Anti human cd3 antibody

Human peripheral blood CD8⁺ T cells (Stem Cell Technologies, 200–0164) were cultured in ImmunoCult XF T Cell Expansion media (Stem Cell Technologies, 10981) at 37 °C with 5% CO₂. Antibodies were immobilized on Dynabeads M-280 Tosylactivated (Invitrogen, 14203) as described previously (Desai et al., 2022). Briefly, a total of 10 μg of antibody consisting of 2 μg of anti-human CD3 antibody (Bio X Cell, BE0001–2) and 8 μg of experimental antibody in 1 mL of 1X PBS was incubated at room temperature for 2 days with rotation. The beads were subsequently blocked with 10 mM glycine buffer for 1 h with rotation, followed by washing twice with PBSB. Next, the CD8⁺ T cells were plated at a density of 50,000 cells/well in a 96-well plate to a volume of 150 μL. Then, 50 μL of bead solution was added at a ratio of 4:1 or 8:1 of conjugated beads:T cells. After incubating at 37 °C with 5% CO₂ for 96 h, the cells were stained with BV510 anti-human CD8 (BioLegend, 344732) and APC anti-human CD25 (BioLegend, 302610) antibodies at 1:200 dilution each on ice for 5 min. Cells were then centrifuged at 300 xg for 4 min, washed with 1X PBS, and analyzed via flow cytometry.

Blood filters containing cells from deidentified donors were supplied by Hoxworth Blood Center at the University of Cincinnati under exemption granted Cincinnati Children’s Hospital Medical Center Institutional Review Board. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation. Isolated peripheral blood mononuclear cells were processed with the EasySep Human Naive CD4⁺ T Cell Isolation Kit (#19555; STEMCELL) to negatively isolate human CD4⁺/CD45RO⁻ cells (naive T cells), and the purity was confirmed by flow cytometry (Fig. S1 B). Isolated naive T cells were cultured in RPMI media with L-glutamine (#SH30027.01; HyClone) supplemented with 10% FBS and 25 µM 2-ME before activation. For bead activation, 100 µl anti-mouse pluriBeads (#31-GTaMS-10; pluriSelect) were incubated with 24 ng anti-human CD3 antibody (#BE0001-2; Bio X Cell) and 52 ng of either anti-human CD28 antibody (#BE0248; Bio X Cell) or mouse IgG (#12-371; Millipore) with rotation for 3–4 h at room temperature (RT). The beads were centrifuged and washed with PBS twice. Antibody-bound pluriBeads were directly added to the cultures of human CD4⁺ T cells, and the cultures were shaken for 4 h and swirled every 10 min until the cells were bound to the beads.