Malaria-driven expansion of adaptive-like functional CD56-negative NK cells correlates with clinical immunity to malaria

Natural Killer (NK) cells likely play an important role in immunity to malaria, but whether repeated malaria modifies the NK cell response remains unclear. Here, we comprehensively profiled the NK cell response in a cohort of 264 Ugandan children. Repeated malaria exposure was associated with expansion of an atypical, CD56neg population of NK cells that differed transcriptionally, epigenetically, and phenotypically from CD56dim NK cells, including decreased expression of PLZF and the Fc receptor g chain, increased histone methylation, and increased protein expression of LAG-3, KIR and LILRB1. CD56neg NK cells were highly functional, displaying greater antibody dependent cellular cytotoxicity than CD56dim NK cells, and higher frequencies of these cells were associated with protection against symptomatic malaria and high parasite densities. Importantly, following marked reductions in malaria transmission, frequencies of these cells rapidly declined, suggesting that continuous exposure to malaria is required to maintain this modified, adaptive-like NK cell subset. Overall design: Approximately 1,000,000 cells from 10 patient samples were stained with Human TruStain FcX Fc Blocking Reagent (BioLegend, 422302) for 10?min at room temperature. The cells were stained for 30?min at 4?°C. Cells were then washed twice with PBS supplemented with 2% FCS and 2?mM EDTA (Sigma) before resuspending in PBS and counting. Approximately 10,000 cells per sample were loaded onto the 10x Chromium controller. ATAC-Seq was performed on 2 samples from the same Ugandan child: one in 2013 when malaria transmission was high, and one in 2015 when transmission had decreased due to insecticide spraying.

自然杀伤（Natural Killer, NK）细胞在疟疾免疫中或发挥关键作用，但反复疟疾感染是否会重塑NK细胞应答仍未明确。本研究对264名乌干达儿童队列中的NK细胞应答开展了全面表征。研究发现，反复疟疾暴露与一群非典型CD56阴性NK细胞的扩增显著相关，这群细胞在转录组、表观基因组及表型层面均与CD56dim NK细胞存在显著差异，具体表现为PLZF与Fc受体γ链表达下调、组蛋白甲基化水平升高，以及LAG-3、KIR与LILRB1的蛋白表达上调。CD56阴性NK细胞具有高度功能性，其抗体依赖性细胞毒性（antibody dependent cellular cytotoxicity）强于CD56dim NK细胞；且该细胞亚群的高频率与症状性疟疾的防护效应及高寄生虫载量呈正相关。值得注意的是，在疟疾传播水平显著降低后，这群细胞的频率迅速下降，提示维持这种经过修饰的适应性样NK细胞亚群需要持续的疟疾暴露。

实验整体设计如下：从10份患者样本中获取约1×10⁶个细胞，使用人源TruStain FcX Fc封闭试剂（BioLegend，货号422302）于室温下孵育10分钟。随后将细胞置于4℃下染色30分钟。细胞经含2%胎牛血清（FCS）与2mM乙二胺四乙酸（EDTA，Sigma）的磷酸盐缓冲液（PBS）洗涤两次后，重悬于PBS中并进行细胞计数。每份样本取约10,000个细胞上样至10x Chromium控制器。此外，本研究对同一名乌干达儿童的2份样本开展了ATAC测序（ATAC-Seq）：一份采集于2013年（彼时疟疾传播水平较高），另一份采集于2015年（彼时因杀虫剂喷洒，疟疾传播已显著降低）。