Hi, I'm working in the detection of CTLA4 on human Treg cells.
Our assay measures intracellular expression of CD152 by fow cytometry, in CD4+/CD25hi/CD127lo T cells, in a 20 hour stimulation assay.
My question is if anybody has experience on:
- CTLA4 induction with PHA. In our hands it works better than Dynabeads CD3/CD28. Other mitogen/s can be better?
- Use whole blood instead of PBMC. In our hands % increase is higher with whole blood
- Other conditions (incubation time...) are advisable?
Thank you!
Non-specific stimulators are more potent, but less physiological, than activating T cells through specific molecules (i.e. CD3, CD28 etc). You may try ConA and PMA/ionomycin as well.
Whole blood is the best condition to stimulate T cells because they are still in their optimal milieu (assay can be accepted ex-vivo).
I would perform an expression kinetic setup up to 96h.
I would suggest to perform functional assays on suppressive capacity of Tregs or testing some transcription factors (like FoxP3) or at least cytokine secretion (TGF-b or IL-10) profile. Because, upregulation of CD25 and CTLA-4, downregulation of CD127 is also evident when T cells are simply activated. 20h is too early for a naive T cell to fully differentiate.
Good luck.
Thank you so much!
Your suggestions are very usefull. We are using the test to check for deficiencies affecting CTLA4 espression, that is the reason to try to get as much signal as possible.
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