Treg Migration and Suppression Assay

Splenocytes (1 × 10⁶ cells/well) from FoxP3-eGFP/B6 mice (The Jackson Laboratory, Bar Harbor, ME) were plated in 5 Nm pore size transwells (Cell Biolab, Inc., San Diego, CA, USA). RPMI 1640 with 10% FBS and antibiotics (Life Technologies, Grand Island, NY, USA) was added to the outer well in the presence or absence of 100-500ng/ml mouse Ccl22 (R&D Systems, Minneapolis, MN, USA). Cells were allowed to migrate for 2 hrs before harvesting cells from the bottom well (Klarquist et al, 2010 (link); Justus et al, 2014 ). Cells from top and bottom wells and a splenocyte sample not included in the migration assay were subjected to immunofluorescent staining to detect Treg, using 145-2C11, RM4-5, and PC61 antibodies to CD3, CD4, and CD25, respectively (BD Biosciences, San Jose, CA, USA). Splenocytes were sorted using a BD FACS Aria III sorter (Becton Dickinson, Franklin Lakes, NJ, USA). Sorted Treg from reporter mice were combined with effector cells from TCR transgenic mice recognizing gp100_22-35 (Pmel1 mice) or human tyrosinase_368-376 (h3TA2 mice) in IFN-gamma ELISPOT plates in the presence of cognate peptide. The resulting suppression by Treg cells was quantified by comparing to the spot numbers produced by the same number of effector cells without Treg cells.