FTVI treatment increases Treg fucosylation. Fucosylation is characterized by the presence of sLeX residues, as assessed by flow cytometry, with antibody HECA-452 (BD Biosciences, San Jose, CA), raised against CLA, shown to be sLeX³⁰. The left side of each contour plot shows the isotype control, and the right side the results of staining (percentage CLA-positive cells). (A) Positive control. CB CD34⁺ cells show an endogenous rate of 56.5% CLA+ cells that increases to 100% on treatment with FTVI enzyme. (B) Expanded Tregs. Untreated ex vivo-expanded Tregs show minimal endogenous fucosylation of 6.6% that increases to 65.5% on treatment with FTVI. (C) Fucosylation of Tregs leads to increased ability to bind E-selectin ligand. Expanded Tregs were treated with FTVI enzyme for 30 minutes, followed by biotin CLA–streptavidin phycoerythrin. Unlabeled selectins (E, P, L) with APC anti-Fc were used to study selectin binding ability in untreated Tregs (left) or fucosylated Tregs (right). Significant increase in the binding ability of fucosylated Tregs to E-selectin ligand was demonstrated (41% vs 2%) (upper). A high level of endogenous expression of P-selectin ligand was seen in untreated Tregs (81.7%) that increased on fucosylation (92.9%) (lower). No change was seen in the total L-selectin ligand expression (middle). (D) Increased E-selectin binding in fucosylated Tregs. Average values from 3 independent experiments show that the extent of fucosylation is related to the binding ability of E-, P-, or L- selectin. (E) Fucosylated Tregs suppress immune response in alloMLR similar to untreated Tregs. No differences in the degree of suppression of alloMLR was identified at the varying ratio of T-effector (Teff) and Treg (untreated or fucosylated). The x-axis denotes the varying ratio of Teff:Treg. Y-axis denotes counts per minute (mean ± SEM; n = 3).