Innate lymphocyte function in malaria

通过反复暴露于恶性疟原虫而自然获得的抗体对控制血液期疟疾至关重要。虽然已经研究了抗体依赖的中和寄生虫与宿主的相互作用、补体激活和激活Fc受体介导的吞噬作用，但在疟疾的背景下尚未建立NK细胞的抗体依赖的细胞毒性(ADCC)。我们已经证明，从疟疾流行地区的成年人中分离出的免疫球蛋白在体外可以诱导原代人类NK细胞对感染的红细胞产生ADCC。NK细胞裂解感染恶性疟原虫的红细胞，并在体外抑制ADCC依赖的生长抑制试验中抑制寄生虫的生长。用定量血红蛋白释放法测定红细胞溶解能力。活体显微镜显示，在与未感染的红细胞混合培养时，NK细胞对感染的红细胞具有很高的选择性。在没有抗体的情况下，NK细胞对感染的红细胞的天然细胞毒性可以忽略不计。在与NK细胞共同孵育的过程中，用活体成像检测感染红细胞中颗粒酶B的活性。 透射电子显微镜和扫描电子显微镜显示，在马里疟疾流行地区的成人血浆存在下，NK细胞与感染的红细胞之间存在紧密的相互作用。在未感染的红细胞中没有观察到这种相互作用。在一些NK：感染的红细胞膜结合物中，红细胞膜的完整性明显丧失。此外，还观察到较小的圆形小体，其大小与寄生虫性空泡(PV)的大小一致。流式细胞仪分析表明，这些假定的PV与临床免疫的马里人的血浆发生强烈反应。为了测试这些抗体是否是PV，我们设计了一株表达PF-EXP2的恶性疟原虫菌株。EXP2是转运蛋白的一个跨膜亚单位，与红细胞的胞浆相连。将GFP标签添加到暴露在红细胞胞浆中的EXP2末端。与NK细胞和马里血浆孵育后，在红细胞内检测到GFP+PVs。与NK细胞孵育较长时间后，游离静脉曲张积聚。开发了一种方法来破坏感染的红细胞膜，并通过细胞分选来分离PV。在马里血浆存在下，PVs能被原代单核细胞有效地吞噬。这项工作表明，人类的免疫系统首先依赖于NK对红细胞膜的破坏，释放出PVs，然后单核细胞吞噬PVs，从而准备好干净地处置感染的红细胞。 马里队列中900多名受试者的人类白细胞抗原和杀伤细胞Ig样受体(KIR)基因的全部基因类型已通过基于序列的分型确定，并分析了与疟疾疾病的相关性。对疟疾的抵抗力或易感性与几个HLAI类和HLAII类等位基因显著相关。提示CD8和CD4T细胞在恶性疟原虫感染的免疫应答中起一定作用。虽然CD4Th1细胞可以通过帮助B细胞做出贡献，但考虑到红细胞没有HLAI类，CD8T细胞可能扮演的角色还不太清楚。

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.