Innate lymphocyte function in malaria

通过反复接触恶性疟原虫自然获得的抗体对于控制血液期疟疾至关重要。虽然已经研究了寄生虫-宿主相互作用的抗体依赖性中和、补体激活和Fc受体介导的吞噬作用的激活，但是在疟疾的背景下尚未建立NK细胞的抗体依赖性细胞毒性（ADCC）。我们已经表明，从生活在疟疾流行地区的成人中分离的IgG诱导ADCC的原代人NK细胞对感染的红细胞在体外。NK细胞裂解恶性疟原虫感染的红细胞，并在体外测定中抑制寄生虫生长，以进行ADCC依赖性生长抑制。通过定量血红蛋白释放试验测量红细胞溶解。如活显微镜所示，NK细胞的裂解对与未感染RBC混合培养的感染RBC具有高度选择性。在不存在抗体的情况下，NK细胞对感染的红细胞表现出可忽略的天然细胞毒性。在用报告荧光探针加载红细胞后，在与NK细胞共孵育期间通过活体成像检测感染的红细胞中的颗粒酶B活性，所述报告荧光探针通过颗粒酶B的蛋白水解切割而激活。 透射电子显微镜和扫描电子显微镜显示NK细胞和感染的红细胞之间的相互作用，在血浆中的成年人生活在疟疾流行地区的马里。在未感染的红细胞中未观察到这种相互作用。在一些NK：感染的红细胞结合物中，红细胞膜完整性的丧失是明显的。此外，观察到较小的圆形体，其大小与寄生虫液泡（PV）的大小一致。流式细胞仪分析表明，这些推定的PV强烈反应与临床免疫马里个人的血浆。为了测试这些体是否是PV，恶性疟原虫菌株被工程化以表达Pf-EXP 2。EXP 2是转运蛋白的跨膜亚基，与红细胞的胞质溶胶连接。将GFP标签添加到暴露于红细胞胞质溶胶的EXP 2的末端。用NK细胞和Mali血浆孵育后，在红细胞血影内检测到GFP+ PV。在与NK细胞更长时间孵育后，游离PV积累。开发了一种方案来破坏感染的红细胞的膜并通过细胞分选分离PV。在Mali血浆存在下，PV被原代单核细胞有效吞噬。这项工作表明，人类免疫系统配备了一个干净的处理感染的红细胞，首先NK依赖性损伤红细胞膜，释放PV，然后吞噬PV的单核细胞。 通过基于序列的分型确定了马里队列中900多名受试者的HLA和免疫细胞Ig样受体（KIR）基因的全基因型，并分析了其与疟疾疾病的相关性。观察到对疟疾疾病的抗性或易感性与HLA I类和HLA II类的几个等位基因的显著关联。这表明CD 8和CD 4 T细胞在对恶性疟原虫感染的免疫应答中起作用。虽然CD 4 Th 1细胞可以通过帮助B细胞发挥作用，但考虑到红细胞不具有HLA I类，CD 8 T细胞可能具有的作用不太清楚。

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.