Anti hla dr pe cy7

Upon cisplatin and CM treatment, macrophages were harvested using accutase (Sigma Aldrich, #A6964) and subsequent gentle scrapping of the culture wells. The harvested macrophages were washed once with PBS 2 mM EDTA and stained for 20 min with the following human antibodies: anti-HLA-DR-PE-Cy7 (Clone: L243, BioLegend, San Diego, CA, USA, #307616), anti-CD86-BV421 (Clone: IT2.2, BioLegend, #305426), anti-CD206-PE (Clone:19.2, BD Biosciences, Franklin Lakes, NJ, USA, #555954), and anti-CD163-BV510 (Clone: GHI/61, BioLegend, #333628). All antibodies were properly titrated before. Consecutively, the cells were also stained with the viability dye Sytox Bue (Invitrogen, #S34857). The cells were then acquired using flow cytometry (FACS Canto II, BD Biosciences) and the obtained .fcs data were analyzed with FlowJo 10 software (purity check, Supplementary Figure S2; changes in macrophage marker expression, Supplementary Figure S3).

To identify monocytes, the following procedure was performed. Freshly isolated PBMC were stained with viability marker PromoKine 840 (PromoCell, Heidelberg, Germany); then, samples were washed with FACS buffer (PBS, 2% FBS) and incubated with a mix of pre‐titrated directly conjugated mAbs. The mix included the following: anti‐CD14‐APC, anti‐CD16‐AF488, anti‐HLA‐DR‐PE‐Cy7, anti‐PD1‐BV421, anti‐TIM‐3‐BV785, anti‐CD15‐PE‐Cy5, anti‐CD11‐PE‐Cy5, anti‐CCR2‐BV605 (BioLegend, San Diego, CA), anti‐CD38‐BUV496, anti‐CXCR3‐BUV395 (Becton Dickinson, San José, CA), anti‐PD‐L1‐PE (Thermo Fisher, Eugene, OR). Samples were acquired by using CytoFLEX LX (Beckman Coulter, Hialeah, FL). To identify circulating mature and immature monocytes, thawed PBMC were stained with LIVE‐DEAD Aqua, anti‐HLA‐DR‐PE‐Cy7, anti‐CD14‐APC, anti‐CD13‐PE, anti‐CD64‐FITC. Table 2 reports mAbs clones, catalog numbers, type of fluorochrome used, and mAbs dilutions. Mitochondrial mass was analyzed by staining cells with MitoTracker green (MT Green, Thermo Fisher) (De Biasi et al, 2019). Mitochondrial membrane potential was analyzed by staining cells with 1,1ʹ,3,3ʹ‐tetraethyl‐5,5ʹ,6,6ʹ‐tetrachloroimidacarbocyanine iodide (JC‐1, Thermo Fisher) (Cossarizza et al, 2019). Cells were acquired using Attune NxT Acoustic flow Cytometer.

The purity of generated DCs was assessed as CD14⁻CD11C⁺ by staining 1 × 10⁶ cells with anti-CD11C-APC and anti-CD14-FITC (BD Biosciences, San Diego, CA, USA).
Immature moDCs were resuspended to 1 × 10⁶ cells/ml and loaded with peptides±HB_100-108 (40 μg/ml for each peptide) or 40 μg/ml of free HB_100-108 at 37°C, 5% CO₂. After one hour, pulsed DCs were washed and cultured overnight. The following monoclonal antibodies (mAbs) were used to characterize the maturation and activation status of DCs: anti-HLA-ABC-PE, anti-HLA-DR-PE/Cy7, anti-CD80-PE, anti-CD86-APC, anti-CD83-APC, anti-CD40-Alexa Fluor 700, and anti-CCR7-PE/Cy7 (BioLegend, San Diego, CA, USA). Isotype-matched fluorescent antibodies were used as negative controls. Cells were incubated with antibodies at 4°C for 30 minutes. After washing, samples were detected by a FACSCalibur analyzer (BD Biosciences, San Jose, CA, USA). The data were analyzed by FlowJo software (TreeStar).