Hello everyone!
I have the following question: I tried to expand CD4 Tregs in vitro to get enough material for WB or any other application. This is my first trial with them.
So, I`ve got FoxP3+ CD4 T-cells from mice (I use FoxP3-GFP mice) by FACS and activated them in vitro for 3 days with DynaBeads as beads/T-cells 1/2 ratio with rhIL2 ~100 U/ml. By that time the cells were mostly alive, looking good and being >65% GFP+. I`ve removed beads from the culture and rested the cells for another 6 days on rhIL2 100 U/ml dividing them every 2-3 days.
When I checked them, only ~23% were GFP+ and they didn`t secrete any TGFb or IL10 upon PMA/Ionomycin stimulation, but expressed quite high level of IL2 and TNFa (even GFP+).
I`ve checked protocols, but mostly all of them say that I just need to sort cells and activate them and then culture with IL2 100-1000 U/ml (quite a huge variation range).
What did I do wrong?
I`m quite fresh with this expansion method, so I`d be very grateful for suggestions.
Sarkar et al 2014 used a solid protocol for isolating foxp3 gfp+ cells from spleen. With bead based activation plus high dose of il2, you may get massive proliferation of foxp3 deficient cells, including cd8+ t cells. My hunch is that when you sort the cells based on gfp positivity, you should use very stringent gating strategy. This should reduce the initial contamination so that after expansion, you have less than 10% contamination. If you want to share a picture of the gating strategy that you used, I can perhaps recommend a better gate for your next sort.
Lora Benoit
So, one of the possible reasons may be contamination with conventional CD4 T-cells, right?
Acrually, it is possible. I can`t say that the gating strategy was very strict.
yes, highly probable or CD8+ T cells. can you run facs on this to confirm? If the gate you used was not super stringent for GFP+, this will happen for sure.
Lora Benoit
BTW, is it critical to culture CD4 Tregs on that much high IL2 - 1000 U/ml?
Will they not expand on lower IL2 100 U/ml? Not so big deal, but in my previous small experience, virus-specific CD8 T-cells cultured on high IL15 (100 ng/ml) for polyclonal expansion showed lower cytokine response upon peptide stimulation compared to similar cells cultured on lower IL15 (50 ng/ml or less). Maybe such a high IL2 will affect those expanding CD4 Tregs?
Hi Dmytro,
The different protocols for Treg expansion include a very broad range in IL-2. You are at the upper limit of IL-2 concentration and you are correct to assume that if you use higher IL-2 levels, you are most likely going to observe expansion of other T cell populations (in the context of bead activation). That is the basis of CD25 /T reg function.
Lora Benoit
Hi, thanks for the recommendations!
I`ve made much more strict gating and observed ~90% FoxP3+ CD4 T-cells by the first week of culture as well as quite acceptably retained FoxP3 expression after the first restimulation. Although later I`ve failed to divide cells on time and they gradually lost FoxP3 to only 65%, but they still expanded acceptably well and retained their suppressive activity.
Here I also found a nice protocol - In Vitro Expansion Improves In Vivo Regulation by CD4+CD25+ Regulatory T Cells
I`ve used rhIL2 ~200 U/ml. Later maybe I`ll try higher dosage, but overall I`m satisfied :)