【论文】人NK细胞在整个寿命期间表现出主要的表型和功能变化
Human NK cells display major phenotypic and functional changes over the life span
Magali Le Garff‐Tavernier ， Vivien Béziat， Julie Decocq， Virginie Siguret ，Frédérique Gandjbakhch ，Eric Pautas ，Patrice Debré ，Hélène Merle‐Beral ，Vincent Vieillard
First published: 21 July 2010|https://doi.org/10.1111/j.1474-9726.2010.00584.x|Cited by: 115
Dr Vincent Vieillard, INSERM UMR‐S 945, Laboratoire d’Immunologie Cellulaire et Tissulaire, Hôpital Pitié‐Salpêtrière, Paris, France. Tel.: +33 142177524; fax: +33 142177490; e‐mail: vincent.vieillard@upmc.fr
Summary 概述
衰老通常与感染性疾病和癌症的易感性增加有关，部分与免疫衰老的发展有关，免疫衰老是影响免疫系统所有细胞区室的过程。尽管许多研究已经研究了年龄对自然杀伤（NK）细胞的影响，但他们的结论仍存在争议，因为研究对象的多样化健康状况导致了不一致的发现。为了澄清这种情况，我们对来自健康受试者的NK细胞进行了第一次广泛的表型和功能分析，比较了来自新生儿（脐带血），中年（18-60岁），老年（60-80岁）的NK细胞，和非常古老的（80-100岁）科目。我们发现脐带血中的NK细胞显示出与不成熟相关的特异性特征，包括KIR和LIR-1 / ILT-2的低表达以及NKG2A和IFN-γ的高表达。另一方面，来自老年受试者的NK细胞保留了其主要的表型和功能特征，但其成熟特征更加突出。这些包括CD56bright子集的显着下降，LIR-1 / ILT-2的特异性增加，以及在非常老的受试者中IL2激活后NK细胞功能的完美恢复。我们得出结论，保持NK细胞特征直到非常高龄可能有助于延长寿命和成功衰老。
Aging is generally associated with an increased predisposition to infectious diseases and cancers, related in part to the development of immune senescence, a process that affects all cell compartments of the immune system. Although many studies have investigated the effects of age on natural killer (NK) cells, their conclusions remain controversial because the diverse health status of study subjects resulted in discordant findings. To clarify this situation, we conducted the first extensive phenotypic and functional analysis of NK cells from healthy subjects, comparing NK cells derived from newborn (cord blood), middle‐aged (18–60 years), old (60–80 years), and very old (80–100 years) subjects. We found that NK cells in cord blood displayed specific features associated with immaturity, including poor expression of KIR and LIR‐1/ILT‐2 and high expression of both NKG2A and IFN‐γ. NK cells from older subjects, on the other hand, preserved their major phenotypic and functional characteristics, but with their mature features accentuated. These include a profound decline of the CD56bright subset, a specific increase in LIR‐1/ILT‐2, and a perfect recovering of NK‐cell function following IL2‐activation in very old subjects. We conclude that the preservation of NK cell features until very advanced age may contribute to longevity and successful aging.
Introduction前言
衰老是一种自然的成熟后过程，与传染病和癌症的死亡率和发病率的增加有关，至少部分归因于免疫缺陷(Miller, 1996;Hakim et al.， 2004;Pawelec & Larbi, 2008)。免疫衰老的复杂过程影响免疫系统的先天和适应性两臂。它与多种因素有关，包括胸腺复合体，导致T细胞库收缩的记忆T细胞积累，以及反映体液免疫缺陷的B细胞产量下降(Aw et al.， 2007;Hakim & ess, 2007)。同时，各种证据表明，衰老对先天免疫系统有显著影响。自然杀伤(NK)细胞作用的几种变化已经被描述:细胞数量和功能都随着年龄的增长而变化(Solana et al.， 1999;Plackett et al.， 2004)。然而，其他研究报告，衰老并不显著影响NK细胞毒性(Min et al.， 2005;DelaRosa等，2006;Solana et al.， 2006)。有关年龄影响的研究结果存在差异，主要是由于研究对象的选择标准不同;采用严格的选择标准排除感染、炎症或癌症患者，重点关注非常健康的老年人的方案报告了NK细胞毒性的保存(Mocchegiani & Malavolta, 2004)。
Aging is a natural postmaturational process associated with increased mortality and morbidity from infectious diseases and cancer, attributable at least in part to defects in immunity (Miller, 1996; Hakim et al., 2004; Pawelec & Larbi, 2008). The complex process of immunosenescence affects both the innate and adaptive arms of the immune system. It has been linked to various factors, including thymic involution, the accumulation of memory T cells, which leads to contraction of the T‐cell repertoire, and a decline in B‐cell production, reflecting defective humoral immunity (Aw et al., 2007; Hakim & Gress, 2007). At the same time, a variety of evidence indicates that aging exerts significant effects specifically on the innate immune system. Several alterations in the role of natural‐killer (NK) cells have been described: both the number of cells and their functions change as people age (Solana et al., 1999; Plackett et al., 2004). Other studies, however, report that aging does not significantly affect NK cytotoxicity (Min et al., 2005; DelaRosa et al., 2006; Solana et al., 2006). The discrepant findings about the effects of age seem due mainly to the different selection criteria for the elderly populations studied; protocols with strict selection criteria that exclude individuals with infections, inflammation or cancer and focus on very healthy older people report the preservation of NK cell cytotoxicity (Mocchegiani & Malavolta, 2004).
NK细胞是抵御感染和恶性肿瘤的第一道防线。这些细胞是异质的，在它们的增殖潜能、归巢特性、功能能力和对不同细胞因子的反应方面各不相同。根据CD56表面表达的相对密度可分为两个主要亚群。外周血NK细胞由10%左右的CD56brigh和90%左右的CD56dim NK细胞亚群组成，相对独立(Caligiuri, 2008);激活后,CD56bright NK细胞增殖,产生各种细胞因子(例如交互一些γ干扰素、肿瘤坏死因子高β和IL 10)和趋化因子(例如MIP量1α和咆哮)但显示细胞毒性最小活动;相比之下，CD56dim NK细胞增殖少，产生的细胞因子相对较少，细胞毒性强(Freud & Caligiuri, 2006)。最近的实验证据表明，NK的发展是从CD56bright向CD56dim表型发展(Chan et al.， 2007;Romagnani等，2007;Yu et al.， 2009)。随着我们对NK细胞生物学认识的提高，我们越来越清楚地认识到，来自细胞表面受体的抑制信号和激活信号之间的平衡决定了它们的反应。当刺激信号超过抑制信号达到临界阈值时，NK细胞会做出反应，通过穿孔蛋白/颗粒酶或死亡受体(Fas, TRAIL)相关通路杀死某些受感染或转化的细胞，并产生影响宿主免疫反应的细胞因子(Vivier et al.， 2008)。在免疫自我监测的正常情况下，NK细胞具有识别MHC类‐I分子为同源配体的抑制受体;这些受体包括杀手免疫球蛋白样受体(KIR)、LIR‐1/ILT‐2、CD94/NKG2A异二聚体受体，以及LAIR‐1，后者识别胶原为配体(Lebbink et al.， 2006)。然而，细胞溶解并不是因为MHC类‐I分子的简单缺失，而是需要一个激活受体。其中几件物品已经被描述,包括NKG2C NKG2D,自然细胞毒性受体(NKp30、NKp44和NKp46),和NKp80(Vivier & Anfossi,2004;波蒂诺等人，2005;尼尔,2005;Bryceson等，2006)。
NK cells are the first line of defense against infections and developing malignancies. These cells are heterogeneous and differ in their proliferative potential, homing characteristics, functional capacities, and responses to different cytokines. They can be divided into two major subsets based on their relative density of CD56 surface expression. Peripheral blood NK cells are comprised of around 10% CD56brigh and 90% CD56dim NK cell subsets, which are relatively distinct (Caligiuri, 2008); upon activation, CD56bright NK cells proliferate, and produce a wide range of cytokines (e.g. IFN‐γ, TNF‐β, and IL‐10) and chemokines (e.g. MIP‐1α and RANTES) but display minimal cytotoxicity activity; in contrast, CD56dim NK cells have little proliferation, produce relatively lower amounts of cytokines, and are highly cytotoxic (Freud & Caligiuri, 2006). Recent experimental evidences have demonstrated that NK development proceeds from a CD56bright to CD56dim phenotype (Chan et al., 2007; Romagnani et al., 2007; Yu et al., 2009). As our understanding of NK cell biology has improved, it has become clear that the balance between inhibitory and activating signals originating from cell‐surface receptors dictates their responses. When stimulatory signals outweigh inhibitory ones by a critical threshold, NK cells respond, killing certain infected or transformed cells via perforin/granzyme or death receptor (Fas, TRAIL)‐related pathways as well as producing cytokines that influence the host’s immune responses (Vivier et al., 2008). Under normal circumstances of immune self‐surveillance, NK cells have inhibitory receptors that recognize MHC class‐I molecules as their cognate ligands; these receptors include killer immunoglobulin‐like receptors (KIR), LIR‐1/ILT‐2, and the CD94/NKG2A heterodimeric receptor, as well as LAIR‐1, which recognized collagen as ligand (Lebbink et al., 2006). However, cytolysis is not because of the simple absence of MHC class‐I molecules but requires an activating receptor. Several of these have been characterized, including NKG2C, NKG2D, the natural cytotoxicity receptors (NKp30, NKp44, and NKp46), and NKp80 (Vivier & Anfossi, 2004; Bottino et al., 2005; Lanier, 2005; Bryceson et al., 2006).
据我们所知，这项研究是对人类NK细胞的第一次广泛分析，从新生儿到健康老年人，其中25人年龄在60至80岁之间，被称为老年人（平均年龄68.8±7.2岁）和超过80岁的30岁，被称为非常老（平均年龄87.1±4.9岁），经过50名成年对照受试者（年龄18-60岁）和20个CB样本。我们评估了这些样品中大量NK受体的表型表达，以及它们在细胞因子刺激之前和之后的功能。我们的研究结果提供了关于NK细胞稳态和动态随时间变化的重要线索。
To our knowledge, this study is the first extensive analysis of NK cells from individuals over the human life span from newborns to healthy old people, 25 of them aged from 60 to 80, referred to as old (mean age 68.8 ± 7.2 years) and 30 older than 80 years, referred to as very old (mean age 87.1 ± 4.9 years), passing by 50 adult control subjects (aged 18–60 years), and 20 CB samples. We assessed the phenotypic expression of a large panel of NK receptors in these samples, as well as their functional capacities before and following cytokine stimulation. Our findings provide important clues about NK cell homeostasis and dynamics over time.
Results 结论
Changes in the proportions of lymphocyte subsets across the life span
在整个生命周期中淋巴细胞亚群比例的变化
流式细胞术检测CD3+ T, CD19+ B,和CD3−CD56+ NK子集脐带血(CB)和血液从老很老的主题以及成人控制。这些淋巴细胞类似于CB子集的频率和成人外周血控制,异常的 CD3+ T 细胞,显著降低(P < 0.001)(图1)。相比之下,这些细胞的子集,绝对值显著增加在CB样本,与成人相比,控制,所述(Thornton et al .,2003)。相比之下，对于这些细胞亚群中的每一个，与成人对照相比，CB样品中的绝对值显着增加（如Thornton等，2003）。 与它们相比，旧受试者B细胞的绝对值显着降低（P <0.05）（图1a），但CD3 + T细胞的频率和绝对值保持在一起，与成人对照组相似（图1b））。 同时，CD3-CD56 + NK细胞的频率显着增加，但仅在非常老的受试者中（P <0.05）（图1c）。
Flow cytometry measured CD3+ T, CD19+ B, and CD3−CD56+ NK subsets in cord blood (CB) and blood from old and very old subjects as well as from adult controls. The frequency of these lymphocyte subsets was similar in CB and peripheral blood from the adult controls, with an exception for the CD3+ T cells, which are significantly decreased (P < 0.001) (Fig. 1). By contrast, for each of these cell subsets, the absolute values were significantly increased in CB samples, when compared to the adult controls, as described (Thornton et al., 2003). In comparison to them, the absolute value of B cells in old subjects was significantly lower (P < 0.05) (Fig. 1a), but the frequency and the absolute values of CD3+ T cells remained together similar to the adult controls (Fig. 1b). Concomitantly, the frequency of CD3−CD56+ NK cells was significantly increased but only in the very old subjects (P < 0.05) (Fig. 1c).
Figure 1: Proportion of lymphocyte subsets. Frequency and absolute values of (a) CD19+B cells, (b) CD3+ T cells, and (c) CD3−CD56+ NK cells from peripheral blood. (d) FACS profile of CD56bright subpopulation of NK cells gated on CD3−CD56+ NK cells. Cells were collected from cord blood (CB, crosses), adult controls (Ctl, squares), old subjects (Old, triangles), and very old subjects (V.Old, diamonds). Horizontal bars represent the median. P values refer to the comparison between the adult controls and either the CB or the old or very old subject groups. *P < 0.05, **P < 0.01, ***P < 0.001.
图一：淋巴细胞亚群的比例。 （a）CD19 + B细胞，（b）CD3 + T细胞和（c）来自外周血的CD3-CD56 + NK细胞的频率和绝对值。 （d）在CD3-CD56 + NK细胞上门控的NK细胞的CD56bright亚群的FACS谱。 从脐带血（CB，杂交），成年对照（Ct1，正方形），老对象（老，三角形）和非常老的对象（V.Old，钻石）收集细胞。 水平条代表中位数。 P值是指成人对照与CB或旧的或非常老的受试者组之间的比较。 * P <0.05，** P <0.01，*** P <0.001。
