In my ELISPOT of tumor antigen-specific T cells we can see a very nice reduction in IFN-y (about half) in the presence of anti-Class I , suggesting a CD8-mediated response to peptide(s) in these pools.
We see no reduction in IFN-y in the presence of anti-Class II, confirming the CD8-mediated response.
However all the IFN-y is obliterated to baseline in the presence of both Class-I & Class-II ABs.
If IFN-y only drops by about half in the presence of anti-Class I, that suggests that some of the gamma is coming from the CD4s. So if that’s the case, why is there no reduction in the presence of anti-Class II?
I would love to hear any thoughts on this
Many thanks
Melanie
What exactly is in your wells? Are these TIL populations being tested against peptide-pulsed PBMC? Or something else? Are they pools of peptide in each well or single-peptide? Are the stimulator and effector cells autologous or allogeneic?
It's an interesting result for sure but need more context!
Hi Govinda, These are the non-adherent fraction of PBMCs that were stimulated 3X with peptide-loaded DCs. They were plated 1:2 with peptide-loaded PHA blasts (1 T cell:2 PHA blasts). The PHA blasts were loaded with pools of peptide (4 peptides per pool). The stimulator & effector cells are autologous (both from the same donor).
When you block MHC I, I'm assuming you add the antibody (or whatever blocker) before the co-culture right or do you add the blocker in with the co-culture? Because the most likely explanation I can think of is CD8 IFNg production that is dependent on CD4 help. So if you block both MHC I and II, you block both direct CD8 IFNg production and those that depend on CD4 help.
I was thinking something similar. We often find that there's background IFNg in ELISPOT unless we rest them for a period without IL-2. I'm wondering if there's cytokine-stimulated interferon release that's being mediated by IL-2 (or other cytokines) coming from CD4. It would certainly explain why blocking the TCR with the class I antibody didn't completely eliminate IFNg release.
Thank you Govinda & Shen-An, we plate our T cells without IL-2 so the only IL-2 in the culture would be coming from the activated T cells. We usually only see background if we include IL-2 in the medium or when they're plated with APCs - but in the presence of APCs a specific response is still higher than background.
I add the blocking AB at least an hour prior to adding the T cells.
I realise that a reduction in IFN-y in the presence of Class-I blocking ABs suggests CD8-mediated IFN-y production but I was curious as to why the IFN-y was only reduced by half in the presence of the Class I blocking ABs when it is hardly reduced at all in the presence of Class II ABs. 50% reduction in the presence of a-Class-I suggests that there's another source of IFN-y that is not the CD8+ cells. The logical choice is the CD4 cells. But if that's the case, why is there very limited reduction in IFN-y in the presence of a-Class-II and obliteration in the presence of both? I shall keep thinking & looking for answers :)
That's why I say I can only think it's CD4 mediated CD8 T cell produced IFNg. Meaning that you didn't block the MHC I on the CD4s. (The only other guess is that the IFNg is coming from the blasts themselves. Since CD4 has more stimulatory activity back onto the APC then CD8s.)
Part of the unknown is that you assume that all the T cells recognize only the loaded peptides. But that's most likely not true. Also. there is no guarantee that the peptides loaded into the blasts STAY in the blasts.
Depending on the goal of your experiment, you might need to control some of these variables.
Great thoughts, thank you Shen-An! That makes a lot of sense!
There is no gamma coming from the PHA blasts, we plated PHA blasts only & saw no/very, very little gamma. Which is kind of odd given that they're activated T cells...
Thanks for your input :)
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