Hla e pe cy7 3d12

After treatment with selinexor, leptomycin B or brefeldin A (BFA, GolgiPlug, BD Bioscience) for the required timepoint, Fc receptors were blocked on CLL cells with 10% human serum for 15 min then cells were stained with CD19-PE (HIB19, Biolegend) or CD19-PB, CD5-PerCP (UCHT2, Biolegend) or CD5-APC, ULBP-1-PE (170818, R&D Systems), ULBP-2/5/6-PerCP (165903, R&D Systems), CD54-PB (HCD54, Biolegend), B7H6-APC (875001, R&D Systems), ecto-calreticulin-A488 (EPR3924, Abcam), MICA/B-PE/Cy7 (6D4, Biolegend), CD20-PE (2H7, BioLegend), CD38-A488 (HIT2, BioLegend), FAS-FITC (DX2, Biolegend), DR4-PE (DJR1, Biolegend), DR5-APC (DJR2-4, Biolegend), HLA-E-PE/Cy7 (3D12, Biolegend) and total HLA class I proteins (W6/32, Biolegend) for 30 min at 4 °C. Assessment of CLL cells that have recently egressed from the lymph nodes was performed as previously described [30 (link), 31 (link)]. Patient CLL cells were rested for 1 h at 37 °C, then stained with CD19-BV510 (HIB19), CD5-APC, HLA-E-PE/Cy7 (3D12), pan-HLA (W6/32) and CXCR4-PE (12G5, Biolegend). Cells were then acquired on a BD FACS Aria II.

Following mAbs were used for detection of surface markers: anti-CD11b APC (clone: ICRF44), CD48 BV421 (TÜ145), HLA-ABC FITC (G46-2.6), CD62L APC (DREG-56), CD66 PE (B1.1; all from BD Biosciences, CA, USA), HLA-C (DT9, Merck Millipore), HLA-ABC Alexa Fluor 647 (W6/32) and HLA-E PE-Cy7 (3D12; both from Biolegend), B7-H6 (875002), ULBP1 PerCP (170818), ULBP-3 PE (166510), ULBP-2/5/6 APC (165903; all from R&D Systems, MN, USA), CD56 PE-Cy7 (N901, Beckman Coulter, IN, USA), MICA/B viobright FITC (6D4, Miltenyi Biotech, Bergisch Gladbach, Germany), and Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, CA, USA). Anti-B7-H6 (17B1.3) was kindly provided by Prof E Vivier (CMIL, Marseille, France), and anti-PVR (CD155; M5A10) and Nectin-2 (CD112; L14) were received from the lab of Prof. A Moretta (Genova, Italy). Goat serum and α1-antitrypsin were obtained from Sigma-Aldrich (MO, USA), CL097 from InvivoGen (San Diego, CA), GM-CSF from R&D Systems (MN, USA), anti-PR3 from Abcam (UK), and Batimastat and cathepsin G inhibitor from Calbiochem (CA, USA). In experiments where the extracellular FITC signal was quenched, a Fluorescin/Oregon green polyclonal antibody (ThermoFisher, Sweden) was used.