DataSheet_3_Detailed phenotypic and functional characterization of CMV-associated adaptive NK cells in rhesus macaques.pdf

Previous research on adaptive NK cells in rhesus macaques suffered from the lack of specific antibodies to differentiate between inhibitory CD94/NKG2A and stimulatory CD94/NKG2C heterodimeric receptors. Recently we reported an expansion of NKG2C receptor-encoding genes in rhesus macaques, but their expression and functional role on primary NK cells remained unknown due to this deficit. Thus, we established monoclonal antibodies 4A8 and 7B1 which show identical specificities and bind to both NKG2C-1 and NKG2C-2 but neither react with NKG2C-3 nor NKG2A on transfected cells. Using a combination of 4A8 and Z199 antibodies in multicolor flow cytometry we detected broad expression (4-73%) of NKG2C-1 and/or NKG2C-2 (NKG2C-1/2) on primary NK cells in rhesus macaques from our breeding colony. Stratifying our data to CMV-positive and CMV-negative animals, we noticed a higher proportion (23-73%) of primary NK cells expressing NKG2C-1/2 in CMV+ as compared to CMV- macaques (4-5%). These NKG2C-1/2-positive NK cells in CMV+ macaques are characterized by lower expression of IL12RB2, ZBTB16, SH2D1B, but not FCER1G, as well as high expression of IFNG, indicating that antibody 4A8 detects CMV-associated adaptive NK cells. Single cell RNA seq data of 4A8-positive NK cells from a rhCMV-positive macaque demonstrated that a high proportion of these adaptive NK cells transcribe in addition to NKG2C-1 and NKG2C-2 also NKG2C-3, but interestingly NKG2A as well. Remarkably, in comparison to NKG2A, NKG2C-1 and in particular NKG2C-2 bind Mamu-E with higher avidity. Primary NK cells exposed to Mamu-E-expressing target cells displayed strong degranulation as well as IFN-gamma expression of 4A8+ adaptive NK cells from rhCMV+ animals. Thus, despite co-expression of inhibitory and stimulatory CD94/NKG2 receptors the higher number of different stimulatory NKG2C receptors and their higher binding avidity to Mamu-E outreach inhibitory signaling via NKG2A. These data demonstrate the evolutionary conservation of the CMV-driven development of NKG2C-positive adaptive NK cells with particular molecular signatures in primates and with changes in gene copy numbers and ligand-binding strength of NKG2C isotypes. Thus, rhesus macaques represent a suitable and valuable nonhuman primate animal model to study the CMV-NKG2C liaison in vivo.

此前针对恒河猴适应性自然杀伤（NK）细胞的研究，因缺乏可区分抑制性CD94/NKG2A与刺激性CD94/NKG2C异二聚体受体的特异性抗体而受阻。本团队此前曾报道恒河猴体内编码NKG2C受体的基因发生扩增，但受限于上述抗体缺失问题，原代NK细胞上NKG2C受体的表达模式与功能角色仍未明确。为此，我们开发得到单克隆抗体4A8与7B1：二者特异性一致，均可结合NKG2C-1与NKG2C-2，但在转染细胞实验中均不与NKG2C-3或NKG2A发生反应。通过将4A8与Z199抗体联用进行多色流式细胞术检测，我们在本繁育群体的恒河猴原代NK细胞中，检测到NKG2C-1和/或NKG2C-2（合称NKG2C-1/2）的广泛表达，表达比例介于4%至73%之间。将实验动物按巨细胞病毒（CMV）感染状态分为阳性组与阴性组后，我们观察到：CMV阳性恒河猴体内表达NKG2C-1/2的原代NK细胞比例（23%~73%）显著高于CMV阴性个体（4%~5%）。CMV阳性恒河猴体内的NKG2C-1/2阳性NK细胞具有如下特征：IL12RB2、ZBTB16与SH2D1B的表达水平较低，而FCER1G的表达无显著变化，同时IFNG的表达水平较高；这表明抗体4A8可识别与CMV相关的适应性NK细胞。对一只恒河猴巨细胞病毒（rhCMV）阳性个体的4A8阳性NK细胞进行单细胞RNA测序的结果显示：该群体中多数适应性NK细胞除表达NKG2C-1与NKG2C-2外，还可转录NKG2C-3，有趣的是，这类细胞同时也表达NKG2A。值得注意的是，与NKG2A相比，NKG2C-1，尤其是NKG2C-2，与Mamu-E的结合亲和力更高。将原代NK细胞与表达Mamu-E的靶细胞共培养后，来自rhCMV阳性个体的4A8阳性适应性NK细胞表现出强烈的脱颗粒现象与IFN-γ表达上调。因此，尽管抑制性与刺激性CD94/NKG2受体存在共表达，但多种刺激性NKG2C受体的存在，以及它们与Mamu-E更高的结合亲和力，可抵消NKG2A介导的抑制性信号通路。上述数据证实，在灵长类动物中，由CMV驱动的具有特定分子特征的NKG2C阳性适应性NK细胞发育过程具有进化保守性，且该过程伴随NKG2C亚型的基因拷贝数与配体结合能力的差异变化。因此，恒河猴是研究体内CMV-NKG2C相互作用的合适且极具价值的非人灵长类动物模型。