Anti hla dr

To evaluate the surface markers of hucMSCs, hucMSCs at passage three were incubated with antibodies anti-CD34 (catalog number: 343503, BioLegend, San Diego, CA, USA), anti-CD73 (catalog number: 344015, BioLegend), anti-CD29 (catalog number: 303004, BioLegend), anti-CD14 (catalog number: 397706, BioLegend), anti-CD105 (catalog number: 323203, BioLegend), anti-CD19 (catalog number: 302205, BioLegend), anti-CD45 (catalog number: 304005, BioLegend), anti-HLA-DR (catalog number: 327005, BioLegend), and anti-CD90 (catalog number: 328108, BioLegend). Fluorescence was detected using a flow cytometer (NovoCyte 1300; ACEA, San Diego, CA, USA) to identify hucMSCs.
To evaluate cell differentiation, hucMSCs at passage three were cultured in adipogenic (catalog number: A1007001, Gibco, Grand Island, USA), chondrogenic (catalog number: A1007101, Gibco), or osteogenic medium (catalog number: A1007201, Gibco) for 3 weeks. Subsequently, the cells were fixed with 4% paraformaldehyde, and stained with oil red O (catalog number: HY-D1168, MedChemExpress, New Jersey, USA), alcian blue (catalog number: HY-D0001, MedChemExpres), or alizarin red (catalog number: HY-120601, MedChemExpres), respectively. The stained cells were observed under a light microscope (Olympus, Tokyo, Japan).

All samples were processed within 12 h from collection. Whole blood was lysed prior to staining using the RBC Lysis solution (Qiagen, Germany) according to manufacturer's specification. Immune populations were identified by staining with fluorophore-conjugated anti-CD11b, anti-CD33, anti-CD3, anti-HLA-DR, anti-CD45, anti-CD68, anti-CD206, anti-CD163, (BioLegend), anti-CD14, anti-CD15 (eBioscience) antibodies on ice for 30 min. Fluorescence data was acquired using BD Accuri C6 (BD Biosciences), Cyan and/or CytoFLEX (Beckman Coulter) cytometers. Normalised population statistics –including the median fluorescence intensities (MFI) were determined using FlowJo (BD Biosciences, formerly developed by FlowJo LLC).
Where indicated cell death was assessed by propidium iodide staining of cells after 72 h incubation with Gemtuzumab ozogamicin (Gift from Pfizer).

mDCs were resuspended in ice cold PBS supplemented with 2 mM EDTA, 0.1% BSA, and 0.01% sodium azide, blocked with 10% mouse serum (Sigma) and stained with anti‐CD83 (BD), anti‐CD86 (BD), and anti‐HLA‐DR (Biolegend). Surface expression was evaluated relative to unstimulated stained controls on a BD CANTO II and analyzed using FlowJo software (Treestar).

Flow cytometric analyses were performed with Gallios (Beckman Coulter) flow cytometers, and the data were analyzed with the FlowJo (Treestar) software packages.
MSCs were incubated with anti-CD90, anti-CD105, anti-CD73, anti-CD44, anti-CD11B, anti-CD34, anti-CD19, anti-CD45 and anti-HLA-DR antibodies to examine MSC surface markers, which were purchased from BioLegend. Anti-CD3, anti-CD4, anti-CD8, anti-TNF-α and anti-IFN-γ antibodies were used to examine stain T cells. Propidiumiodide (PI; BD) and Annexin V (BD) were used to stain apoptosis cells. Anti-CD86, anti-F4/80 and anti-CD45 antibodies were used to stain macrophages.

To determine HLA I-restricted epitopes from CD8⁺ T cells, a B cell panel containing autologous and allogeneic BCL was used. BCL were pulsed with the specific peptide for 1 h at 10 μg/mL, washed, and then cocultured with CD8⁺ T cell clones in an IFN-γ ELISpot assay. To determine HLA II-restricted epitopes from CD4⁺ T cell clones or lines, autologous BCL were blocked with 10 μg/mL of either anti-HLA-A/B/C (BD Biosciences), anti-HLA-DP (Abcam), anti-HLA-DQ (BioLegend), anti-HLA-DR (BioLegend), or anti-HLA-DP/DQ/DR (BioLegend) antibodies for 30 min and then pulsed with the specific peptide for 1 h at 10 μg/mL. Pulsed BCL (1 × 10³ cells/well) were then washed 3 times with R10, mixed with CD4⁺ T cells (1 × 10⁴ to 2 × 10⁴ cells/well), and tested in IFN-γ ELISpot assay in the presence of 10 μg/mL of anti-HLA antibodies.