Anti-CD20 rituximab IgG1, IgG3, and IgG4 but not IgG2 subclass trigger Ca2+mobilization and cytotoxicity in human NK cells

NK cell-mediated Ab-dependent cellular cytotoxicity (ADCC) is increasingly recognized to play an important role in cancer immunotherapy, transplant rejection, and autoimmunity. However, several aspects of the molecular interactions of IgG subclasses with the Fc-gamma receptor IIIA (Fc gamma RIIIA)/CD16a expressed on NK cells remain unknown. The aim of the current study was to further analyze the role of IgG subclasses andFCGR3AV158F single nucleotide polymorphism (SNP) on Ca(2+)signaling and NK cell-mediated ADCC against Daudi target cells in vitro. NK cells were isolated from donors with differentFCGR3ASNP. The affinity of rituximab IgG subclasses to CD20 expressed on Daudi cells showed similar dissociation constant as tested by flow cytometry. Induction of Ca(2+)signaling, degranulation, intracellular cytokine production, and ADCC was demonstrated for IgG1 and IgG3, to a lesser degree also for IgG4, but not for IgG2. Compared to NK cells carrying the low-affinity (FF) variant for theFCGR3AV158F SNP, binding of IgG1 and IgG3 to NK cells carrying the high-affinity (VV) and VF SNP variants was two- to threefold higher. Variations ofFCGR3ASNP among the eight tested donors (1 VV, 3FF, and 4VF) revealed no significant differences of Ca(2+)signaling and degranulation; however, ADCC was somewhat weaker in donors with the low-affinity FF variation. In conclusion, this is the first study correlating Ca(2+)signaling and NK cell-mediated ADCC triggered by the four IgG subclasses with theFCGR3AV158F SNP. Our findings indicate important differences in the interactions of IgG subclasses with Fc gamma RIIIA/CD16a but no major impact ofFCGR3ASNP and may therefore help to better correlate the functional properties of particular engineered therapeutic antibodies in vitro with individual differences of their clinical efficacy.