Anti pd l2 apc

Expression of PD-1/PD-L1/PD-L2 was assessed retrospectively in prospectively collected blood samples. The following combination of human monoclonal antibodies were used according to the manufacturers’ instructions to identify different immune cell populations (CD3⁺, CD4⁺ and CD8⁺ T lymphocytes): anti-CD3-FITC, anti-PD-L1-PE, anti-CD8-APC Cy7, anti-PD-1-PE Cy7, anti-PD-L2-APC and anti-CD4-PerCp (BioLegend; San Diego, CA, US). The PBMC fraction was blocked for 5 minutes with a FAB anti-IgG. Samples were incubated for 45 minutes with the appropriate antibodies (2.5 μl) at room temperature and protected from light exposure. After incubation, 2 ml of a 1:1 solution of PBS-Fetal bovine serum (FBS) were added to each sample. Samples were then centrifuged at 1100 rpm for 5 minutes. The supernatant was decanted and cells were fixed in paraformaldehyde (1%).
Gating strategy were set using fluorescence minus one (FMO) for PD-1 /PD-L1 &PD-L2. The samples were acquired in a FACS Aria II Flow Cytometer (BD, Biosciences, San José, Cal, USA) and analyzed with FlowJo software 10.1 (Tree Star. Ashland, Or, USA). The leukocyte population was gated based on morphological parameters on a forward vs side scatter (FSC/SSC) plot.

About 24 and 48 h after the last irradiation, tumor cells were harvested for analyses by flow cytometry. For cell death detection and analysis of PD-L1 or PD-L2 surface expression, 0.5–1 × 10⁵ tumor cells were blocked with Fc Block (anti-CD16/32 antibodies, Affymetrix, USA), stained with 7-Aminoactinomycin D (7-AAD Biolegend, USA), AnnexinV-FITC (AxV, Life Technologies and Sigma-Aldrich, USA), and anti-PD-L1-PE-Cyanine7 (Affymetrix, USA) or anti-PD-L2-APC (Biolegend, USA) for 30 min at 4°C in the dark and analyzed using flow cytometry (Gallios, Beckman Coulter, USA). Before use, the anti-PD-L1 (clone MIH5, dilution 125 ng/ml) and anti-PD-L2-APC (clone TY25, 1/100, Biolegend, USA) antibodies were titrated, and the isotype control for every condition was subtracted from the measured PD-L1 or PD-L2 mean fluorescence intensity. Cells negative for AnnexinV-FITC and 7-AAD (AxV⁻/7-AAD⁻) were identified as vital, cells positive for AxV but negative for 7-AAD (AxV⁺/7-AAD⁻) as apoptotic and cells positive for 7-AAD (7-AAD⁺) as necrotic.

Venous blood samples (1 mL) were collected from individual patients and healthy individuals immediately after visiting the hospital. To determine the frequency of T cells (CD3/CD4/CD8), natural killer (NK) cells, and monocytes (CD14), blood samples were stained with the following antibodies: Pcy5-conjugated anti-CD3, FITC-conjugated anti-CD4, P-phycoerythrin-conjugated anti-CD8 (BD Biosciences, CA, USA), and PE-conjugated anti-CD16/CD56 (Beckman Coulter, CA, USA).
The frequency of different types of immunocompetent cells was characterized by flow cytometry as previously described (PMID: 8404602). Briefly, at least 1 × 10⁵ cells were analyzed using BD FACS Canto^TM II flow cytometer (BD Bioscience, CA, USA). The staining of T cells or monocytes was performed with a single antibody or a combination of the following antibodies: anti-Tim-3-APC (R&D Systems), anti-PD-1-PE (eBiosciences), anti-PD-L1-BrilliantViolet421 (Biolegend), and anti-PD-L2-APC (Biolegend). The isotype-matched immunoglobulins were used as controls. The tests were performed at least in triplicate.