【作者】 张彩；

【导师】 田志刚；

【作者基本信息】 山东大学 ， 免疫学， 2004， 博士

【摘要】 目的 NK细胞是天然免疫系统的主要效应细胞,具有广泛的生物学功能,位于机体抵抗肿瘤和病毒感染的第一道防线;不需要预先刺激即可识别并杀伤肿瘤和病毒感染的细胞,同时又能通过早期分泌多种细胞因子(如IFN_γ、GM-CSF和TNF-β)和趋化因子来调节获得性免疫应答,因此,是连接天然免疫和获得性免疫的桥梁。NK细胞及其分泌的细胞因子不仅参与调节天然免疫和获得性免疫,而且参与调节造血系统的功能,在血液系统疾病,尤其是异基因骨髓移植中,在抑制和预防GVHD,促进GVT效应和促进血液和免疫系统重建方面起重要作用。同时,NK细胞对自身免疫性疾病的调节亦引起了人们的重视。因此,尽管关于NK细胞的发育分化和识别功能的研究远远滞后于T细胞和B细胞,近年来,关于NK细胞功能和调节的研究引起了人们极大的兴趣,并有了突飞猛进的发展,成为免疫学研究的一大热点。 肿瘤的免疫监视包括天然免疫监视和获得性免疫监视两大方面,CTL是获得性免疫的代表性效应细胞,其识别与杀伤功能具有高度的特异性,只杀伤表面带有特异性抗原和相应组织相容性复合体的靶细胞,而此过程涉及数种免疫分子及分子伴侣的参与,任一分子的丢失均可导致CTL杀伤功能的丧失,致使肿瘤细胞可以从CTL的免疫监视中逃逸。NK细胞是机体重要的天然效应细胞,在抵抗肿瘤和病毒感染过程中起关键作用。NK细胞的杀伤不受NtC的限制,在T细胞免疫启动之前,就与M_φ一起构成了细胞免疫系统的第一道防线。正常情况下NK细胞和CTL的免疫监视功能相互补充,相互协调,共同抑制肿瘤细胞的发生发展。由于NK细胞在病变早期即能迅速发挥杀瘤效应,因此在肿瘤免疫监视中发挥着举足轻重的作用。更多还原

【Abstract】 Objective NK cells constitute an important component of the innate immune system, providing surveillance against certain viruses, intracellular bacteria and transformed cells. NK cells exert cell-mediated cytotoxicity and act to regulate innate and acquired immune responses through the release of various cytokines ( such as IFNγ, GM-CSF and TNF-β) and chemokines. Therefore, they stand as a bridge between innate and adaptive immune responses. Unlike T cells, NK cells killing of virus-infected or malignant transformed cells do not need pre-sensitization and are independent of a MHC restricted manner, thus take a vital position in both innate and acquired immunity. Therefore, NK cells are now the hot items in immunological researches and are considered as promising reagents in adoptive transferring treatment of multi-cancers especially haematopoietic disorders.NK cells typically kill target cells that do not express self MHC class I molecules. Tumor cells that lose MHC class I antigen expression obviously escape detection by cytotoxic CD8+ T cells. At the same time, they are at a greater risk of elimination by NK cells. However, Tumors have developed different strategies to counteract immune responses mounted by their hosts. Tumors can escape from immune recognition by down-regulation of MHC class I molecules, the expression of Fas-L to kill responsive lymphocytes, the production of immunosuppressive cytokines such as TGF-β. It is of major interest in understanding the ways in which tumors and hosts interact. Understanding tumor escape mechanisms may be the key to successful immunotherapy for cancer.As we know, cytotoxic T lymphocytes (CTL) recognoze MHC class I molecules as well as peptide presented on target cells through TCR and exert cytolytic activity. On the contrary, the susceptibility of tumor targets to natural killing is inversely related to target-cell expression of MHC class I molecules. This observation formed the basis for the’missing-self ’ hypothesis, which proposes that NK cells survey the MHC class I expression by inhibitory receptors on target cells and eliminate cells with aberrant MHC class I expression, In the absence of MHC class I molecules expression (’missing self’), NK cells are released from the negative influence of MHC class I molecules and kill the target.However, the existence of target cells with normal MHC class I expression that are sensitive to NK cells suggests that there may be other mechanisms to regulate NK cell activity. The activating receptors also play a major role and are believed to be necessary for the initial activating of NK cells. The well-characterized example to date is the NKG2D receptor and its ligands. Thus, NK cell effector functions are regulated by integrated signals across the array of stimulatory and inhibitory receptors engaged upon interaction with target cell surface ligands.NKG2D is an activatory immunoreceptor whose expression was first recognized on NK cells, but was subsequently found on CD8+aPT, y5T and Macrophages, making it one of the most widely distributed NK receptors currently described. In human, the NKG2D receptor binds to the stress-inducible polymorphic nonclassical MHC molecules MICA (MHC class I chain-related A), MICB and the MHC class I-related UL16-binding proteins-1, 2, 3 (ULBP-1, 2, 3), which are up-regulated strongly in many types of tumor cells. Recent studies reveal that the expression of MIC and ULBP on human tumor cells is sufficient to overcome the inhibitory effects of MHC class I expression on NK cell killing. These observations indicate that NKG2D provides first line surveillance against stressed or abnormal cells that have been induced to express one of its ligands.Interferon is an important regulator of the immune response to tumors. Type I interferons (IFNa and IFNP) exert many biological functions including antiviral activity, anti-proliferation and anticancer effects. They do not directly kill tumor or infected cells, but exert immunoregulatory functions on immune cells to eliminate transformed or virus infected cells . Type II interferon, immune interferon, IFN-y is a multi-functional cytokine produced by Thl and NK cells. It increases the expression of Fc receptors for IgG on macrophages and PMNs as well as increasing MHC class I and II expression on a wide variety of cells. This enhances the phagocytic function of these cells as well as increasing the antigen presenting capabilities of professional antigen presenting cells . More importantly, IFN-y functions to regulate Thl responses that are critical to CTL responses and IgG antibody production. Also, It is a critical component of the endogenous and manycytokine-induced antitumor immune responses. As well known, IFNa acts as an important regulatory factor to NK cells and can up-regulate the function of NK cell. But the direct effect of IFN-y on NK cells is still unclear although it exerts an important role during the effector stage of NK lysis to tumor or virus-infected cells.In this study, on one hand, we observed the cytotoxicity of three NK cell lines (NK92, NKL and YT) to different tumor cells and the different effects of IFN-y and IFNa on function of NK cells as well as the expression of NK cell receptors, especially the NKG2 family. The aim is to investigate whether cytokines, such as IFN-y and IFNa, can regulate the balance of activating and inhibitory signals provided by NK cell receptors and to find some new strategies to manipulate the expression of NK cell receptors and their ligands and alter the balance of inhibitory and activating signals in favor of activation . On the other hand, we observed the expression of NKG2D ligand-MICA on some tumor cell lines and tumor tissues, and analysed the relation to NK lysis. Also, we created the prokaryotic expression vector pBV220/sMICA by gene engineering technology and obtained a product of rsMICA with a purity of 95%. Then we observed the different effect of membrane MICA (mMICA) and soluble MICA (sMICA) on expression of NKG2D and the influence on NK cell receptor repertoire. The aim is to explore the the anti-tumor mechanisms of NK cells ahd the meaning of the expression of MICA on mechanisms of tumor escape from immune surveillance.Methods (1) The cytotoxicity and ability of proliferation of human NK cells were detected by MTT method. Human freshly-isolated NK cells were obtained from healthy volunteers by isolating peripheral blood mononuclear cells followed by MACS isolation. (2) The expression of NK cell receptors ( NKG2D, NKG2A/B, KIR2DL1 and KIR2DS1) was measured by RT-PCR or by FACS. The expression of MICA was measured by RT-PCR, or Immunohistochemical method (SABC technique). Cytotoxic molecules levels such as TRAIL, Perforin and FasL were assayed by RT-PCR, P-actin was amplified as a control. (3) sMICA cDNA was obtained from human cervical carcinoma cell line Hela by RT-PCR method. After sequencing, the prokaryotic expression vector pBV220/sMICA was created by gene engineering technology. The expression of sMICA in E.coli was analyzed by SDS-PAGE.Results1. The cytolitic ability of three NK cell lines to tumor cellsAmong three NK cell lines, NK92 exerts highest cytotoxicity. NK92 and NKL express NKG2D and NKG2A as well as CD94, but YT doesn’t. Both NK92 and NKL cells exert higher cytotoxicity to tumor cells with MICA expression, while tumors without MICA expression do resist against NK lysis.2. The effect of IFN-a and IFN-y on NK lysisIFN-a can promote the cytotoxicity of NK92 and NKL to nearly all tumor cells except PG cells. IFN-y (>1000u/ml) inhibited the NK lysis to tumor cells with MICA expression and lowered the proliferation of NK cells slightly. While the inhibitory effect to tumor cells without MICA expression was not found.3. The effect of IFN-a and IFN-y on expression of NK cell receptorsIFN-a enhanced the mRNA expression of activting receptor NKG2D, but down-regulated the mRNA expression of inhibitory receptors NKG2A and KIR2DL1.While IFN-y inhibited the gene expression of NKG2D, but up-regulated the gene expression of NKG2A and KIR2DL1. These results were confirmed by FACS that IFN-a and IFN-y exert opposite effect on expression of NKG2D and NKG2A to NK cell lines (NK92 and NKL) and to freshly isolated peripheral blood NK cells. Meanwhile, we found IFN-a and IFN-y have the same effect on expression of NKG2D to freshly isolated peripheral blood T cells.4. The expression of MICA in tumor cell lines and tumor tissuesThe expression of MICA mRNA and protein was found in most tumor cell lines and tumor tissues, but was not found in paracancer specimens.5. Construction of the vector pBV220/sMICA and expression in E.coli of rsMICA Both the vector pBV220 and the sMICA fragment were degested with EcoR I and BamHI restriction endonucleases. Purified fragment was cloned into corresponding sites of the pBV220 vector. Then, the recombinant vector was transformed to E.coli DH5a. The positive clones were screened by PCR, identified the sMICA cDNA by enzymes digestion and sequenced. Expression of sMICA was induced with 42°C hot-induction for 3.5h. SDS-PAGE analysis suggested that the quantities of sMICA was about 20%. The purity of sMICA was 95% after purified by RP-HPLC.6. Changs of NK cell receptors on NK cells when contact occurred between NK cellsand tumor cellsWhen NK92 cells were cocultured with MICA+ tumor cells (Hep2 and Hela) for 30min, the mRNA expression of NKG2A and KIR2DL1 was weakened obviously. Whencocultured for lh, the expression of NKG2D mRNA was increased significantly and this effect sustained to 21h. At 24h, NKG2D mRNA was lowered. The changes of NKG2D expression were also found on NK92 by FACS. No similar changes were found when cocultured with MICA" tumor cell (PG).7. Changs of Cytotoxic molecules levels on NK cells when contact occurred between NK cells and tumor cellsWhen NK92 cells were cocultured with MICA+ tumor cells (Hep2) for 30min, the mRNA expression of Cytotoxic molecules TRAIL and Perform was increased. This effect was significant when cocultured for lh and 3h, and was weakened for 24h. The expression of FasL in NK92 cells enhanced when cocultured with Hep2 for 3h, but decreased for 24h.8. The effect of rsMICA on expression of NK cell receptors and on NK lysisrsMICA ( lpg/ml or 5ug/ml) down-regulated the mRNA expression of NKG2D, NKG2A/B and KIR2DL1 when cocultured for 24h and the effect was especially significant on NKG2D expression. Further, we found that the cytotoxicity of NK92 to MICA+ tumors reduced significantly after cocultured with rsMICA for 24h, while no changes were found to MICA" tumor cells. Conclusions1. IFN-a promotes NK lysis through up-regulating the expression of activating receptor NKG2D and down-regulating the expression of inhibitory receptors NKG2A and KIR2DL1.2. IFN-y has an opposite effect on expression of NK cell receptors and has a negative effect on activation and cytotoxicity of human NK cells through altering the balance of activating and inhibitory receptors on NK cells in favor of inhibition, which may serve to limite NK cell activation in vivo.3. mMICA and rMICA exert opposite effect on expression of NKG2D and other NK cell receptors. mMICA can enhance the cytotoxicity of NK cells against tumor through up-regulating the expression of NKG2D. While sMICA down-regulates the immune response of NK cells through reducing the expression of NKG2D, which may promote tumor immune evasion.4. During the efector stage of NK lysis, the effect of TRAIL and Perforin was predominant.We postulated theories for the first time that "IFN-a and IFN-y exert opposite effect on NK lysis especially on NKG2 family" and that "the balance of mMICA and sMICA更多还原