Anti cd3 mab coated beads

The suppressive function of iTreg cultured in vitro was examined with a carboxyfluorescein succinimidyl ester (CFSE) inhibition assay as previously published^{14 (link)}. Briefly, purified PBMCs were labeled with CFSE (Invitrogen) and stimulated with anti-CD3 mAb-coated beads (Dynal) ± cultured iTreg (1:2 to 1:16 iTregs/PBMCs). Four days later, cells were harvested and stained with CD4 and CD8 antibodies. Suppression was accounted from the Division Index (FlowJo, TreeStar). iTregs suppressed CD4 and CD8 T cell responses equivalently and only CD8 data are presented.

Different subsets of Tregs were treated or not, washed three times, and added at ratio 1:3 (1.25 × 10⁵ Tregs: 5 × 10⁵ CD4⁺CD25⁻ T cells) to CD4⁺CD25⁻ T cells at final concentration 2 × 10⁶/ml. The cells were co-cultured on anti-CD3 mAb (5 μg/ml) pre-coated 24-well plates for 48 hr. The PKC-θ inhibitor, AEB071, was provided by Novartis (Ridgefield, CT) and dissolved in DMSO. T cells were pretreated for 30 min with 10 μM at 37 °C and washed three times. Cytokine secretion was determined by ELISA as previously described^{20 (link)}, using Human IL-17 (R&D Systems Inc, Minneapolis, MN) and IFN-γ Cytoset^tm (Biosource; Camarillo, CA). The capacity of expanded Tregs to suppress target cell proliferation in vitro was assessed by using 5-carboxyfluorescein-diacetate-succinimide ester (CFSE) inhibition assay^{31 (link)}. Peripheral blood mononuclear cells (PBMCs) were purified, labeled with CFSE (InVitrogen), and stimulated with anti-CD3 mAb-coated beads (Dynal) in 96-well round-bottom plates with or without expanded cells (Treg:PBMC at ratios of 1:4-1:64). Assays were harvested on day 4. Cells were stained with anti-CD4 and/or −8 mAb and data acquired by LSRII. Data was analyzed using the proliferation platform in FlowJo (Treestar, Ashland, OR), and suppression determined from the Division Index.