Anti hla dr bv421

PBMCs and other cells were stained with anti-human mAbs: anti-CD3 FITC, anti-CD4 BV605, anti-CD8 BV421, anti-CD14 BV711, anti-CD19 BV605, anti-CD1d APC, anti-HLA-DR BV421, IFN-γ PE, and IL-4 PE (all from BD Biosciences, Vienna, Austria). APC-labeled human CD1d tetramers loaded with the α-GalCer analogue PBS-57 were obtained from the MHC Tetramer Core Facility (Emory University Vaccine Center, Atlanta, GA). Anti-human ADRB2 (AbD Serotec, Oxford, UK) was coupled with alexa fluor647 fluorochrome by using a protein labeling kit (Life Technologies, Paisley, UK). Viability was assessed with Zombie Nir viability dye (BioLegend, London, UK) or with fixable viability dye eFluor506 (eBiosciences, Vienna, Austria). The corresponding isotype control mAbs were used to assess staining specificity.

Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (GE-171440-02, Merck). Separated cells were subsequently collected from the interphase, washed with isolation buffer (2.23 g/l d-glucose, 2.2 g/L sodium citrate, 0.8 g/L citric acid, 0.5% BSA in PBS), and further centrifuged at 500 g for 10 min at RT. The cell pellet was resuspended in FBS, counted, and aliquoted 1:1 in FBS with 20% dimethyl sulfoxide (DMSO, Sigma-Aldrich). The samples were then stored in liquid nitrogen at − 160 °C until use.
Freshly thawed PBMCs (1 × 10⁶) were washed with RPMI and stained with Zombie NIR Dye (Biolegend) for living cell identification following the manufacturer’s instructions. Then, Fc receptors were blocked with beriglobin (50 μg/ml; CSL Behring) for 10 min at 4 °C. The antibody panel for MDSC analysis was made up of anti-CD15 FITC (1.25 µl/test, HI98 clone), anti-CD14 PerCP-Cy^TM5.5 (0.5 µl/test, Mφ29 clone), anti-CD11b PE-Cy7 (0.5 µl/test, ICRF44 clone), anti-CD33 APC (1.25 µl/test, WM53 clone), and anti-HLA-DR BV421 (0.5 µl/test, G46-6 clone, all from BD Biosciences). PBMCs were fixed with 0.1% PFA and then were analyzed as described above for murine blood cells.

After stimulation, PBMCs were labeled with viability marker LIVE/DEAD Near-IR fluorescent reactive dye (Thermo Fisher) for 30 min and subsequently stained for 20 min with the following surface markers: anti-CD3-PerCP (BioLegend), anti-CD4-BV786, anti-CD8-BV510, anti-CD27-BV605, anti-CD38-PE, and anti-HLA-DR-BV421 (BD Bioscience). Cells were then fixed and permeabilized with the Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher) and stained for 30 min with intracellular markers: anti-IFNγ-APC, anti-TNFα-PE-Cy7 and anti-Ki-67-FITC (BD Bioscience). The complete list of antibodies, conjugated proteins and dilutions can be found in Supplementary Table 1. All incubation processes were performed at room temperature in darkness. Fluorescence Minus One (FMO) controls of the four analyzed cell markers (CD27, CD38, HLA-DR, and Ki-67) were included in each run to accurately distinguish negative from positive populations. Samples were resuspended in 100 μl of PBS-0.1%BSA (Bovine Serum Albumin, Sigma Aldrich) and acquired in a BD LSRFortessa flow cytometer (BD Bioscience) using FACSDiva software (BD Biosciences) with compensated parameters.