Hi Jian,

The effects you are seeing in your cultures with TGBF alone (no IL-2) may be simply due to impaired prolifeartion (see ref1) or T cell death; are you including a viability dye in your flow panel for detection of Foxp3+ status?

Ref 1 Article Long-Term Proliferation and Survival of In Vitro-Activated T...

Dear Jian Li,

I agree, this is rather unexpected!

My guess would be that your protocol is suboptimal regarding the CD3/28 stimulation or the TGFb dose, and as Jessica mentionned, IL2 might impact more on proliferation or survival rather than differentiation.

Regarding TGFb,

In general, IL2 has mainly an effect on survival, rather than on Foxp3 induction, and it has been proposed that TGFb has tendency to be pro-apoptotic, and you would need to use IL2 to compensate its effect.

But when I played with adding TGFb alone, with higher dose my cells actually survived better, so in vitro, it is not totally clear what TGFb does in term of death.

Normally I use 3 to 5ng/mL of TGFb to induce iTregs, with 20ng/mL IL2 and it works good. Maybe you can titrate TGFb to check, in case your batch of cytokine has weak effect and you would need to increase the concentration...

Regarding IL2, actually with a good CD3/CD28 stimulation, your cells will naturally produce quite a lot of IL2, even in presence of TGFb. So, putting IL2 is helping, but I would bet it will not change much if you put IL2 or not. However, it might be that your TCR stimulation is not strong enough, and then most of your cells would die, and by adding IL2 you could actually expand/make survive the Foxp3- cells more than the Foxp3+ cells. One trick is to coat anti CD3 and anti CD28 on special culture plates that are 'untreated' (non NUNC), in which case the antibodies stick better to the plate prior to the culture. I let the antibodies stick for 3 hours in PBS; then wash, and then the activation is much stronger/better.

Hope this helps! I would say that the in vitro differentiation culture/protocols is a very nice system to manipulate T helper cells populations, but it is not totally well understood and reproducible what each combination of cytokines does, especially in term of proliferation and death, and it can be very cahotic due to the variation in strength of TCR activation. The only important thing is to manage to set up a strong working condition for your differentiation, then you can modify one parameter at a time in the same experiment.

Hi Philippe, Thanks for your answer! I typically use 0.5ng/ml TGFb to get 50% of iTreg among CD4+ T cells. And I can see some of the T cells are losing CD4. Is it because weak TCR stimulation? We are using COSAR plates for anti-CD3/CD28 coating. These plates are tissue culture treated. And we usually coat the plate with 1ug/ml antibodies at 37oC for 4 hours. Is it gonna help if I increase the concentration of antibodies for coating? Thank you so much. I really appreciate it.