I am currently investigating the potential of using tumour lysate as a source of antigen in cancer vaccines.
I immunized mice with microparticles (MP) containing either EL4- oder EG-7-lysate and polyI:C. 6 days post-immunization I performed an IFNy ICS and ELISpot, where I restimulated with different peptides. In ELISpot, I often observed very high background in unstimulated splenocytes from lysate-immunized mice. Interestingly the same splenocytes did not show any substantial background in ICS.
Do you have any idea how this could be? The only difference between the two splenocytes was I depleted them of red blood cells before using them for ELISpot. Another thing could be incubation time (5h for ICS, 18h for ELISpot), but I don't see how that could activate T cells.
I would appreciate some input if you have any ideas.
Greetings,
Isabel
Hi Isabel, I have a lot of experience with tumor lysate as an antigen source & using ELISPOT & ICS Flow to try to detect specific responses :)
Did you use IL-2 in your incubation medium for both assays?
I included IL-2 in my ICS incubation medium but it is crucial to leave it out of your ELISPOT medium to avoid high background (non-specific, cytokine-mediated IFN-y production).
I hope this helps :)
Hey Melanie, thanks for your answer and suggestion :)
I actually did not use IL-2 in incubation medium. In both assays unstimulated splenocytes were only cultured in IMDM medium (+ FCS and P/S).
Interesting.
How many cells are you plating per ELISPOT well? You might need to titrate the cell #s plated to allow you to see a difference between the unstimulated & stimulated cells?
I saw higher than expected SFU in my unstimulated, lysate-primed human T cells but we were re-stimulating with lysate, not peptides. I plated DCs with the T cells to present lysate antigens to the T cells. So that explained the higher than usual level of background IFN-y. But there were still more SFU in the wells containing T cells+lysate-loaded DCs, than the wells with T cells + DCs that were not loaded with lysate.
I'm wondering if it's something to do with how you're preparing your splenocytes. Feel free to send me your protocol if you'd like me to take a look: [email protected]
Check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339961/