FIG 5.

DBL2χ, DBL3χ, and DBL5ε antigen reversal lowers opsonization of FCR3-CSA for primary monocytes. (A) Representative flow plots of PBMCs collected from malaria-naive Americans before (Pre) and after (Post) depletion of T cells, B cells, and NK cells to negatively select for primary monocytes. Propidium iodide labeling confirmed ≥99% viability of monocytes. Each plot shows at least 50,000 events. (B) Giemsa smears of enriched primary monocytes before (Parasite −) and after (Parasite +) phagocytosis of IEs. Bar, 10 μm. Photomicrographs were collected as stated in the legend for Fig. 3A. (C) Representative flow plots of enriched primary monocytes after coincubation with ethidium-labeled FCR3-CSA IEs preincubated with pooled IgGs from either malaria-naive Americans (left) or malaria-exposed multigravida Malian women (right). Following a 2-h coincubation with IEs and ACK lysis treatment to remove noninternalized erythrocytes, CD14⁺ monocytes that phagocytosed IEs showed high ethidium fluorescence. Phagocytosis was measured as the proportion of CD14⁺ monocytes above the autofluorescence level (horizontal bar). Each flow plot shows 5,000 events. (D) Purified IgG pools (1.25 mg/ml) from Malian women, Malian men, and American adults were pretreated with 100 μg of recombinant antigens (or no antigen) for 1 h. 3D7, FCR3, and FCR3-CSA IEs were incubated with pretreated IgGs for 1 h and then incubated with CD14⁺ monocytes. Means ± SEM of phagocytosis from three independent experiments (using monocytes from three separate donors) are shown.