Ulbp 2 5 6 percp

Ramos, SU-DHL-4 and JeKo-1 cells were incubated with selinexor (50-2000nM), leptomycin B (50nM) or DMSO control for 16 hours in the presence of QVD. Cells were then surface stained with antibodies against activating NK cell ligands Vimentin-A488 (clone 280618, R&D Systems), 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) and MICA/B-PE/Cy7 (6D4, Biolegend); the inhibitory NK cell ligand HLA-E-PE/Cy7 (3D12, Biolegend) and pan-HLA class-I molecules (W6/32, Biolegend) for 30 min at 4°C. CLL cells were incubated with selinexor (50-2000nM) or DMSO control for 40 hours in the presence of QVD then incubated with 10% human serum at 4°C for 15 minutes before CD5+CD19+ CLL cells were surface stained with anti-HLA-E-PE/Cy7 (3D12, Biolegend) for 30 min at 4°C. Normal PBMC were incubated with selinexor (500-2000nM) or DMSO control for 16 hours then surface stained with anti-HLA-E-APC (3D12, Biolegend), anti-CD3-PerCP (UCHT1, Biolegend), anti-CD19-PE (HIB19, Biolegend) and anti-CD56-PE/Cy7 (HCD56, Biolegend) for 30 min at 4°C. Cells were then acquired on a BD FACS Aria II (BD Biosciences) using FACSDiva software (BD Biosciences) as above.

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.