HEK293T cells were transfected with full length FcγRIIb, FcγRIIaH131 or FcγRIIaR131 expressing plasmids. After 24 h of expression, cells were dispersed by StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific) and seeded to 96-well plate with 5×104 per well followed by 6 h of adherence. NK003 variants and 5×104 CD40 reporter cells were resuspended in 200 μL of complete RPMI 1640 medium and added to the well. GFP expression in CD40 reporter cell was detected after 6 h or 24 h by flow cytometry (LSR Fortessa, BD) and mean fluorescence intensity (MFI) was calculated by FlowJo software.

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