NK cell-induced damage to P . falciparum -infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum -infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMβ2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P . falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P . falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK–iRBC synapses and free vesicles similar in size to GFP + PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P . falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P . falciparum infection. Author summary The parasite Plasmodium falciparum is the main contributor to malaria disease that causes more than 600,000 deaths/year, primarily among young children. To control disease, it is critical to understand how clinical immunity develops in people exposed to malaria. Antibodies acquired during repeated malaria exposure are sufficient to protect against disease. One of the mechanisms for protection is antibody dependent killing of parasite-infected red blood cells (RBC) by natural killer (NK) cells, a type of white blood cell. Here we show how NK cells detect and bind to parasite-infected RBC and damage the RBC plasma membrane. We identified two separate NK cell receptors that promote stable interaction only with infected RBC and identified the two proteins that these receptors recognize on RBC. Interactions between NK cells and infected RBC were captured by live imaging, which showed that selective NK-mediated damage to the infected RBC membrane is followed by the release of vesicles (“parasitophorous vacuoles”, or PV) that contain parasites. PV are readily detected by antibodies from malaria-exposed individuals and stimulate monocytes to engulf the PV by phagocytosis. This study provides insights into how NK cell activity contributes to reducing parasite load and promotes malaria resistance in people in malaria-endemic areas.