Pd 1 pe

To study the immune cells in distant tumors, right tumors were harvested from mice in different groups (n = 5) and stained with Viobility 405/520 Fixable Dye (Miltenyi), CD45.2 APC-CY7 (Biolegend, Clone: 104), CD3e FITC (Biolegend, Clone: 17A2), CD8a PE-vio615 (Miltenyi, Clone: REA601), PD-1 PE (Biolegend, Clone: 29F.1A12), TIM3 APC (Miltenyi, Clone: REA602), CD4 VioBlue (Miltenyi, Clone: REA604), and Foxp3 Alexa Fluor 700(Biolegend, Clone: MF-14.1A12) antibodies, according to the manufacturer's protocols.
Briefly, tumor tissues were cut into small pieces and digested with collagenase and DNase. Then, cell suspension was filtered through a 75-μm cell mesh and resuspended in PBS (pH 7.4) with 0.5% FBS for further analysis. Flow cytometric analysis was performed using a FACS LSRFortessa flow cytometer (BD).
Tumor-infiltrating cytotoxic T lymphocytes (CTL) and helper T cells were CD45⁺CD3⁺CD4⁻CD8⁺ and CD45⁺CD3⁺CD4⁺CD8⁻, respectively. Then, the expressions of PD-1 and TIM-3 in cytotoxic T lymphocytes were analyzed. Further, CD4⁺ helper T cells were classified into regulatory T cells (Tregs) (CD3⁺CD4⁺Foxp3⁺) and effective T cells (CD3⁺CD4⁺Foxp3⁻).

To quantify induction of myeloid-derived suppressor cells (MDSCs) and nonclassical monocyte (NCM), monocytes were resuspended in MACS buffer and stained with the following markers: CD11b:PE-Cy7, HLA-DR:BV421, CD16:BV785, PD1:PE (BioLegend, #101216, #307636, #302046, #329906), CD14:FITC, CD3-PerCP (Invitrogen, 11-0149-42, 67-0036-T100). To compare PD-L1 levels following IFN-γ stimulation, monocytes were incubated with EVs derived from GBM cells that had been incubated +/−IFN-γ and were stained with PD-L1:Alexa 596 (BD Pharmingen). In either case, cells were incubated with the antibodies at room temperature for 30 min. After washing with MACS buffer, cells were then fixed in 2% paraformaldehyde. All flow cytometry experiments were carried out using the LSRII flow cytometer with data analyzed by FlowJo (BD Life Sciences).

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.

Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (BeckmanCoulter, U.S.A.). Fluorescein-conjugated mouse anti-human antibodies were used, including CD3-Alexa Fluor 700, CD3-BV650, CD8-BV786, CD8-PerCP/Cy5.5, CD45RA-APC/CY7, CCR7-PE/CY7,CD27-PE, CD28-BV421, CD69-APC/CY7, CD103-BV605, CXCR3-BV510, HLA-DR-APC, CD39-BV421, PD-1-PE, CD127-PE/CY7, CD62L-PE/CY7, Perforin-APC, Granzyme B-PE, IFN-γ-PE, and IL-4-APC (Biolegend, UK). For cell-surface staining, single-cell suspensions were stained on ice for 30 min in PBS with 1% fetal bovine serum (FBS). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (BD Biosciences, U.S.A.). To detect intracellular cytokines, CD8⁺T cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA; 1 μg/mL; Sigma) and ionomycin (1 μg/mL; Sigma), and 4 h with GolgiStop (1 μL/mL; BD Biosciences) in a round-bottom 96-well plate. Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.).

Cells were washed with PBS and stained first with LIVE/DEAD stain followed by combinations of cell surface markers, including: CD3 APC-Fire750, CD4 PE-CF594, CD8 PerCP-Cy5.5, CD45RA PE-Cy7, CD27 APC, PD-1 PE, CD39 BB515, CCR7 BV421 (all BioLegend), TIGIT-BV421, CD57-BB515 (BD Biosciences). For staining of transcription factors surface Foxp3 Transcription Factor Staining Buffer (Invitrogen) was used, according to manufacturer’s instructions, to fix and permeabilize cells before staining with T-bet-BV786 (BD Biosciences) and Eomes-PE-eFluor 610 (Invitrogen). Samples were fixed with 2% PFA, acquired on either a CyAn (Beckman Coulter) or LSR Fortessa (BD Biosciences) flow cytometer and analysed using FlowJo v9.9.6 software (FlowJo, Ashland, OR, USA).

Monoclonal antibodies for CD8-APC (clone: HIT8a), CD56-PerCP(clone: 5.1H11) and PD-1 PE (clone: EH12.2H7) were purchased from Biolegend, CD4-PE-Texas Red (clone: S3.5) was purchased from eBioscience. 50 μl of fresh heparinized whole blood from the patients was incubated with the indicated antibodies (10 μL) for 15 min, lysed with FACSTM lysing solution (BD Biosciences, San Jose, CA, USA), washed with phosphate-buffered saline, fixed and eventually detected with a BD FACSAria with BD FACS Diva (BD Biosciences, San Jose, CA, USA) software support. The data were analysed using FlowJo software (Tree Star, Ashland, OR, USA).

Cells were washed with 1xPBS then stained for surface markers with fluorophore-conjugated antibodies in 1x PBS for 30 min at 4°C protected from light. The following antibodies were used for staining: CD3-APC-Cy7, CD4-PE-Cy7, CD8-Alexa-Fluor-700, CD69-Brilliant Violet-605, PD-1-PE, CD71-Per-CP-Cy7, LFA-1-PE, CD11a-FITC (all BioLegend). Cells were stained concurrently with 1 μL/mL Live/Dead dye (ThermoFisher Scientific). Cells were washed with 1x PBS then fixed with fix/perm buffer set (BD Biosciences) for 20 min at 4°C protected from light. Cells were stained for intracellular markers using fluorophore conjugated antibodies in permeabilization buffer (1% FCS, 0.1% saponin in 1x PBS). The following intracellular antibodies were used: GLUT-1-APC (Abcam), TNF-α-FITC and IFN-γ-V450 (Becton Dickinson). Samples were run on a BD Fortesssa. FlowJow v10 software was used for the analysis.