Hi everyone, can you help me to answer this question.
I am culturing human dendritic cells (DCs) from blood monocytes,
And I cocultured immature DCs and mature DCs with J558-CD40 Ligand cell lines for 24 hours, and I checked IL12p70 from floating cultured media.
And my data shown IL12p70 from immature DCs cocultured with J558-CD40L was higher than mature DCs cocultured with J558-CD40L, I have tried for several times, and data was similar.
Could you give me some explanation?
Thank you.
P/s: J558-CD40 ligand cell lines: this cells express very high CD40ligand on their surface.
You beat me to this response Payal. I will let you take it from here.
Thanks Payal Watchmaker,
I used TNF alpha: 50ng/mL for maturation for 48 hours.
after maturation, I coculture DCs and immature DCs with J558-CD40Ligand, and then I colected floating media and checked IL-12p70.
Hi,
I am sure you have all the controls, but just in case:
Are you sure your immature DC are truly immature (based on CD86, CD83, CCR7-not sure the moDCs express this later one though) ? And this before and after the co-culture ? What is their CD40 expression ?
Also have you tested other maturation markers than IL-12 to make sure your TNF-treated DCs are mature ?
The immature DCs you co-culture are "fresh" from the day or been left without stimulus for 48h (in parallel with the mature ones) ?
It seems very unlikely but is it sure that the cell line does not produce IL-12 ?
You may also check this paper in JCI 1998:
Inhibition of IL-12 Production by 1,25-Dihydroxyvitamin D3
They seem to use the same system.
Sorry it is not really an answer but more questions...
Good luck :)
Cuong,
By definition, matured DC under non-type-1 polarizing conditions have a decreased capacity to produce IL-12p70 upon maturation, a term sometimes referred to as DC exhaustion. So, the pattern you are getting would be what i would expect. If you add IFNg to your maturation condition you will get a different story. See our papers in Cancer Research 2004, and J.Immunology 2014. They might be useful for you as you move forward with various references etc...
Good luck.
Robbie
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