The Study Of Unique Features Of Liver NK Cells

Natural killer (NK) cells are an important component of the innate immune system. Unlike T and B cells, NK cells can rapidly respond to tumor cells and viral infected cells without the necessity of priming, mainly through producing various cytokines and direct cytotoxicity to exert effector functions, and thus provide the first line of defense against pathogen invasion. Recently, accumulating studies have found that NK cells are a heterogeneous population, rather than a subset with uniform phenotype and function. The heterogeneity and classification of NK cells have been reported in both human and mice. In mice, according to the tissue-distribution features and maturational stages, NK cells can be classified into distinct NK-cell subsets with different phenotype and function. Categorizing NK cells will help us understand the basic biological properties of NK ceils, and more importantly, it also has profound implications with respect to the clinical application of NK cells.The liver is an immunological organ of great importance in the body. Its special blood supply and anatomical structure need fine adjustment of local immune system to maintain homeostasis, and also result in the predominant innate immunity of the liver. The liver contains various types and a large number of innate immune cells, and NK cells as a component of innate immune cells are also enriched in the liver. Furthermore, the phenotype of liver NK cells is very special. An unusually high frequency of phenotypically immature NK cells exists in the liver, whereas the predominant NK-cell population at other peripheral sites is phenotypically mature. Although the phenomenon has been reported ten years ago, at present, it still lacks the knowledge about the unique NK cells in the liver. Do they develop from the bone marrow (BM) or directly develop from the liver? Do they have unique biological roles within the liver? Which factors can maintain and influence their existence? These questions are not yet addressed, which is due to the limited knowledge about the fundamental phenotypic and functional features of liver-specific NK cells and the lack of a specific marker to define this unique subset. One of the aims of this study is to broaden the knowledge of liver-specific NK cells in phenotype and function, and search for a specific positive marker for this subset.NK cells play important roles in the early resistance to viral infections. In a variety of viral infection models, it has been observed that NK cells in the liver can sharply increase and be activated shortly after stimulation. However, despite revealing this phenomenon, most studies focused on the roles of NK cells in the subsequent antiviral response or the progression of inflammation, whereas little attention has been paid to the underlying mechanism by which NK cells accumulate in the liver at early phase. This is another question we will address in this study.In view of above-mentioned points, our study launches from the following two aspects:I. The features of liver-specific NK cells1. Liver contains relatively high frequency of DX5-NK cells.Firstly, we examined DX5expression on NK cells from liver, spleen, peripheral blood, BM and inguinal lymph node (iLN) of wild-type C57BL/6mice. The results indicate that nearly half of liver NK cells are DX5negative, but more than90%NK cells in other tissues and organs are DX5positive.2. Liver DX5-NK cells are distinct from other NK-cell subsetsWe sorted hepatic DX5-and DX5+NK cells for gene chip analysis, and comparative transcriptome analysis between these two subsets revealed substantial differences. The differentially expressed genes include genes encoding activating/inhibitory receptors, cytokines and receptors, chemokines and receptors, adhesion molecules, cytotoxic effectors, transcription factors and so on, the finding of which may shed new light on the further study of liver-specific NK cells. We also used flow cytometry to compare the expression levels of numerous surface molecules between these two subsets. These data not only confirm the results obtained from gene chip analysis, but also further reveal that liver DX5-NK cells are phenotypically immature. Analysis of functional assays indicates that liver DX5-NK cells have lower capacity in cytotoxicity and producing IFN-y than liver DX5+NK cells.Additionally, we compared the phenotype of liver NK-cell subsets with analogue NK-cell subsets in other organs. The results reveal that DX5-NK cells in the liver are not the same with DX5-NK cells existing in other organs at small amounts. However, DX5+NK cells of the liver phenotypically resembled those of other organs, suggesting that previous findings of unique phenotype of liver NK cells are due to the existence of DX5" NK cells in the liver.3. The migratory and developmental features of liver-specific DX5-NK cells We studied the migratory and developmental features of liver NK-cell subsets by adoptive transfer experiments. We found that the transferred liver DX5" NK cells selectively resided in the recipient liver, maintaining their initial phenotypic features, but donor DX5+NK cells distributed in both liver and spleen of recipient mice. Therefore, we consider that liver-specific DX5-NK cells are liver-resident and will not further differentiate into phenotypically mature NK cells without stimuli; in contrast, DX5+NK cells are highly migratory, distributed all over the body, and represent the majority peripheral NK cells.4. CD49a is a specific marker for liver-specific DX5-NK cellsTranscriptomic analysis reveals that liver DX5-NK cells express CD49a at high levels. By flow cytometry analysis, we further found that liver DX5-NK cells were mostly CD49a positive, but liver DX5+NK cells and NK cells from other organs seldom expressed CD49a. Liver-specific CD49a+NK cells were also identified in RAG-1deficient and BALB/c mice. Collectively, CD49a molecule can be used as a specific marker for liver-specific NK cells.5. The unique function of liver-specific NK cellsIt has been reported that liver NK cells are able to mediate hapten-specific hypersensitivity response, the course of which is dependent on CXCR6, but it remains unclear which NK-cell subset mediate the memory response. Our results show that CXCR6is expressed at high levels on liver-specific NK cells, and liver-specific NK cells have the capacity to mediate hypersensitivity response, whereas the widely distributed NK-cell subset cannot. Moreover, hapten-carrier cells can get into the liver, which facilitates liver-specific NK cells to exert immunological memory function.Conclusion I:The present study systematically describes the phenotypic, functional, migratory and developmental features of liver NK cells, which will facilitate future study of the relation between tissue environment and NK cells. It also provides clues to the developmental pathways of NK cells outside the BM, and supplements our knowledge on the fundamental biological features of NK cells.II. CD62L contributes to the recruitment and maturation of NK cells in the liver during viral infection 1. The expression features of CD62L on resting NK cellsWe examined CD62L expression on NK cells from various tissues and organs of normal C57BL/6mice. The results indicate that the expression levels of CD62L are lowest on hepatic NK cells (less than50%) among the examined tissues and organs, and the second lowest on BM NK cells. In contrast, the majority of NK cells in the spleen, peripheral blood and iLN are CD62L positive. Comparative phenotype analysis between CD62L-and CD62L+NK cells reveals that maturation-associated markers are expressed at relatively higher levels on CD62L+NK cells. We also examined the kinetics of CD62L expression on NK cells at different ages, and found that its expression was acquired and gradually increased along with age. In addition, by adoptive transfer experiments, we found that the transferred CD62L-NK cells could become CD62L positive, but donor CD62L+NK cells were still CD62L positive, indicating that the acquisition the CD62L expression is an irreversible process.Taken together, these results demonstrate that CD62L-and CD62L+NK cells have distinct tissue-distribution properties under normal conditions, and CD62L+NK cells represent a relatively mature NK-cell subset compared to CD62L-NK cells.2. Poly I:C induces the change of hepatic NK cellsWe injected mice with poly I:C to mimic RNA virus infection, and found that NK cells in the liver markedly increased and were activated18hours after injection. Moreover, NK cells in the liver were more mature in phenotype than resting hepatic NK cells. By adoptive transfer experiments, we found that poly I:C stimulation accelerated hepatic NK-cell differentiation from CD62L-to CD62L+, and at the same time prompted peripheral NK-cell recruitment to the liver.3. CD62L is required for poly I:C-induced the change of hepatic NK cellsWe treated mice with CD62L blocking antibodies before poly I:C injection, and found that both accumulation and maturation of NK cells in the liver induced by poly I:C were inhibited, suggesting that poly I:C-induced the change of hepatic NK cells is dependent on CD62L.4. Blockade of CD62L inhibits the change of hepatic NK cells in response to adenovirus infection To assess more broadly whether the above phenomenon and mechanism caused by poly I:C occurs during viral infection, we used DNA virus, namely adenovirus, to infect mice. We consistently found that NK cells in the liver rapidly increased and CD62L expression was significantly upregulated following adenovirus infection. Moreover, blockade of CD62L prior to infection greatly inhibited NK-cell accumulation and maturation in the liver.Conclusion II:The present study demonstrates the change of hepatic NK cells in response to virus or viral mimics. This change is mainly reflected in the sharp increase in quantity and phenotypic maturation, which is highly dependent on CD62L molecule. These findings may provide new insight into the mechanisms of NK-cell involvement in human viral hepatic diseases. Furthermore, we also demonstrate the relation between CD62L expression and NK-cell maturation, and conclude that CD62L expression is gradually acquired by NK cells during NK-cell development. The finding that the conversion of CD62L-to CD62L+NK cells can happen in the organs besides BM, such as liver and spleen, providing an important supplement to the NK-cell development in an "extra-BM" pathway.