Hi,
I want to induce Th17 cells from C57BL6 mouse splenocyte CD4+ T cells in vitro, and now I'm preparing for setting up protocol.
I planed to induce CD4+ T cells in anti-CD3, anti-CD28, TGFb, IL-6, IL-23 condition for 3 days and to analyze by FACS.
Recently, I found a research paper, "CD28 Co-Stimulation Down Regulates Th17 Development(PLOS ONE, 2009)" and it made me confused because anti-CD28 antibody is commonly used for in vitro Th17 differentiation for co-stimulation.
This is my question. Is anti-CD28 antibody indispensable for Th17 differentiation in vitro?
A few pointers:
- IL-2 inhibits Th17 differentiation. In contrast to Th1 and Th2 differentiation, which occurs while the cells are undergoing massive proliferation, Th17 differentiation really happens while the cells start running out of food (after day 3 - more like day 5 or when the media color turns to yellow).
- make sure you don't add too much TGFb, which promotes Treg differentiation. All Th17 culture protocols also generate a little Treg differentiation. Check for it and minimize TGFb.
- lastly, some medias are better then others. See below:
Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells. Marc Veldhoenet al. J.Exp. Med 2008.
Hi
You should check TGF+IL6 combination and IL-23 for human and check time kinetics of Th17 cells differentiation in-vitro conditions.
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