Antihuman cd28 clone 28.2

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.

Treg suppression assays with blood samples from the subset of eight patients and eight control subjects were performed as described.24 In brief, PBMCs were stimulated with 5 μg mL⁻¹ purified low endotoxin/sodium azide‐free mouse antihuman CD3 (clone UCHT1; BD Pharmingen) and 2·5 μg mL⁻¹ antihuman CD28 (Clone 28.2) (BioLegend, London, U.K.) for 96 h. Cellular proliferation was measured with a Wallac 1450 MicroBeta^® TriLux (Perkin Elmer, Brunn am Gebirge, Austria) via tritiated thymidine incorporation (1 μCi per [³H]thymidine; Amersham Biosciences, Piscataway, NJ, U.S.A.) added for the final 16 h of the 96 h incubation. For analysis, the 1 : 1 ratio of effector T cells and Tregs was normalized to the proliferative rate of stimulated effector T cells alone (set to 100%).