I am currently having consistency issues when generating Th17 cells in vitro.
In general the viability in our cultures is pretty bad (around 30%) and the percentage of Th17 cells is never higher than 30%.
We are basically doing 4 days of culture of FACS-sorted naive CD4+ CD44low CD25- CD62L+ cellls with coated anti-CD3 (5ug/mL) + soluble anti-CD28 (1ug/mL).
We use two different culture conditions:
1/ human TGFb (5ng/mL) + IL-6 (25ng/mL) + IL-1b (20ng/mL) + anti-IL-4 (10ug/mL) + anti-IFNg (10ug/mL)
2/ human TGFb (5ng/mL) + IL-6 (25ng/mL) + IL-1b (20ng/mL) + anti-IL-4 (10ug/mL) + anti-IFNg (10ug/mL) + anti-CD25 (5ug/mL) + anti-CD122 (5ug/mL) + IL-23 (20ng/mL)
We usually plate 50000 cells in 200uL of RPMI in 96 well round bottom plates and transfer the cells to a new plate after 48 hours, without changing the medium or adding any cytokine.
After 4 days we restimulate the cells with PMA and ionomycin before intracellular staining for IL-17.
I saw in the litterature that people sometimes let the cells rest for 4 days without any stimulus (after the regular 4 days culture period) before analysing IL-17 production.
Is that something that helps getting the numbers and percentages of IL-17+ cells higher?
I also saw that the use of IMDM instead of RPMI can help in the generation of IL-17-producing cells because it contains aromatic amino acids: http://www.ncbi.nlm.nih.gov/pubmed/19114668
Colleagues told me that the coated anti-CD3 can induce apoptosis of the Th17 cells if they stay too long in contact with it.
Any other advice?
Thanks for your help.
So first, about 30% IL-17+ is not bad. In any Th culture, only a portion of the cells will be detectable as secreting cytokine (and whether this is due to kinetics or variegation in the population is not clear).
Regardless, your overall protocol is fine. We usually use anti-IL-2 rather than anti-CD25/CD122, but I don't think that will make a major difference. Of note though is that when you block IL-2 signaling you will decrease viability while increasing IL-17+ cells, so it's a trade-off.
The other variable to alter is the balance between TGFb and IL-6. The activity of cytokine batches, particularly TGFb, can vary. We usually titer each new batch. 5 ng/ml is on the high side; we often use 1-2 ng/ml. And IL-6 we will often go up to 50-100 ng/ml. This can vary with conditions and source of reagents, so it's best to empirically test.
As for timing of the culture, we generally do 3 days on anti-CD3/CD28 with cytokines, and then 2 days off stimulation, but supplementing cytokines (for Th17, half concentration of IL-6, + the anti-cytokine Abs, you could add IL-23 here too). Another note is that IL-23 will not do much in the first couple of days of culture because naive cells do not express receptor.
You may try MACS instead of FACS for sorting
You may be missing an important cytokine.
Th17 Polarization of CD4 cells
1.
Coat 60 × 15 mm of plastic petri dishes with anti-mouse CD3ε, clone 145-2C11 (2
μg/ml). Incubate at 37°C for 2 hours or 4°C overnight. Aseptically decant antibody
solution from the plate. Wash plate 3 times with sterile PBS. Discard liquid.
2.
Plate CD4 cells at 1 x 10
6
/ml. Culture cells for 3 days in presence of anti-mouse CD28,
clone 37.51 (5 μg/mL), IL-6 (50 ng/mL), TGF-β1 (1ng/mL), anti-mouse IL-4 (10 μg/mL),
anti-mouse IFN-γ (10 μg/mL), and 300 nM of FICZ. Alternatively, mouse IL-23 (5 ng/
ml) can be used in place of FICZ
This is biolegend's protocol
Th17 polarization is difficult and not well understood. The majority of publications looking at Th17 cell differentiation achieve less than 30% of IL17A+ CD4+ T-cells.
- Proliferating naive T-cells do not differentiatiate into Th17 cells until several days of culture. Large amounts of IL2 are generated during proliferation and inhibit differentiation. A T cell extrinsic mechanism by which IL-2 dampens Th17 differentiation.Anderson AC, Sullivan JM, Tan DJ, Lee DH, Kuchroo VK. J Autoimmun. 2015 Feb 25.
- TGF-β1 is used in both Th17 and Treg differentiation . Hence, the need for low levels of TGF-β1 and high levels of IL-6 to inhibit iTreg development and favor Th17 differentiation. Unfortunatly, many studies on in vitro differentiated Th17 cells fail to assess the percentage of Treg cells induced in their culture. Regulatory T cells vs Th17: differentiation of Th17 versus Treg, are the mutually exclusive? Zheng SG.Am J Clin Exp Immunol. 2013 Feb 27;2(1):94-106.
Hope this helps. Good luck.
There is a very nice paper on Nature Protocols on how to get Th17 that works pretty well:
In vitro th17 development: http://www.nature.com/protocolexchange/protocols/284
So first, about 30% IL-17+ is not bad. In any Th culture, only a portion of the cells will be detectable as secreting cytokine (and whether this is due to kinetics or variegation in the population is not clear).
Regardless, your overall protocol is fine. We usually use anti-IL-2 rather than anti-CD25/CD122, but I don't think that will make a major difference. Of note though is that when you block IL-2 signaling you will decrease viability while increasing IL-17+ cells, so it's a trade-off.
The other variable to alter is the balance between TGFb and IL-6. The activity of cytokine batches, particularly TGFb, can vary. We usually titer each new batch. 5 ng/ml is on the high side; we often use 1-2 ng/ml. And IL-6 we will often go up to 50-100 ng/ml. This can vary with conditions and source of reagents, so it's best to empirically test.
As for timing of the culture, we generally do 3 days on anti-CD3/CD28 with cytokines, and then 2 days off stimulation, but supplementing cytokines (for Th17, half concentration of IL-6, + the anti-cytokine Abs, you could add IL-23 here too). Another note is that IL-23 will not do much in the first couple of days of culture because naive cells do not express receptor.
is it possible to perform organotypic cell coculture with th17 cells in DMEM?
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