We studied human Tregs expansion in alloanergization (MLR with Bela blockade). After three days, we washed away Bela. If I performed Treg suppression assay right away, I always saw the CD4+CD25+ (assume Tregs by Miltenyi isolation, 90%) more proliferative than the fresh effector cells. In contrast, the former lab members successfully published the finding. I personally favor to rest the allo-stimulated cells for one more week and then isolate Treg for suppression assay, but it will be not consistent with the established lab protocol. Recently I have tried to restimulate the 3-day MLR cells with Bela for more three days and then perform some suppression assay for Tregs. It seems to work in some donors. I suspect that proliferating CD4+CD25+ allo-stimulated cells are not always Tregs. Welcome any comments.
Hi Ruan,
I agree with your suspicion that CD4+CD25+ allo-stimulated cells are not always Tregs. Allo-stimulated Tregs might be different but natural Tregs isolated directly from peripherlal blood are CD4+CD25highCD127 (IL-7R) low. CD25 without CD127 is not sufficient for Treg cell characterization since CD25 is also an activation marker for T cells that is increased upon proliferation. Also, natural Tregs (and expanded natural Tregs) are mostly double positive for Foxp3 and Helios, not sure whether you expect this in your allo-induced cells, but you might want to check in your 3-day cultures the percentage of 'real' Tregs by including markers CD127, Foxp3 and Helios. Hope this helps.
Best,
Christian
Hi, Christian,
Thanks for your reply. I have not checked Helios expression in allo-expanded Tregs yet. It is a great suggestion and I plan to check it.
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