When we expand Tregs, we use IL-2, CD3/CD28 and rapamycin in our culture media.  I think with that you can expect some Treg proliferation in this experiment, especially if the host has been pre-sensitized to the viruses you are looking at.   I would use soluble CD3/CD28 rather than plate bound.

I hope this helps!

Dear Josh,

Thanks for your reply. Actually the goal of my experiment is not to expand Tregs; but to see if Tregs proliferate in response to viral antigens. So, these Tregs would be responding in response to antigens. My question is keeping this thing in view.

Hi Ashish,

If you want to only study the Treg response, I think you may have to separate the Tregs from all the other T cells and co-culture them with antigen-presenting cells you can show work well to activate naive or effector T cells. Otherwise, I don't see how you'll be able to interpret whether anything you could measure is caused by TCR stimulation of Tregs by viral antigens. If you add viral antigens to whole PBMCs and wait three days, it seems to me that there will be too many variables and indirect explanations for why the Tregs proliferate, or don't. Or, perhaps I don't understand the goal of your experiment?

Luke

Dear kumar, Prof. Marita troye blomberge from Stockholm University is the best one to answer your query 

So I think you are saying you would like to look at the viral antigen specific T reg in PBMC of patients who have recovered from natural viral infection. I'm not clear why you want to do this. Do you want to know what % of PBMC are T reg (this won't tell you if they are viral antigen specific or not), or if you want to test antigen specificity of Treg (non antigen specific T reg can be induced by bystander effect from naive CD4 T cells next to T reg in culture. Also adding cytokine and cd3/Cd28 is what you use in culture to make T reg so you probably want to avoid this except in your positive control). I'm also not clear what type of T reg you are talking about. This is important they all have different survival cytokine requirements and markers. Finally, established T reg don't need professional antigen presenting cells. Any APC should do. B cells for example. Even naive cells will respond in a mixed lymphocyte culture. You don't need DC. 

So, overall, if you want to look at if you have antigen specific T reg you would need to do classic antigen PBMC stimulation experiments with and without T reg depletion (positive bead selection). Test if antigen specific T cell memory response increase in absence of T reg but are suppressed when they are added back in. Check if this is dose dependent. Also test an unrelated antigen response to confirm specificity.  Use transwell experiments to show if contact dependent CD25+. Use blocking antibody to show if IL10 or TGFb dependent. 

Best

Victoria

Hi,

You should use  anti-human CD3/CD28 and also rhIL-2 in PBMC culture for Treg stimulation with high concentration  (more than 500 or 700 IU/ml).

Best

I do believe that Prof. Marita TroyeBlomerg would have the best answer, please contact her at (Stockholm university, Department of Immunology and molecular biosciences, WenerGren Institute)