Anti human cd3 bv605

For surface staining, cells were incubated at room temperature with human Fc block (BD Biosciences, San Diego, CA, USA) and followed by staining with directly conjugated mAbs for 30 min at 4 °C. The mAbs used were anti-human CD3-BV605, CD4-V500, CD8-APC-H7, CD45RA-BV421, CCR7-PerCp-Cy5.5, PD-1-PE-Cy7, CD160-Alexa Fluor 488 (BD Biosciences), CD4-FITC, TIM-3-BV421, 2B4-PerCp-Cy5.5 (BioLegend, San Diego, CA, USA), and TIGIT-APC (eBioscience, San Diego, CA, USA). Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences). FlowJo Software (Tree Star, Ashland, OR, USA) was used in data analysis.

Cryopreserved PBMCs were thawed, washed, and stained with Live/Dead Aqua Blue (Life Technologies), followed by surface staining with antihuman CD3 BV605, anti-human CD4 BV650, anti-human CD8 BV711, anti-human CD27 PerCP-eF710, CD45RO PE-CF594 (BD Biosciences), anti-human CD98 PE (BD Biosciences), anti-human CD71 APC, anti-human PD-1 PE-Cy7, and anti-human T-Bet BV421 (all from BioLegend unless otherwise noted). Anti-human CD14, anti-human CD16, and anti-human CD19 all tagged with V500 (BD Biosciences) were added in dump. For median fluorescence index determination, manual target channeling was performed with 1× beads (SpiroTech). Samples were run on modified 5 laser BD LSR Fortessa X-20. Data were analyzed on FlowJo (FlowJo).