Hey Nana,

the precursor cells in the bone marrow are not iDCs. These are more like a  precursor cell which can differentiate into different cell types (Macrophage, DC, mast cell) dependent on which factors you add to the culture.

iDCs are normally found in the gut or mesenteric lymph node. They have a special tolerogenic phenotype like low MHC, low costimulatory molecules (CD80/CD86), expression of tolerogenic markers like CD103, CTLA4, IDO. Also the cytokine profile is different. Normal DCs respond to a bacterial stimuli with inflammatory cytokines like IL-12, IL-6 etc. but tolerogenic DCs respond with anti-inflammatory cytokines like IL-10, TGFb. Further, tolerogenic DCs induce regulatory T cells whereas normal DCs induce effector T cells.

tolerogenic, immature and regulatory are describing often the same DC cell type.

Hope that helps a bit!

regards

Simone

Hey Nana,

the precursor cells in the bone marrow are not iDCs. These are more like a  precursor cell which can differentiate into different cell types (Macrophage, DC, mast cell) dependent on which factors you add to the culture.

iDCs are normally found in the gut or mesenteric lymph node. They have a special tolerogenic phenotype like low MHC, low costimulatory molecules (CD80/CD86), expression of tolerogenic markers like CD103, CTLA4, IDO. Also the cytokine profile is different. Normal DCs respond to a bacterial stimuli with inflammatory cytokines like IL-12, IL-6 etc. but tolerogenic DCs respond with anti-inflammatory cytokines like IL-10, TGFb. Further, tolerogenic DCs induce regulatory T cells whereas normal DCs induce effector T cells.

tolerogenic, immature and regulatory are describing often the same DC cell type.

Hope that helps a bit!

regards

Simone

Hi Simone, really overjoyed for your quick reply. It did help me to make it clear. May I ask some additional questions?1. What's the type of DC when induced by IL-4&GM-CSF, intermediate?Neither mDC nor iDC? 2. Are tolerogenic markers of CD103 and CTLA4 high expressed? I need some positive marker in my experiment to prove the successful acquisition of iDC. I also notice somebody recommends CD45RB, do you have more advice? 3. The in vitro protocol to get iDC is reported to combine IL-10 and TGF-β，but  their amount is variable by different papers. Is it accessible to get iDC via this way? THX so much!

Best regards

Nana

Keep in mind that iDC and mDC describe the cell activation status independently of the lineage. From primary blood cells we can generate in vitro differentiated DC using IL-4 +GM-CSF ...etc...For instance after a week of differenciation of DC in vitro from CD34+ or monocytes, you will have basically immature DCs that you can activate using LPS or anti-CD40L to induce mature DC. Mature DC show an increased expression of surface costimulation molecules and production of pro-inflammatory cytokines, the addition of IL-10 or TGFb reverts this maturation/activation process and leads to tolerogenic DC ...I'll try to find a very nice and simple paper and send it to you. I can send my thesis manuscript detailing DCs and talking about iDC versus mDC with lineages details etc , but it's a french manuscript! 

Hey Nana,

when you generating DCs with IL4 and GMCSF they are normally conventional DCs. As Haifa mentioned, the same is true for mouse DC culture. Normally the DC culture from bone marrow takes 12d. Then you have fully maturated conventional DCs, expressing high MHC and costimulatory molecules. Harvesting DCs at d6 you get immature DCs, with low MHC and low costim. Treating these immature DCs with LPS induces a rapid maturation (so comparable to d12 DCs). Immature DCs  do not express tolerogenic markers. As described above you can get tolerogenic DCs by adding TGFb. Then these cells express the tolerogenic markers. It is not a matter of how high these markers are expressed, either they are expressed and then the DCs are tolerogenic or they are not expressed. About the TGFb amount I have no recommendation bc I never tried the in vitro assay. Also I have no experience about CD45RB as a tolerogenic marker.

Good luck with your studies!!!

Simone

Dear Nana,

You can also add Vitamin D3 (or its derivatives) to your DC culture to obtain tolerogic DCs.

Examples of these protocols are described for human DCs by the group of Bart Roep at the Leiden University. Below you will find a recent review by them describing the functionality of the tolerogenic DCs http://www.ncbi.nlm.nih.gov/pubmed/23717310

Alternatively, for mouse DC there are several papers describing the in vitro culture conditions including Vit D3, for example: http://www.ncbi.nlm.nih.gov/pubmed/24663679 and http://www.ncbi.nlm.nih.gov/pubmed/12520519

There are also protocols including Dexamethasone (with/without Vit D3) in the in vitro cultures to obtain tolerogenic DCs.

Good luck with your experiments,

Regards,

Rod

Dear Haifa，that's nice of you, but I can't read French, LOL. Now there's only one addtional question. As Simone mentioned, CD103 and CTLA4 can be positive tolerogenic markers for DCreg, do you ever try? Does it work? Or any other positive marker For DCreg you suggest? THX!!

Hello Nana

As already mentioned above, when you generate dendritic cells with GM-CSF and IL-4, from CD14+ human cells, they are normally, conventional immature dendritic cells, with low HLA-DR, CD80/CD86, in RPMI with 10% FBS. I have already seem some phenotypes modification, regarding the medium used to culture the DC. You can stimulate the maturation of these

The first person who will uncover the specific tol-DC marker may pretend for Nobel prize =))

But, if seriously, low MHC number and IDO expression are still most useful markers for immature (i.e. tolerogenic DCs). TRAIL, Galectin-1 and DC-SIGN are also of interest.

The term DCregs is now used for immature DC (iDC), regulatory DC, and tolerogenic DCs!

@Alexey, thx for your answer! Nobel prize, haha…...There're really full of doubts in this area, sigh. I've tried some markers, but no big difference was observed in the so-called positive marker like cd103, cd274, cd 178.While decrease in stimutory signals were got. Lose confidence in the following exp.