Hla e pe

Mononuclear cells were obtained from the bone marrow of newly diagnosed MM patients (n = 12), immediately after bone marrow aspiration. Next, myeloma cells were isolated using the CD138⁺ MACS system (Miltenyi Biotec Inc, Auburn, CA, USA). Isolated myeloma cells were then cryopreserved, enabling them to be used as an irradiated antigen-source for DCs, and as autologous primary myeloma cells. Isolated primary myeloma cells also were stained with mAbs against human CD38-PE, CD138-FITC, CD19-PE-Cy5.5, MHC class I-FITC, MICA-PE, MICB-PE, ULBP1-PE, ULBP-2/5/6-PE, HLA-E-PE, PD-L1-APC, CD112-PE, CD155-PE, and ICAM-1-APC (R&D System, USA) to determine morphology, the surface staining steps were described above. The samples were acquired using FACS Canto (USA), and the data were analyzed using FlowJo.

For Adipose derived MSC characterisation cells were labelled with CD90—Fluorescein isothiocyanate (FITC)(Thy-1), CD105 FITC (Endoglin), CD73 FITC (lymphocyte-vascular adhesion protein 2/ 5′-nucleotidase/NT5E), CD13 FITC, CD29 FITC, CD44 FITC, CD34 FITC (hematopoietic stem cells and endothelial cells markers), CD271/NGFR FITC, Human Leukocyte Antigen (HLA)-DR FITC, CD45—(leukocyte marker) phycoerythrin (PE), CD19 PE, CD106 PE (VCAM), CD11b PE (integrin α M), CD166 PE, CD31 PE (R&D systems). MSC were also labelled with IgG1K antibodies against VCAM, iCAM (CD54 PE), PD-L1 (CD274 PE), HLA-ABC PE, HLA-G PE, HLA-E PE and IgG2bK antibody HLA-DR FITC (R&D systems, United States) to assess homing capabilities after priming. Cell surface markers for PBMCs assessed T cell markers CD3 PE, T _helper CD4 FITC, T _cytotoxic CD8 PE, B cell CD19 PE, CD25 PE and early activation marker CD69 FITC antibodies (R&D systems, United States). All samples were run on FACSCalibur and analysed using CellQuest.