Tim 3 af700

Multi-color flow cytometry analysis was performed on PBMCs from all time points by staining for 30 minutes at 4°C with CD3-V450, CD8-FITC or APC, ICOS-PE, HLA-DR-PerCP-Cy5.5, CD25-PE-Cy7, CD45RA-PerCP-Cy5.5, CD62L-FITC, CD127-V450, PD-1-PE, Tim-3-AF700, CD4-APC-Cy7 (BD Biosciences, San Jose, CA), CCR7-PE-Cy7 (R&D Systems, Minneapolis, MN), CTLA-4-FITC (LSBio, Seattle, WA) and FoxP3-APC (eBioscience, San Diego, CA) for T cells. For natural killer (NK) cells, CD3-V450, CD16-APC-Cy7, CD56-PE-Cy7 and Tim-3-AF700 (BD) were used. For myeloid-derived suppressor cells (MDSCs), CD33-PE, CD11b-APC-Cy7, HLA-DR-PerCP-Cy5.5, CD14-V450 and CD15-APC (BD) were used. 1×10⁵ cells were acquired on an LSRII (BD), and data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR). The appropriate isotype controls were used, and dead cells were excluded from the analysis.