Anti cd3 clone ucht1

Human primary CD4⁺ or CD8⁺ T cells were enriched from healthy donor–derived buffy coats using RosetteSep Human T Cell Enrichment Cocktail (STEMCELL Technologies), according to the manufacturer’s instructions. For T cell activation, 96-well plates were coated with PBS in the presence or absence of 1 μg/ml anti-CD3 (clone UCHT-1; R&D Systems) for 4 h at 37° C. Cells were plated at 0.1 × 10⁶ per well and cultured in a humidified incubator (5% CO₂, 37°C) for 72 h, harvested, and stained with viability dye (Fixable Viability Stain 450; BD Biosciences). Programmed death 1 expression on anti-CD3–treated T cells, but not on resting cells, confirmed the activated status of these cells (data not shown). Resting and activated T cells were incubated for 1 h at room temperature with ILDR2-hFc or control Ig (human IgG1; Synagis) at 100 μg/ml in FACS buffer (0.5% BSA, 2 mM EDTA, 0.05% NaN₃ in PBS), and ILDR2-hFc binding was detected using PE-conjugated anti–human IgG (Jackson ImmunoResearch). Cells were washed three times, gated for singlets (forward scatter [FSC]-H versus FSC-A) and for live cells, and ILDR2-hFc binding was determined within this gated population. Data acquisition was performed with MACSQuant Analyzer 10 (Miltenyi Biotec) and analyzed using FlowJo software (v10).