University of Southampton, Southampton, UK.
Southampton University Hospital, Southampton, UK.
Lancet. 2015 Feb 26;385 Suppl 1:S45. doi: 10.1016/S0140-6736(15)60360-9.
Selected populations of murine natural killer (NK) cells possess memory features to haptens, cytokines, and viruses. Liver-specific adhesion molecules CXCR6 and CD49a have been identified as surface markers in mice. In people, expansion of long-lived terminally differentiated NKG2C+ populations occur in the blood after viral infection. We aimed to compare intrahepatic and blood NK cell receptor expression to determine the existence of CD49a+ and CXCR6+ NK cells in human liver and define the maturation status of NKG2C+ NK cells at this site.
Tissue samples were taken from the liver margin of 39 patients with hepatic metastases and flushed with chelating buffer followed by collagenase or mechanical digestion. Paired peripheral blood samples were taken from 15 patients, the remainder being unpaired. Mononuclear cells were isolated by ficoll separation and cell surface staining performed for CD3, CD56, CD16, CD57, CD117, CD161, CD158a, CD158b, CD49a, CD49b, CXCR6, NKG2C, and NKp46. Statistical analysis to compare intrahepatic and blood NK cell receptor expression included the median, IQR, and Mann-Whitney U test.
Frequencies of NK cell precursors were similar in the liver and the blood (0·91% [0·62-3·26] vs 0·87 [0·41-1·52]); however, expression of all later markers of maturity were reduced including CD16 (47% [40·4-61·4] vs 88·7 [82·2-93·2], p<0·0001), CD57 (30·7% [25·0-53·9] vs 73·4 [70·4-87·6], p=0·0003), and KIR (11·2% [7·5-14·5] vs 26·7 [17·3-30·8], p<0·0001). Expanded hepatic CD16- NK cells were particularly immature with reduced CD57 and increased CD161 compared with the blood. NKG2C+ NK cells were found in similar frequencies in liver and blood. The hepatic NKG2C+ population was terminally differentiated, as in the circulation, but demonstrated a three-fold increase in KIR expression compared with NKG2C- counterparts, which was not seen in the blood. As in previously published research in mice, CD49a+ and CXCR6+ NK cells were liver resident (6·5% [3·9-14·6] liver vs 2·1 [1·3-4·3] blood, p<0·0001, and 65·3 [48·1-75·2] vs 4·5 [1·43-12·12], p=0·0039, respectively). Both populations were immature, with reduced KIR expression.
We have shown that the liver contains an expanded population of immature CD16- NK cells. These cells might traffic from the blood and then differentiate into hepatic-specific CD49a+ and CXCR6+ NK cells. The function of these subsets is unknown but their immaturity hints against memory. Terminally differentiated NKG2C+ cells show KIR expansion in the human liver and probably represent an antigen-experienced population, raising the question of whether the liver is a site of NK cell memory acquisition.
MRC Clinical Research Fellowship.
已鉴定出小鼠天然杀伤 (NK) 细胞中的某些群体具有对半抗原、细胞因子和病毒的记忆特征。CXCR6 和 CD49a 等肝脏特异性黏附分子已被鉴定为小鼠的表面标志物。在人类中，病毒感染后血液中会出现长寿命终末分化的 NKG2C+群体的扩增。我们旨在比较肝内和血液 NK 细胞受体表达，以确定人类肝脏中是否存在 CD49a+ 和 CXCR6+NK 细胞，并确定该部位 NKG2C+NK 细胞的成熟状态。
从 39 例肝转移患者的肝边缘取组织样本，并用螯合剂缓冲液冲洗，然后用胶原酶或机械消化。从 15 例患者中采集配对的外周血样本，其余为非配对样本。通过 ficoll 分离分离单核细胞，并进行 CD3、CD56、CD16、CD57、CD117、CD161、CD158a、CD158b、CD49a、CD49b、CXCR6、NKG2C 和 NKp46 的细胞表面染色。比较肝内和血液 NK 细胞受体表达的统计分析包括中位数、IQR 和 Mann-Whitney U 检验。
肝内和血液中的 NK 细胞前体频率相似（0·91% [0·62-3·26] 比 0·87% [0·41-1·52]）；然而，所有成熟后期的标志物表达均降低，包括 CD16（47% [40·4-61·4] 比 88·7% [82·2-93·2]，p<0·0001）、CD57（30·7% [25·0-53·9] 比 73·4% [70·4-87·6]，p=0·0003）和 KIR（11·2% [7·5-14·5] 比 26·7% [17·3-30·8]，p<0·0001）。与血液相比，肝内 CD16- NK 细胞特别不成熟，CD57 和 CD161 减少。在肝脏和血液中发现了相似频率的 NKG2C+NK 细胞。肝内 NKG2C+群体为终末分化，与循环中相同，但与 NKG2C- 相比，其 KIR 表达增加了三倍，而在血液中则没有。与之前在小鼠中发表的研究一样，CD49a+和 CXCR6+NK 细胞是肝脏驻留细胞（6·5% [3·9-14·6] 肝比 2·1% [1·3-4·3] 血，p<0·0001，和 65·3% [48·1-75·2] 比 4·5% [1·43-12·12]，p=0·0039）。这两个群体都不成熟，KIR 表达减少。
我们已经表明，肝脏中含有一个扩展的不成熟 CD16-NK 细胞群体。这些细胞可能从血液中迁移而来，然后分化为肝脏特异性的 CD49a+和 CXCR6+NK 细胞。这些亚群的功能尚不清楚，但它们的不成熟表明它们没有记忆。终末分化的 NKG2C+细胞在人类肝脏中表现出 KIR 扩增，可能代表一个抗原经验丰富的群体，这就提出了肝脏是否是 NK 细胞记忆获得的部位的问题。
MRC 临床研究奖学金。
