I would like to culture splenocytes in order to expand their Treg population using TGF-B, CD3 antibodies, and CD28. All the protocols I've found are for pre-sorted T cells using an isolation kit and/or make no mention of TGF-B, only IL-2. Can anyone direct me to a good protocol for increasing Tregs in cultured splenocytes using TGF-B?
For iTreg studies in the past I have used:
approx 4ng/mL TGF-b and approx 25 U/mL of IL-2 ( IL-2 sourced from BD) in the past on sorted splenocytes. Confirmed by Foxp3 staining post stimulation.
Here's the paper:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0024226
Hi You can expand nTregs with anti-CD3 and TGFb (5ng/ml) and 40ng/ml IL-2
I pre-coat 24 well plates with 500ul of 0.5ug/ml a-CD3 and a-CD28 (BD) over night. Add enriched cd4 T cells (negative isolation kit from Invitrogen) with the TGFb and IL2 concentrations above (resuspended at 1X10^6/ml) add 2 mls /well. Examine foxp3 expression after 4 days. Conversion is 20-40% of CD4s.
You might consider expanding Tregs in vivo using IL-2/JES6.1 antibody complexes. See: http://www.ncbi.nlm.nih.gov/pubmed/16484453.
One of the characteristics of Tregs is their anergy. That means that Treg proliferation is very weak in comparison to conventional T cells. Therefore, isolation of Tregs is mandatory or you will expand only T effector cells.
You can also add some Retinoic Acid to increase the Treg numbers. However, you should also consider how stable these Tregs will be at the end of your cultures.
For Isolation and expansion of CD4+CD25+ Tregs using magnetic beads please find some info attached.
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