Anti pd l1 pe

To study the activation/maturation markers, the cells were stained for 20 min at 4 °C with an anti-MHCII eFluor450, an anti-CD40 APC, an anti-CD80 FITC, an anti-CD86 PE, an anti-mPDCA PEeFluor610, and anti-CD11c PC7 antibodies. All antibodies were purchased from eBioscience, San Diego, CA, USA, and each was diluted in PBS containing 0.5% of BSA (bovine serum albumin, Sigma-Aldrich, St. Louis, MO, USA), 2 mM EDTA (Fisher Chemical, Leicerstershire, UK), and 1/20 FcR Bloking Reagent (Miltenyi Biotec GmbH, Bergish Gladbach, Germany). In steady state, pDCs and cDCs were distinguished through the expression of CD11c+mPDCA+ for pDCs and CD11c+mPDCA− cells for cDCs. To study tolerogenic markers, the same staining protocol was performed using anti-PDL1 PE (Biolegend, San Diego, CA, USA), anti-CTLA4 APC (Miltenyi Biotec GmbH, Bergish Gladbach, Germany), and anti-CD11c PC7 (eBioscience, San Diego, CA, USA) antibodies.

One hundred microliters of whole blood were stained with anti-CD3-PECy7 (BioLegend, San Diego, CA, USA; clone HIT3a), anti-CD8-PECy5 (BioLegend; clone SK1), anti-PD-L1-PE (BioLegend; clone 29E.2A3), anti-CD14-PECy7 (BD Bioscience, San Jose, CA, USA; clone M5E2), anti-CD41a-FITC (Immunotools, Friesoythe, Germany; clone HIP8), and anti-CD62P-APC (Immunotools; clone HI62P). The cells were then incubated for 15 min in the dark before adding 2 mL of BD FACS lysing solution 1X (BD Bioscience) for 10 min. Finally, the cells were washed with 2 mL of PBS 1X and resuspended in 400 µL of PBS 1X before acquisition by flow cytometry (MACSQuant Analyzer 10 flow cytometer; Miltenyi Biotec, Bergisch Galdbach, Germany). Negative gates for each marker were defined by fluorescence minus one (FMO) controls.

For surface labeling, the same protocol of seeding and treatment as described above for cell cycle analysis was followed. LCLs and DLBCLs cells were collected and washed with DPBS. Then, they were labeled for 15 min in the dark at RT with anti-PD-L1-PE (Biolegend) (Table S1). Intracellular PD-L1 staining was performed on LCLs and DLBCLs cells treated with native fucoidan or F1/F2 fractions using the IntraPrep Permeabilization Reagent kit (Beckman Coulter) according to the protocol recommended by the supplier. Acquisitions were performed on FACSCalibur. Results were analyzed with Kaluza Analysis 2.1 Software. Fold change was calculated based on the mean fluorescence intensity ratio of PD-L1 on its isotypic control, and then normalized to the control (untreated cells).

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.

The staining method used for flow cytometry of exosomes coupled to beads was modified based on the methods described by Morales–Kastresana [36 (link)] and Theodoraki [37 (link)]. Briefly, 10 µg exosome protein was coincubated with 1 µg biotin-labeled anti-CD63 antibody (353,018, Biolegend) for 2 h at room temperature. Next, 15 µL of streptavidin-coated magnetic beads (MBL International) was added and the compounds were gently agitated on a shaker for 2 h at room temperature. The samples were washed once with dilution buffer from the kit and then coincubated with 10 µL of the detection antibody anti-PD-L1 PE (329706, Biolegend) or the labeled isotype control antibody (400314, Biolegend) for 1 h at room temperature. The complexes were resuspended in 100 µL PBS after washing them three times with dilution buffer for antigen detection (Beckman Coulter CytoFlex). The lower edge of the “positive gate” was set at the point where  < 2% of the isotype control was positive.