I want to investigate cytokines produced by human gamma delta T cells by flow cytometry in PBMCs after isolation by microbeads. My stimulus would be PMA and ionomycine followed by inhibition by brefeldin A. Does anybody have experience with the timing of stimulation (how long) and when to add brefeldin and for how long? I do not want to culture the cells for a long time and cytokines I would stain for are IL-17A and IL-10.
I've never seen much IL-10 or IL-17 from human gamma/delta T cells (from blood derived, purified gamma/deltas or pyrophosphate-expanded Vd2 cells), having tried 4hr, 24hr and 72hr stimulations, using a range of usual mitogens (PHA, PMA/Ionomycin, SEB, etc) and pyrophosphate antigens, by both ELISA and flow cytometric detection methods. I've chatted with others in the field about this, and the consensus seems to be that - for Vdelta 2 cells anyway - these cells don't seem to produce IL-10 'naturally', and they require a LOT of prompting in the form of exogenous cytokine cocktails to produce IL-17. Papers reporting IL-10 production from Vd2 cells generally involve clones, and don't seem to be representative of the general blood population.
I would suggest however, that it might be worthwhile looking at other gamma/delta T cell subsets for these cytokines, as a colleague of mine recently demonstrated significant IL-17 production by the Vd3 subset (Mangan et al, Cutting Edge: CD1 restriction and Th1/Th2/Th17 cytokine secretion by human Vd3 T cells. JI, article in press).
Another issue to always keep in mind is the stimulators you're using and whether they require processing/presentation/accessory cells. I know gamma/delta cells are not MHC-restricted, but we found that stimulations in the presence of accessory cells always seem to work better.
Also, BFA (in our hands) was preferable to monensin for longer term stimulations (we tested up to 18hrs, and it didn't have much effect on cell viability). We finally settled on addition of BFA for the last 4hrs of stimulation.
I hope this helps.
Thank you Margaret, I would stimulate with PMA/Iono+Bref. For your info, there has been een publication about IL-17 in gamma delta's (total) in SA (Kenna et al., Arthritis Rheum. 2012). In my study I would analyze them in patients with have a defect in inflammasome regulation, producing higher levels of cytokines IL-1b (also IL-6, TGF-b, IL-10 has been reported). So I would expect gamma delta cells to be influenced by this and produce more IL-17.
That sounds interesting - and certainly there could always potentially be circumstances under which gamma delta T cells could be driven to produce particular cytokines. Our findings predominantly involved healthy donors, so please don't let it put you off! I wonder what's going on in AS? A recent paper by Zhou et al (http://www.ncbi.nlm.nih.gov/pubmed/23727634) shows a link between MICA and AS susceptibility- I wonder if that's what's driving the IL-17 production in gamma delta cells... Anyway, best of luck with the work!
- Badges
- Science method