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Regulatory T cells that are isolated by FACS and propagated ex vivo, utilizing anti-CD3/anti-CD28–coated beads and IL-2.
Fresh peripheral blood is collected in sodium heparinized vacutainer tubes. Whole blood was diluted with an equal volume of Hank’s Buffered Salt Solution (HBSS) and subsequently underlayed in each tube with 10 ml Ficoll-hypaque solution. The peripheral blood mononuclear cells (PBMC) layer was collected following density-gradient centrifugation (900 x g for 20 min at
Tregs were isolated by FACS by the selection markers of CD4, CD25, and CD127. Setup controls (5 x 105 cells in 5 μl HBSS) were stained for 30 min at
All remaining cells were stained for collection by adding fluorescently labeled antibodies at the following concentrations: 1 μl/106 cells for CD4-PerCP and CD127-PE, and 0.7 μl/106 cells of CD25-APC. Cells were stained for 30 min at
Following FACS isolation, cells were centrifuged at 420 x g for 5 min at 4oC to remove residual sheath fluid and collection medium. Cells were resuspended in 1 ml X-VIVO 15 medium containing 10% serum and plated at 2.5 x 105 Tregs per well in a 24-well plate.
Tregs were activated with Dynabeads ClinExVivo anti-CD3/anti-CD28-coated microbeads at a 1:1 bead to cell ratio. CD4+CD127lo/- cells were immediately treated with rapamycin to a final concentration of 100 ng/ml and maintained (assuming consumption) for 7 days. On day 2, the culture volume was doubled and IL-2 was added to a final concentration of 300 U/ml. Cells were resuspended, counted and fresh medium and IL-2 were added on days 2, 5, 7, 9, and 12 and maintained at 300U/ml assuming consumption. On day 9, cells were restimulated with fresh anti-CD3/anti-CD28-coated beads at a 1:1 bead to cell ratio.