Innate lymphocyte function in malaria

National Institute Of Allergy And Infectious Diseases

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Abstract

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. Target cell lysis by NK cells requires two processes controlled by different receptors, namely adhesion to target cells and degranulation to release lytyic effector molecules. Our laboratory has previously shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of so-called adaptive NK cells, which have an 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. In this project, antibody-blocking experiments with iRBC were applied to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via NK receptors CD2 and integrin M2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. To validate these results and obtain genetic evidence, we initiated a collaboration with Dr. Duraisingh (Harvard U.). Using CRISPR/Cas9, individual genes in erythrocytic precursors were knocked out. These cells were then differentiated into late-stage erythrocytes. A CD58-KO clone consistently showed reduced interaction with and sensitivity to NK cells. Furthermore, blocking experiments with the CD11b (alphaM) antibody showed greater inhibition with the CD58-KO erythrocytes than it did with the Cas9+ wild-type erythrocyte control. These results suggest some redundancy of CD2 and Mac-1 for conjugation of NK cells with erythrocytes, but that their combined engagement with their respective ligands provides an optimal response. 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 NKiRBC synapses and free GFP+ vesicles similar 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. During ADCC NK cells damage iRBC selectively, leaving uninfected RBC alone. This selectivity occurs even when uninfected RBC and iRBC are coated with the same polyclonal anti-RBC IgG serum. Here we show that uninfected RBC did not form conjugates with NK cells, even in the presence of IgG. The basis for the selective recognition of iRBC is unknown. In addition to its role in adhesion, CD2, the receptor for CD58, is also an amplifier of signaling by CD16 on NK cells and by the T cell receptor. CD2 enhances CD16-mediated responses and CD2 expression is upregulated in adaptive NK cells. It is therefore likely that CD2 on NK cells contributes not only to adhesion but also to the ADCC response to infected erythrocytes. NK cell-dependent ADCC does not destroy iRBC and PV but permeabilizes and damages the iRBC plasma membrane. Two reliable readouts of iRBC damage were the release of hemoglobin and the accessibility of F-actin in iRBC to phalloidin. The PV membrane remained structurally intact even for those PV that were released from ghost iRBC after incubation with NK cells. However, damage to the parasite occured, as shown by accessibility of DNA to propidium iodide. In addition, active granzyme B was detected in iRBC during NK-mediated ADCC, much like GzmB delivered into tumor target cells. Granzyme is known to cleave P.f. proteins at the intra-erythrocytic blood stage, as was shown with 2 T cells. Permeabilization of iRBC requires granulysin rather than perforin due to cholesterol depletion from the iRBC plasma membrane at late stages of Plasmodium development. In summary, we show that NK cells recognize P.f.-infected erythrocytes and adhere to them via CD2CD58 and M2ICAM-4 interactions. This adhesion event is followed by damage to the iRBC plasma membrane and the release of parasitophorous vacuoles. These PV show robust binding to IgG from malaria-exposed individuals from endemic areas and this marks the PV for clearance by monocytes through phagocytosis. These findings provide insights into earlier observations that NK cell frequency and function correlated with decreased parasitemia and resistance to malaria and reveal parasite clearance from the blood by NK-mediated ADCC in cooperation with monocytes.