(A) Staining of RBCs infected with a P.f. 3D7 strain enriched for expression of a RIFIN family member (PF3D7_1400600) with human monoclonal Ab MGD21 (green) or variant MGD21-LALA (red). The shaded histogram represents staining with AF488-conjugated anti-human IgG (H + L) antiserum alone. (B) Lysis of RIFIN+ iRBCs incubated for 6 hr in the absence of NK cells or at an NK to iRBC ratio of 5:1 in presence or absence of MGD21 or MGD21 LALA Abs, as indicated. Data are shown (mean ± SD) for NK cells from four independent NK cell donors, as measured by Hb release (ANOVA, p=0.0005). (C) Staining of RBCs infected with P.f. FCR3 strain expressing VAR2CSA with rabbit polyclonal Ab to the DBL3X domain of PfEMP1 VAR2CSA (green) or with control rabbit serum (red). The shaded histogram represents staining with secondary FITC-labeled anti-rabbit IgG alone. (D) Hemoglobin release measured after incubation of NK cells with VAR2CSA-iRBCs, at an NK to iRBC ratio of 5:1 for 5 hr, in the presence of affinity-purified IgG from control rabbit serum or from serum of rabbit immunized with VAR2CSA PfEMP1. Each color represents a single NK cell donor tested in independent experiments (n = 6) (t-test, p=0.0049). (E) Staining of iRBCs expressing P.f. VAR2CSA with human polyclonal Abs to either AMA1 antigen as control (red), or to the DBL domains of PfEMP1 VAR2CSA (green). The shaded histogram represents staining with AF488-conjugated anti-human IgG (H + L) antiserum alone. (F) Parasite GIA-ADCC analyzed by flow cytometry. Enriched trophozoite-stage FCR3 VAR2CSA-iRBCs were incubated with NK cells, at an NK to iRBC ratio of 5 for 6 hr, in the presence of a control rabbit IgG (–), of rabbit anti-PfEMP1 IgG, and of human anti-PfEMP1 IgG, as indicated. A 100-fold excess of uRBCs (relative to the iRBCs input) was added, and incubation resumed for another 42 hr. Inhibition is expressed as percent decrease in parasitemia relative to iRBCs that were incubated with NK cells in the absence of Abs (ANOVA, p=0.0027).