SUCCESS!!

Silvia, I used a new gating strategy sorting the Tregs: with a very tight gate around the lymphocyte compartment and using cd4+CD25 neg as Tconv, and CD4+CD25 high as Tregs. (apparently there are many monocytes that are CD4+ and CD25high! , which were contaminating my Treg population) ) I also used your recommended OKT3 aCD3 Ab, in you recommended concentrations. The result: two donors so far, in triplicate, great proliferation and fantastic dose response.

Thank you!

Label CD4+CD25- responder T cells (Tresp) with CFSE (by incubating cells at 10*7/ml in complete medium with CFSE 10 uM for 15 min at 37°C, then wash extensively).

In 96 well Ubottom plate, seed 25x10*3 Tresp/well alone or with down-scaled numbers of Treg (from 1:1 to 1:32 Treg:Tconv ratios by serial 2-fold dilutions).

Stimulate cells with 10*5 irradiated PBMC/well plus anti-CD3 (OKT3) 30 ng/ml.

Set up each condition in triplicate.

Check Tresp proliferation by flow cytometry after 5-6 days (CFSE will be diluted in daughter cells showing 4-7 peaks).

Let me know it works ;)

Hi Silvia, thanks for your answer. This is very close to what i have been doing, except the CD3 stimulation. Is it soluble a-CD3? does it have to be OKT3?

also how do you isolate/ define your Treg population, I have done CD4+ CD39+, or sorted CD4+ CD25 high, or isolated with CD25 beads, and most of the time when I add Tregs, there is not only no suppression, but MORE proliferation...

I usually enrich Treg with magnetic beads as CD4+CD25+ cells and use the negative fraction as Tresp. I use soluble OKT3 (functional grade purified) from eBioscience but I think that other agonists may work similarly. I usually premix feeders with anti-CD3 before addition to the culture. How is the proliferation of Tresp alone? My impression is that you need a high Tresp activation to see a good Treg suppression. I always use autologous Treg and Tconv but I think that Treg may efficiently suppress allogeneic Tresp. What kind of Tresp are you using? CD4+CD25- work well, CD4+CD25-CD127- may work worse. You may also try special beads from Miltenyi (Treg suppression inspector) to stimulate your cells, you don't need any feeder or anti-CD3 but in my hands those beads are worse stimulators than feeders+aCD3.

I use the CD4+CD25- fraction from the cell sort as responders.

It seems that when I activate them over night with CD3 and CD28 (2.5ug/ml each, plate bound) they proliferate very well . You do not activate the Tconv over night, right?

If I don't activate the Tconv I get poor proliferation and the CFSE looks like a smear, no clear peaks.

I always use autologous Tregs, and the numbers you have suggested are exactly the ones I have derived as well.

My problem is inconsistency. Whenever I get the Tconv to proliferate well, I set up a suppression assays and then they do not proliferate.

But I will try adding the soluble CD3 to the irradiated feeders.

Do you add IL-2 at all? I have found it makes no difference...

I have never pre-activated Tconv before Treg addition and never added IL-2. Be careful with aCD28 because a too strong activation may revert Treg suppression. Indeed I use anti-CD3 at very low concentrations (30 nanograms/ml).

Thank you Silvia.

I am setting up an assay "a la Silvia" today, with sorted Tregs CD4+CD25 high, if this works, I will be very happy and let you know. ;-)

I would like to add one more part to Dr. Piconese's very good answer. You should try to deplete "pathogenic" effector cells first....THEN isolate Tregs. Path effectors when early activated can express CD25, SO you can get cells that do not suppress, without knowing it. If you deplete total lymphocytes of CD40+ cells first (CD4+CD40+ T cells are pathogenic effector cells); you will clean up your assay. This gets to Dr. Hoelzinger's point too. If you dont remove the effector cells first the data is very inconsistent.

didn't work so well... I have some proliferation (45% of the CFSE positives) and a little bit inhibition with sorted cd4+CD25 high (down to 35% of CFSE labeled cells dividing, but not in nice peaks..) with a 5 day incubation

Could it be that cell sorting makes Tconv unhappy?

I also set up the experiment the next day after activating the Tconv on plate bound a-CD3 and a-CD28 , and got NO proliferation.

Next I am going back t the CD26 bead isolation for Tregs after enriching CD4s with beads..

SUCCESS!!

Silvia, I used a new gating strategy sorting the Tregs: with a very tight gate around the lymphocyte compartment and using cd4+CD25 neg as Tconv, and CD4+CD25 high as Tregs. (apparently there are many monocytes that are CD4+ and CD25high! , which were contaminating my Treg population) ) I also used your recommended OKT3 aCD3 Ab, in you recommended concentrations. The result: two donors so far, in triplicate, great proliferation and fantastic dose response.

Thank you!

Very happy for your success!