Mouse anti human cd3 clone ucht1

OT-I CTLs or hCTLs were resuspended in killing assay media (phenol red–free RPMI 1640 containing 2% FBS) and mixed at effector-to-target ratios shown with EL4 (pulsed with 1 μM OVA_257–264 for 1 hour at 37°C) or P815 (mixed with 0.5 μg/mL mouse anti-human CD3 (clone UCHT1, catalog 555330, BD Pharmingen), respectively. For experiments with CK666 or CK689, OT-I CTLs were treated 10 minutes at 37°C with 90 μM of the drug, which was also added to the killing assay media. For experiments without IL-2, hCTLs were washed and cultured in media without IL-2 for 16 hours prior to the assay. After 1.5 hours to 2 hours incubation, the percentage of target cell lysis was determined using the CytoTox 96 Non-Radioactive Cytotoxicity Assay following the manufacturer’s instructions (Promega).

Treg suppression assays were performed, as described.^{26 (link),54 (link)} In brief, PBMCs were isolated with Lymphoprep™ (StemCell Technology, Grenoble, France) and stimulated with 5 μg ml^–1 purified, plate-bound, low endotoxin/sodium azide-free mouse anti-human CD3 (clone UCHT1; BD Pharmingen, San Diego, CA) and 2.5 μg ml^–1 anti-human CD28 (Clone 28.2) (BioLegend, London, UK) for 96 h. Cell proliferation was visualized via tritiated thymidine incorporation (1 μCi per [³H]thymidine) (Amersham Biosciences, Piscataway, NJ), added for the final 16 h of the 96 h incubation with Wallac 1450 MicroBeta® TriLux (Perkin Elmer, Brunn am Gebirge, Austria). For analysis, the 1 : 1 ratio (i.e. that of the co-culture of the same number of T effector cells [CD4+CD25–CD127+] and Tregs [CD4+CD25+CD127–]) was compared with the proliferative rate of stimulated T effector cells alone (normalised and set to 100%) to determine the suppressive capacity of Tregs.