Several studies have suggested that T-cell receptor (TCR) stimulation with anti-CD3 and anti-CD28 can induce FOXP3 expression in human CD4+CD25 -cells. Most of the papers stimulate the cells for 3-5 days to show the transient expression.
Can anyone tell me if in vitro activation of PBMC with ionomycin and PMA for 4 hours in the presence of brefeldin can induce this transient expression of Foxp3 in T cells and lead to erroneous estimation?
Under PMA/ionomycin treatment I have not seen much Foxp3 in the CD25- population. In three days of culture with CD3/28 activation, I do see a small population of Foxp3+ cells in the CD25- gate. However, this was a small percentage of the total CD4+CD25- population and I observed much less Foxp3+ than the CD25+ cells. My Foxp3 staining is using eBiosciences kits, which have recently been suggested to be nonspecific. So, it appears this CD25-Foxp3+ population may also be from nonspecific binding of cells or antibody trapping during cell permeabilization. From my experience, as long as you keep consistent gating and use isotope and non-activated controls, you should be fine using your system. Good luck!
Hi there!. Brodie, where have you find that eBioscience Foxp3 kit is nonspecific? We are using that kit also and it is true that we always have this doubt as we always get an unusually high % of Foxp3+ cells. It would be nice a confirmation to the fact. Thank you.
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