Ki67 fitc

CD4⁺ T cells were sorted into T_CM and T_EM subsets using a BD FACS Aria (BD Biosciences, San Jose, CA) run by BD FACS DIVA software. T_CM were defined as live, singlet, CD3⁺CD4⁺CD27⁺CD45RO⁺CCR7⁺ cells. T_EM were defined as live, singlet, CD3⁺CD4⁺CD27⁻CD45RO^+/dimCCR7⁻ cells, T_SCM were defined as live, singlet, CD3+CD4+CD27+CD45O-CCR7+CD95+ cells as previously described and as demonstrated in Figure 2D[23] (link). Cryopreserved PBMCs were thawed and stained with predetermined optimal concentrations of the following markers: Aqua Amine Reactive Dye (Invitrogen), CD3 AL700, CD8 PERCPCY5.5, CCR5 PE, CCR7 PE-CY7, CD95 PE-CY5 and Ki67 FITC (BD Pharmingen), CD4 eFluor450, CD27 APC-eFluor780 (eBiosciences, San Diego, CA), CD45RO ECD (Beckman Coulter, Chaska, MN).

Samples were stained without further separation to minimize selective losses shortly after collection. Combinations of the directly conjugated monoclonal antibodies CD3-V500, CD4-PerCP, CD56-PE, CD127-BV421, CD25-PE-CY7, CD25-APC, CD62L-FITC, CD69-APC, CD45RA-FITC, CCR7-PE, Ki67-FITC, Foxp3-APC (BD Bioscience, Mountain View, CA, USA), CD16-APC/CY7, HLADR-APC/CY7 (Biolegend, San Diego, CA, USA) and their relative allotypes were used in individual 8-color flow cytometry assays to analyze the immunophenotype of regulatory T cells and effector T cells.
Intracellular staining was performed using the Intracellular Staining Kit (eBioscience, San Diego, CA, USA). The expression of Ki67 was determined in freshly isolated CD4⁺CD25^highFoxp3⁺ regulatory T cells. The cellular secretion and function of cytokines were determined after incubation of cells for 5 h with phorbol myristate acetate (PMA) (100 ng/ml) plus ionomycin (2 ug/ml, all reagents from Sigma Chemical Co., St. Louis, MO, USA) to stimulate maximal production of IL-17 and IFN-γ; GolgiStop (0.7 μl/ml) was added to the samples during the last 4 hours to sequester the proteins in the cytoplasm [22 (link),23 (link)].

MHC class I tetramer was prepared in-house. Surface staining of T cells was achieved by addition of tetramer to whole blood, incubation for 10 minutes at room temperature, followed by addition of antibody co-stains for 20 minutes. Whole blood was preferred to thawed PBMCs for tetramer labelling due to greater consistency and signal intensity. Following lysis of erythrocytes using BD FACS Lysing solution (BD Biosciences), cells were either fixed using 2% formaldehyde (v/v) or permeabilized using the BD Cytofix/Cytoperm kit for intra-cellular staining. The following antibodies were used for surface and intra-cellular staining: CD3-PerCP, CD8-Horizon V500, CD8-APCH7, Ki-67-FITC, Bcl-2-PE, HLA-DR-Horizon V450, HLA-DR-PerCP, Perforin-FITC, Granzyme B-Horizon V450, CCR7-PE, CD27-Horizon V450, CD27-FITC, CD28-PECy7, CD28-PE (all BD Biosciences) and CD38-PECy7 (eBioscience), Granzyme B-PE (Caltag), Granzyme K-PE (Santa Cruz), CD45RA-FITC (Beckman Coulter), PD-1-PE and 2B4-PerCPCy5.5 (Biolegend). Intra-cellular cytokine staining with IFN-γ-FITC, TNF-α-APC, and IL-2-PerCpCy5.5 (all BD Biosciences) was undertaken after in vitro stimulation of PBMCs using peptide for 6 hours. Flow cytometry analysis was performed BD LSRII and BD FACSCanto flow cytometers. Flow cytometry data were analyzed using FlowJo software.

The cetuximab (Bristol-Myers Squibb, New York, NY), trastuzumab (Genentech, San Francisco, CA), pertuzumab (Genentech) and isotype (rituximab, Genentech) antibodies were purchased from the National Institutes of Health Pharmacy. Flow cytometry of haNK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Flow cytometry of tumor cells was performed on a BD FACSVerse flow cytometer (BD Biosciences) and analyzed in BD FACSuite (BD Biosciences). Staining of haNK cells was performed with four panels, and the antibodies used were: Anti-CD56-PErCP-Cy5.5, anti-CD16-APC-Cy7, anti-NKG2D-APC, anti-NKG2D-FITC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-CD158a-PE, anti-CD158d-PE, anti-CD158j-APC, anti-PD-1-PE-Cy7, anti-PD-L1-APC, and anti-MICA-PE; all were obtained from BioLegend (San Diego, CA). Anti-CD56-PE, anti-CD16-PE-Cy7, CD11a-PE, Ki67-FITC, anti-HLA-ABC-FITC and anti-4-1BB-BV421 were obtained from BD Biosciences. In controlled experiments to detect CD16 on haNK cells, the anti-CD16 MAb was CD16-FITC (BD Biosciences) and the isotype control antibody was MIgG1k-FITC (BD Biosciences).

FACS sorted HSCs were fixed and permeabilized using a Cytofix/Cytoperm plus kit (BD Biosciences, NJ, USA), according to the manufacturer instruction. The, cells were stained overnight with Ki67 FITC (BD Biosciences, NJ, USA) at 4 °C, and 10 min with Hoechst 33,342 (Invitrogen, CA, USA). Stained cells were run on a BD LSRII for cell cycle analysis.