Measuring Treg-mediated Suppression with miR-106a

Treg-mediated suppression was measured as previously described ^{37 (link)}. Briefly, CD4⁺CD25⁺ Tregs were isolated by negative selection of CD4⁺ T cells, followed by positive selection of CD25⁺ cells using the EasySep Treg isolation kit (STEMCELL). Treg frequency was previously confirmed by flow cytometry with approximately 85% of enriched CD4⁺CD25⁺ expressing FoxP3 ³⁸ . CD4⁺CD25^Neg effector T cells were labeled with CellTrace Violet (Invitrogen) according to manufacturer’s instructions prior to stimulation with anti-CD3 mAb (1μg/ml; 145–2C11, BioLegend) in the presence of irradiated syngeneic APC to allow for fluorescent monitoring of cell proliferation. Proliferating cells were co-cultured with varying ratios of converted CD4⁺CD25⁺ Treg from WT or TNF^ΔARE/+ which had been transduced with either miRZIP000 or miRZIP106a to determine the impact of selective inhibition of miR-106a on Treg suppressive function. Anti-IL-10R (10μg/ml; 1B1.3a, BioLegend) or recombinant IL-10 (10ng/ml; Peprotech, Rocky Hill, NJ) were added to culture conditions as indicated.