The suppressive capacity of Tr1 cells in vitro was measured by a mixed lymphocyte reaction (MLR) assay. After labeling with 5 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen), CFSE-labeled human PBMCs were incubated with 5 μg/ml anti-human CD3 mAb and 2 μg/ml anti-human CD28 mAb (BD Pharmingen) in RPMI 1640 medium (Invitrogen) containing 10% human AB serum. Irradiated (30 Gy) xenogeneic PBMCs or polyclonally expanded Tr1 cells or xenoantigen-expanded Tr1 cells were added to MLR cultures at 1/1, 1/2, and 1/16 dilutions. After seven days, the cultures were harvested to analyze the suppression capacity of Tr1 cells.
In the coculture system, 20 μmol/l CD39 activity inhibitor polyoxometalate-1 (POM-1; Santa Cruz Biotechnology) was added into the MLR to evaluate the effect of CD39 on Treg-mediated suppression.
The proliferation of PBMCs was assessed depending on the percent-proliferating PBMCs cultured in the absence and presence of Tr1 cells. The percent proliferation of PBMCs in the absence of Tr1 cells was recognized as 100% of proliferation and 0% of suppression. Percent suppression of proliferating PBMCs was determined as %suppression = (percent‐proliferating PBMCs in the presence of Tr1/percent‐proliferating PBMCs) × 100%.
In the coculture system, 20 μmol/l CD39 activity inhibitor polyoxometalate-1 (POM-1; Santa Cruz Biotechnology) was added into the MLR to evaluate the effect of CD39 on Treg-mediated suppression.
The proliferation of PBMCs was assessed depending on the percent-proliferating PBMCs cultured in the absence and presence of Tr1 cells. The percent proliferation of PBMCs in the absence of Tr1 cells was recognized as 100% of proliferation and 0% of suppression. Percent suppression of proliferating PBMCs was determined as %suppression = (percent‐proliferating PBMCs in the presence of Tr1/percent‐proliferating PBMCs) × 100%.