Innate lymphocyte function in malaria

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Antibodies acquired naturally through repeated exposure to P. falciparum are essential for control of blood-stage malaria. While antibody-dependent neutralization of parasitehost interactions, complement activation, and activation of Fc receptor-mediated phagocytosis have been studied, the antibody-dependent cellular cytotoxicity (ADCC) of NK cells has not been established in the context of malaria. We have shown that IgG isolated from adults living in a malaria-endemic region induced ADCC by primary human NK cells toward infected erythrocytes in vitro. NK cells lysed P. falciparum-infected erythrocytes and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. Erythrocyte lysis was measured by a quantitative hemoglobin release assay. Lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs, as shown by live microscopy. In the absence of antibodies, NK cells exhibited negligible natural cytotoxicity towards infected erythrocytes. Granzyme B activity in infected erythrocytes was detected during co-incubation with NK cells by live imaging, after loading erythrocytes with a reporter fluorescent probe that is activated by proteolytic cleavage by granzyme B. Transmission electron and scanning electron microscopy showed tight interactions between NK cells and infected erythrocytes in the presence of plasma from adults living in a malaria endemic region of Mali. Such interactions were not observed with uninfected erythrocytes. In some of the NK:infected erythrocyte conjugates, loss of erythrocyte membrane integrity was evident. Furthermore, smaller, round bodies were observed, which had a size consistent with that of parasitophorous vacuoles (PV). Flow cytometry analysis showed that these putative PV reacted strongly with plasma of clinically immune Mali individuals. To test whether these bodies were PV, a P. falciparum strain was engineered to express Pf-EXP2. EXP2 is a transmembrane subunit of a transporter that connects with the cytosol of erythrocytes. A GFP tag was added to end of EXP2 that is exposed to the erythrocyte cytosol. GFP+ PVs were detected inside erythrocyte ghosts after incubation with NK cells and Mali plasma. Free PVs accumulated after longer incubations with NK cells. A protocol was developed to disrupt the membrane of infected erythrocytes and isolate PVs through cell sorting. In the presence of Mali plasma, PVs were efficiently phagocytosed by primary monocytes. This work showed that the human immune system is equipped for a clean disposal of infected erythrocytes by first NK-dependent damage to the erythrocyte membrane, which releases PVs, and then phagocytosis of PVs by monocytes. The full genotypes of HLA and killer-cell Ig-like receptor (KIR) genes in more than 900 subjects in the Mali cohort have been determined by sequence-based typing and analyzed for association with malaria disease. Significant associations of resistance or susceptibility to malaria disease with several alleles of HLA class I and HLA class II were observed. This suggests that CD8 and CD4 T cells have a role in immune responses to P. falciparum infection. While CD4 Th1 cells could contribute by helping B cells, the role CD8 T cells may have is less clear, considering that erythrocytes do not have HLA class I.