Anti cd3 ucth 1 mab

The suppressive activity of Treg was evaluated by monitoring the inhibition of dye dilution in PBMC stained with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, 5 µM, Molecular Probes, Invitrogen).
Briefly, the PBMC-CFDA-SE+ were pulsed with the anti-CD3 UCTH-1 mAb (5 µg/ml, BD Bioscience) and cultured for 5 days in a 96-well U bottomed plate (1 × 10⁵ cells/well) in the presence (or not) of ex vivo- or in vitro-generated CD8+ T lymphocytes (1 × 10⁵ cells/well). Then, the samples were harvested, washed in PBS, and analyzed by flow cytometry. The dead cells were excluded from analysis by adding 7-aminoactinomycin D (7-AAD) (BD Bioscience) prior to analysis. Suppression activity was expressed as the percentage reduction of the proliferation in the presence of CD8+ Treg lymphocytes compared to the levels of proliferation observed in control cultures of PBMCs cultured in the absence of Treg cells. A suppression activity ≥25% was considered significant. This threshold was chosen based on the results achieved in a large historical cohort of more than 50 healthy subjects of both sexes with age ranging from 18 to 87 years. In healthy donors, CD8+ Treg suppression activity never fell below 25%.

CD8+ T lymphocytes were purified from the tumor immune infiltrate by immunomagnetic beads using microbeads conjugated with mAb-specific for the CD8 antigen (Miltenji Biotec, Bergisch Gladbach, Germany). Due to the paucity of purified cells, no further selection procedures were possible. In the case of patients #5 CD8+CD28− T cell percentage on total purified CD8+ T cells was >80%, so their suppressive activity was tested. Hence, PBMC (1 × 10⁵ cells/well) from a healthy donor, stained with carboxyfluorescein succinimidyl ester (CFDA-SE) (5 μM) (Thermo Fisher), were pulsed with the anti-CD3 UCTH-1 mAb (5 μg/mL, BD Bioscience) and incubated for 5 days in a 96-well U bottomed plate in the presence or not of purified CD8+CD28− T cells (1 × 10⁵ cells/well). Then, the samples were washed in PBS and analyzed by a BD Fortessa X20 flow cytometer (BD Biosciences) using the BD FACS Diva™ software version 8.0 (BD Biosciences) in order to monitor the inhibition of dye dilution. Dead cells were excluded from analysis by adding 7-aminoactinomycin D (BD Biosciences) before the analysis. Suppression activity was expressed as a percentage reduction of proliferation in the presence of tumor infiltrating CD8+CD28− T lymphocytes compared to the levels of proliferation observed in control cultures of PBMCs alone.