【作者】 姜晓君；

【导师】 田志刚；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2013， 博士

【摘要】 肝脏的免疫系统别具特色。这不仅因为双重血供使肝脏直接暴露于胃肠道来源的免疫刺激物中,更体现在肝脏免疫细胞的独特组成上。肝窦特殊的结构有利于选择性驻留循环血液中的某些免疫细胞。同时,肝脏的自身造血作用也可能参与改变肝脏免疫细胞的组成。在个体发育的过程中,胚胎肝脏是造血发生的场所之一。虽然出生后造血干细胞会由胎肝转移到骨髓,但是资料显示,成年肝脏仍然具有造血功能。因此,成年肝脏独立于骨髓的造血作用如何影响肝脏免疫细胞的构成是值得探索的科学问题。肝脏中天然免疫系统处于优势状态,除了存在大量具有吞噬功能的细胞外,肝脏NK细胞和NKT细胞的比例也远远高于全身其它脏器。天然免疫细胞在肝脏富集的机制,以及它们与其它脏器细胞的差别,都引起了学界广泛的关注。肝脏中NK细胞的表型与其他脏器存在明显不同。据报道,肝脏含有大量DX5-表型不成熟的NK细胞。尽管分泌细胞因子的能力和部分细胞毒作用弱于经典DX5+NK细胞,它们可以通过TRAIL相关的方式起杀伤作用。此外,在半抗原介导的过敏性皮炎和部分病毒感染模型中,记忆NK细胞特异性驻留于肝脏。这些现象都表明肝脏NK细胞具有与经典NK细胞不同的特性,暗示着肝脏中DX5NK细胞可能并非处于NK细胞发育的不成熟阶段,而是区别于骨髓发育的NK细胞的另一个亚群。针对以上切入点,本项研究工作分为两大部分：I.成年肝脏造血免疫系统发育分化的研究1.成年肝脏中含有造血干细胞通过流式检测小鼠肝脏单个核细胞,可以发现成年肝脏中存在造血干细胞表型的细胞。转输肝脏单个核细胞给致死照射后的小鼠,也可以避免小鼠死亡。因此成年肝脏确实具有造血能力。2.肝脏单个核细胞转输重建T细胞为主的免疫系统肝脏单个核细胞转输后,受者体内髓系系统和淋巴系统都逐渐恢复,但恢复速度比骨髓转输要慢。特别有趣的是,在肝脏单个核细胞转输早期,受者体内重建出以T细胞为主的免疫系统,B细胞和NK细胞比例极低,而骨髓重建的受体中是以B细胞为主的免疫系统。3.肝脏造血前体细胞具有与骨髓细胞类似的淋巴细胞生成能力纯化肝脏造血前体细胞转输,证实了肝脏造血前体细胞确实具有与骨髓造血干细胞类似的淋巴细胞生成能力。与肝脏单个核细胞转输的结果不同,肝脏造血前体细胞先重建出B细胞而后再产生T细胞。4.肝脏成熟T细胞参与受体免疫系统的重建通过纯化肝脏成熟的T细胞转输,我们发现肝脏T细胞在受者中优先向肝脏归巢,并且能长期存活,维持稳定的细胞群体。此外,转输后扩增的T细胞主要为具记忆表型的CD8T细胞。结论I：成年肝脏中的造血前体细胞以及部分成熟的T细胞都能够产生并维持特定的淋巴细胞群体。肝脏造血前体细胞具有与骨髓造血干细胞类似的T、B细胞生成能力,但两者生成NK细胞的能力差别较大。肝脏驻留的记忆性CD8T细胞在转输后会在受者中大量扩增并优先向肝脏归巢。Ⅱ.肝脏特有NK细胞亚群的研究1.肝脏中存在特有NK细胞群体DX5-NK细胞约占肝脏NK细胞一半左右,而在其它脏器中比例很低。肝脏DX5-NK细胞与骨髓中不成熟的NK细胞存在表型上的差别。利用转输实验,我们也证实了这群细胞能稳定维持群体,不向DX5+NK细胞转化。因此它们是区别于经典NK细胞的特殊亚群,而非不成熟NK细胞。此外,基因芯片结果筛选出CD49a可以作为该亚群的特有标志。2.肝脏特有NK细胞具有介导记忆反应的能力在半抗原诱导的过敏性皮炎模型中,我们发现致敏后小鼠肝脏CD49a+DX5-NK细胞可以向未经处理的naive小鼠传递抗原特异的免疫记忆,而CD49a-DX5+NK细胞不能。3.肝脏特有NK细胞驻留于肝脏并在肝脏局部获得记忆功能通过转输实验,我们证明了肝脏中驻留的CD49a+DX5-NK细胞天生就具有产生免疫记忆的潜能,该种能力是经典的CD49a-DX5+NK细胞所不具备的。利用FITC作为致敏剂追踪半抗原在体内的分布,可以看到半抗原会被肝实质细胞摄入,同时也观察到荷载半抗原的DC和单核细胞停留在肝脏中,为NK细胞在肝脏局部产生免疫记忆提供了物质基础。4.肝脏特有NK细胞的发育不依赖于骨髓骨髓转输能够迅速补充受者体内CD49a-DX5+NK细胞,却不能有效重建肝脏特有CD49a+DX5-NK细胞亚群,提示两者具有不同的发育路径。肝脏造血前体细胞转输实验证实其具有向肝脏特有NK细胞分化的能力。结论Ⅱ：区别其他脏器,肝脏中存在一群特有的CD49a+DX5-NK细胞。该群细胞特异性驻留于肝脏,不参与血液循环,在半抗原致敏后能够获得抗原特异性的免疫记忆。此外,肝脏特有NK细胞群体由肝脏中的前体细胞分化而来,其发育不依赖于骨髓。更多还原

【Abstract】 Liver is a unique immunological site, to which dual blood supply brings immune stimulants from gastrointestinal tract, and features by complex repertoires of immune cells capable of effective defense. The structural organization of sinusoids may selectively retain immune cells from circulating blood. Meanwhile, hematopoiesis of adult liver also probably influences the inclusion of hepatic immune system. Fetal liver is one of the hematopoietic places during ontogeny. Though HSCs is generally considered to migrate from fetal liver to BM, the hematopoietic capacity of liver is still found in adults. Thus, the contribution of liver hematopoiesis to liver immune system is of great interest.The liver is an organ with predominant innate immunity. Besides large population of cells with phagocytosis, NK and NKT cells are in high frequency comparing liver with other organs. The mechanism underlying innate immune domination as well as the different between innate cells in liver and other organs attracts many attentions. Different phenotypes were described in liver NK cells and NK cells in other organs. It has been reported that nearly half of liver NK cells possessed DX5" immature profile, with a weaker secretion of cytokines and cytotoxicity than DX5+classical NK cells, but could kill target cells by TRAIL related pathway. Moreover, memory NK cells in hapten induced contact hypersensitivity and some virus infections were liver resident, implying liver DX5" NK cells might be another NK cell subset rather than a stage during NK cell development.In view of the above point, this study contains the following two aspects:I. Liver lymphopoiesis in adult mice1. Hematopoietic progenitors existed in adult liver.We analyzed the surface marker on liver mononuclear cells (MNCs) and found cells with the phenotypes of hematopoietic progenitors (HPCs) in them. Further study showed that liver MNCs transfer could rescue lethally irradiated mice, demonstrating hematopoietic capacity of adult liver.2. Liver MNCs transfer rebuilt immune system predominated by T cells.Myeloid and lymphoid lineages were slowly rebuilt after liver MNCs transfer. Interestingly, CD3+CD19-T cells were major population in recipients3weeks after liver MNCs transfer, while CD3-CD19+B cells were major population in BM transfer groups.3. Liver MNCs contained HPCs functionally similar to BM cells.Purified liver HPCs similarly reconstituted T and B cells as BM cells. Contrast to T cell predominance after liver MNCs transfer, liver HPCs regenerated T cells later than B cells.4. Liver MNCs contained liver-resident memory CD8+T cells.Hepatic mature T cells recruited to the liver after transfer. They proliferated and maintained population for long time. Most of them were CD8T cells with memory phenotypes.Conclusion Ⅰ:Adult liver contained both HPCs and T cells with lymphopoietic capacity. T and B cells were similarly reconstituted by HPCs in the liver and BM, but NK cell recovery was quite different. Liver resident memory CD8T cells also rapidly proliferated and long-lived after transfer with liver specific retention.Ⅱ. The features of liver unique NK cells1. Liver contained a unique NK cell subset.Nearly half of hepatic NK1.1+NK cells were DX5-NK cells, which were rare in other organs and phenotypically distinct from BM immature NK cells. They maintained DX5-phenotype and did not convert to DX5+NK cells after adoptive transfer, implying they were NK cell subset different from classical NK cells rather than immature NK cells. Besides, we found CD49a was a specific marker for them by genechip.2. Hepatic unique NK cells conferred CHS responses.In hapten induced contact hypersensitivity (CHS), CD49a+DX5-NK cells isolated from sensitized mice liver delivered antigen specific memory to naive mice, while CD49a DX5+NK cells could not.3. Memory NK cells were liver resident and priming in the liver. Liver unique NK cells naturally possessed memory capacity. They resided in the liver and acquired memory ability after hapten sensitization. We traced hapten delivery by FITC, and found hepatocytes could absorb haptens after sensitization. Monocytes and DCs also took part in this process. Thus, liver might be a site where naive NK cells were primed.4. Liver-resident HPCs differentiated into CD49a+DX5-NK cells.Very few DX5-NK cells were detected in the livers of BM transplanted mice, suggesting that DX5-NK cells were not predominantly derived from BM. We transferred purified hepatic precursors and found they could give rise to liver unique NK cells.Conclusion Ⅱ:We identified a unique NK cell subset in the liver characterized by the CD49a+DX5-phenotype. This subset possessed memory potential and conferred hapten-specific CHS responses upon hapten challenge. Importantly, CD49a+DX5-NK cells were liver resident and appeared to originate from hepatic HPCs but not from the BM.更多还原