Igg isotype control

The dLNs were extracted with sterilized surgical equipment and crushed with frosted surfaces in ice‐cold PBS. The cell mixtures were then filtered through 75 mm cell strainers into 15 ml conical tubes. The cells were washed, counted, and then seeded into 48‐well plates. Single‐cell suspensions (1 million cells) were first incubated with anti‐mouse Fc blocking antibodies (BioLegend) for 30 min at 4°C and then coincubated with Fixable Viability Dye (Invitrogen) to exclude dead cells followed by co‐incubated with antibodies against CD45 (BioLegend), CD3 (BioLegend), CD8 (Invitrogen), PD‐1 (BioLegend) and Tim‐3 (BioLegend) for 20 min at 4°C. For intracellular Gzm B (Invitrogen) staining, cells were stimulated for 4 h at 37°C in a medium containing PMA (Sigma, 50 ng ml⁻¹), ionomycin (Sigma, 1 μg ml⁻¹), and brefeldin A solution (Invitrogen), and then the cells were subjected to an intracellular staining protocol (Invitrogen). Intranuclear Ki‐67 (BioLegend) staining was carried out with fixation/permeabilization buffer solution (Invitrogen) according to the manufacturer's instructions. Flow cytometry was performed with an ACEA NovoCyte, and the data were analysed with NovoExpress software. The negative staining threshold was obtained with IgG isotype control (BioLegend).

HLA surface expression on CD38⁺CD138⁺ myeloma cells of patients and MCLs was analyzed using the QIFIKIT bead-based quantitative flow-cytometric assay (Dako, Glostrup, Denmark) according to the manufacturer's instructions as described before.^{21 (link)} In brief, samples were stained with the pan-HLA class I-specific monoclonal antibody (mAb) W6/32 (produced in-house) or IgG isotype control (BioLegend, San Diego, CA, USA), respectively. Surface marker staining for primary samples was carried out with directly labeled CD138, anti-κ, anti-λ, CD19, CD20 (BioLegend) and CD38, CD3 and CD34 (BD, Franklin Lakes, NJ, USA) antibodies. 7-AAD (BioLegend) was added as a viability marker immediately before flow-cytometric analysis on an LSR Fortessa (BD).

Frozen HMNCs were recovered and rested in complete RPMI supplemented with 2ng/mL IL-15 (Miltenyl Biotec) and 25ug/mL DNAse-I (Sigma-Aldrich) for 45 mins at 37°C in 5% CO₂. CD56⁺ NK cells were magnetically isolated from HMNCs using a negative selection kit (Stemcell, Vancouver, Canada) and from peripheral blood samples using a RosetteSep™ kit (Stemcell). Whole PBMCs were isolated by density centrifugation using Ficoll-Paque™ PLUS and CD3⁺ T cells were magnetically isolated from PBMCs using a negative selection kit (Stemcell). PBMCs or CD3⁺ T Cells were stimulated with anti-CD3 (5µg/mL; Tonbo Biosciences, California, USA) and anti-CD28 (2µg/mL; Tonbo Biosciences) and co-cultured with either hepatic or peripheral blood NK cells at a 1:1 (PBMC : NK) ratio in cRPMI supplemented with 2ng/mL IL-15, unless otherwise indicated. A monoclonal antibody (mAb) against CD160 (MBL, Massachusetts, USA) or an IgG isotype control (Biolegend, California, USA) was added at 10µg/mL to assess its role in NK cell killing. After 24 hr the percentage of dead T cells was assessed using the coculture flow cytometry panel (Supplementary Table 2).

For the in vitro neutralization of the α_v-integrin receptor, a total of 1.5×10⁶ dHL-60 cells were placed into 1.5 ml microfuge tubes containing 1 ml of Opti-MEM. The cells were then pre-treated with 1 µg/ml of each of the IgG isotype control or anti-α_v-integrin neutralizing antibody (both from BioLegend) for 1 h at 37°C. Subsequently, the cells were stimulated with rhMFG-E8 (500 ng/ml) or PBS for different periods of time and then analyzed by flow cytometry or western blot analysis.

Each MPA reaction tube included two antibodies: CD14 (monocyte identifier) conjugated to the fluorophore Brilliant Violet (BV) 421 (Clone M5E2, BioLegend, San Diego CA) and CD42b (platelet identifier) conjugated to Allophycocyanin (APC) (Clone HIP1, BioLegend) or IgG isotype control (BioLegend). Six MPA reaction tubes (Protein LoBind Eppendorf, Germany) were included: isotype control, no agonist, positive control (250 μmol/L thrombin receptor activating peptide‐6 (SFLLRN, Sigma‐Aldrich, MO)), and threshold (low) concentrations of the following three agonists: adenosine diphosphate (ADP) 1.5 μmol/L (Chrono‐Log Corp., PA), arachidonic acid (AA) 10 μg/mL (Sodium arachidonate, Bio/Data Corp., PA), and collagen 1.5 μg/mL (Chrono‐Log Corp., PA). Absence of spectral overlap was confirmed by single‐color comp bead controls (BD Biosciences). Samples were fixed and red cells lysed with 800 μL of BD FACSLyse solution (BD Biosciences) following exactly 15 min of incubation. Samples were then stored in the dark at 4°C and analyzed by flow cytometry (BD FACSCanto^™ II, BD Biosciences) at a low flow rate for 10 min per tube, to avoid coincident events (Hui et al. 2015).