stimulated APC to use for ELISPOT. This would only capture part of your tumor-specific T cell response, but having a single antigen in play is much easier to track.
The results are going to be difficult to interpret but I agree simplification would certainly help. You can pulse APCs with tumor lysate or a single antigen but either way I would irradiate them once loaded and before adding the T cells. Otherwise they will also produce cytokines (not sure which ones you are measuring) which will confound the interpretation. The problem with using the tumor lysate is that you will have many non-tumor cells in there and those antigens will compete for presentation to the T cells. A better option would be to pulse with the lysate of the cell line- this is still tricky because many normal proteins will also present and they could interfere with presentation of the tumor antigens. Either way- I would kill off the APCs after loading - they will still be capable of stimulating the T cells but won't make cytokines of their own.
Good luck-
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