Cd3 bv785 clone okt3

For both the in vitro and ex vivo studies PBMC were activated for four hours with CD2/CD3/CD28 T cell activation beads (Miltenyi Biotec, Cologne, Germany) in the presence of GolgiStop and GolgiPlug protein transport inhibitors (BD Bioscience, Franklin Lakes, NJ), and anti-human CD107a APC conjugated antibody (clone H4A3, BD Bioscience). Cell surface marker staining was performed for the in vitro studies using LIVE/DEAD Aqua fixable dead cell stain (Thermo Fisher Scientific), CD4 APC Cy7 (clone RPA-T4, BD Bioscience), CD3 BV785 (clone OKT3, BioLegend, San Diego, CA) CD8α BV650 (clone RPA-T8, BioLegend), and IFNγ PE (clone B27, BD Biosciences). Cell surface marker staining was performed for the ex vivo study using LIVE/DEAD Aqua fixable dead cell stain, CD4 BUV395 (clone SK3, BD Biosciences), CD3 BUV496 (clone UCHT1, BD Biosciences) and CD8α BUV805 (clone SK1, BD Biosciences). Surface and intracellular cytokine staining was performed using the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer’s instructions.

Flow cytometry staining was performed as previously described (57 (link)–59 (link)). In brief, 1 × 10⁶ PBMCs were stained with Zombie (BioLegend) fixable viability dye for 30 min at 4°C, then labeled with antibodies to surface markers in Brilliant Stain buffer (BD Biosciences) for 30 min at 4°C. Subsequently samples were stained with 25 μg/mL cholera toxin B subunit FITC conjugate (CTB-FITC) (Sigma-Aldrich), fixed for 1 h in 2% paraformaldehyde, and stained for 2 h with 50 μg/mL filipin complex from Streptomyces filipinensis (Sigma-Aldrich) before reading the samples on a BD LSRFortessa X-20 cytometer using BD FACSDiva software. Compensation was performed using anti-mouse IgGκ/negative control compensation particles set (BD Biosciences) or OneComp eBeads (ThermoFisher Scientific), with the exception of viability dyes and filipin which were performed with single stained and unstained cells. Data was analyzed using FlowJo (Tree Star).
Antibodies for surface markers: CD45RA-BUV737 (clone HI100, BD Biosciences, 584442) CD27–APC (clone M-T271, BioLegend, 356409), CD4-AF700 (clone OKT4, eBioscience, 56-0048-82), CCR7-BV421 (clone G043H7, BioLegend, 353207), CD69-BV510 (clone FN50, BioLegend, 310936), CD8-BV711 (clone RPA-T8, BioLegend, 301044), CD3-BV785 (clone OKT3, BioLegend, 317330), CD25-PE (clone M-A251, BioLegend, 356104), CD127-PE-Cy7 (clone A019D5, BioLegend, 351320).