Regulatory T cells (University of California, San Francisco)

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 23°C, without brake) and subsequently washed twice in 10 ml HBSS at 420 x g for 5 min at 23°C. Cells were resuspended in phenol red-free HBSS at a final concentration of 1 x 10⁸/ml.

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 4°C with 2.5 μl of each single color compensation antibody (CD4-PerCP (clone SK3), CD127-PE (hIL-7R-M21), CD25-APC (2A3). Compensation controls were washed in 2 ml phenol red-free HBSS and resuspended in 500 μl phenol red-free HBSS for instrument setup. 

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 4°C, washed in 12 ml phenol red-free HBSS and resuspended at a final concentration of 25-30 x 10⁶ cells/ml in phenol red-free HBSS containing 10% human heat-inactivated pooled AB serum for sorting. CD4⁺CD127^lo/-CD25⁺ and CD4⁺CD127^lo/- T cells were sorted using aseptic technique at a cGMP-level clean room facility with a sorting rate of 15-20,000 events per second. The sorted populations were collected into 3 ml of 4°C X-VIVO 15 medium containing 10% human heat-inactivated pooled AB serum.

Following FACS isolation, cells were centrifuged at 420 x g for 5 min at 4^oC 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⁺CD127^lo/- 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.