乳鼠人肝癌异种移植及其细胞免疫排斥机制的研究
摘要
背景和目的
原发性肝细胞癌(HCC)是世界三大癌症相关死亡之一,是最常见的预后较差的肝脏恶性肿瘤。目前研究者建立了各种肿瘤异种移植模型用于人肝癌生物学和化疗研究,但由于其采用的模型动物主要是免疫缺陷鼠或诱导免疫耐受鼠,致使以此模型进行肿瘤免疫治疗的研究受到限制。因而构建免疫力正常的异种肿瘤移植模型,研究肿瘤与宿主的相互作用将有助于我们更好地理解肿瘤的免疫治疗。然而早先的研究证实肿瘤异种移植物仅可以生长于一些免疫豁免位点,如大鼠的颊囊、眼前房,或环孢霉素A处理的小鼠或大鼠的肾囊下生长。异种肿瘤移植物在免疫力正常宿主体内成功存活的主要障碍被认为是T细胞介导的免疫排斥及组织微环境。T细胞介导的异种免疫排斥的重要性已在裸鼠模型中得到证实,而异种肿瘤移植物与组织微环境的关系研究较少。
肝脏作为人肝癌细胞移植的常见位点,近年被公认为一个复杂的免疫与耐受器官。它富含天然免疫细胞,如NK、NKT、枯否细胞和树突状细胞,尤其是NKT细胞,近年被称为免疫系统的瑞士军刀,其活化后可激活各类免疫细胞参与多种免疫反应。此外,经典CD4~+T和CD8~+T细胞在肝脏中的比例也与外周血相反。所有这些天然免疫和获得性免疫细胞决定着肝脏微环境的肿瘤监视,但它们在肿瘤异种移植物存活中所扮演的角色却知之甚少。
本研究尝试在免疫力正常的乳鼠肝脏内移植入人的肝癌细胞来建立人肝癌—小鼠动物研究模型。其理论依据为:①乳鼠(约出生后0～15d)肝内高表达的甲胎蛋白(alpha-fetaprotein,AFP)具有抑制机体抗肿瘤免疫的生物学特性;②乳鼠胸腺尚未发育成熟,外周免疫系统尚未完善,免疫排斥较弱;③选择“免疫特赦器官”—肝脏作为荷瘤器官,易于诱导免疫耐受。在前期的实验中,我们首次将人肝癌细胞HepG2细胞原位移植到具正常免疫力的昆明种乳鼠肝内,构建了人肝癌HepG2移植性小鼠模型。虽然我们证实在免疫力正常的乳鼠肝内移植人HepG2细胞较成鼠成功率高,移植瘤细胞存活时间较长,但是随着乳鼠发育免疫系统的逐渐完善,肝内异种免疫排斥反应增强,肿瘤细胞在移植后7～9天开始坏死。肿瘤细胞在乳鼠肝内遭遇免疫排斥,何种免疫细胞参与了这一过程?肿瘤细胞在肝内被清除,肝内的免疫微环境发生了怎样的变化?尤其是数量丰富的NKT细胞的活化和功能有着怎样的改变?分化程度不同的异种肿瘤细胞,在乳鼠肝内的命运是否一样?CsA免疫抑制的荷瘤乳鼠肿瘤组织病理切片中并未发现淋巴细胞的浸润和肿瘤细胞的坏死,其对细胞介导的异种免疫排斥有着怎样的影响?
围绕这些问题,本研究将生长特性不同的人肝癌细胞株(HepG2和HCCLM3)移植到免疫力正常的乳鼠体内,探讨人肝癌细胞在乳鼠肝内的存活情况、肝内细胞介导的免疫排斥反应、NKT细胞活化及功能的改变。为更好地阐明这一点,我们同时采用成鼠及低剂量CsA处理的乳鼠和成鼠做移植受体,进行对比研究。本研究为HCC异种模型的建立提供了一种新的尝试,为NKT细胞介导的肿瘤异种排斥呈现了一个新的视角。而且它有助于更好地了解CsA的免疫抑制机制,无论是在临床还是动物模型的研究中。
研究方法
1、DMAHAS体外标记细胞与细胞毒性分析
1)DMAHAS标记人肝癌HepG2细胞并荧光成像;
2)DMAHAS标记细胞荧光释放分析;
3)MTT和中性红法检测DMAHAS处理HepG2细胞的活力;
4)考马斯亮蓝法测定细胞总蛋白;
5)PI流式细胞术检测细胞周期与凋亡;
2、乳鼠和成鼠人肝癌原位移植
1)肿瘤细胞培养与体外DMAHAS标记;
2)低剂量CsA(10 mg/kg)处理小鼠及其血清浓度检测;
3)人肝癌细胞小鼠肝内原位移植;
4)异种移植物组织冰冻切片分析及荧光成像;
5)异种移植物组织常规HE染色及病理分析;
3、荷瘤乳鼠及成鼠不同细胞免疫排斥机理的研究
1)人肝癌细胞HCCLM3小鼠肝内原位移植;
2)低剂量CsA(10 mg/kg)小鼠皮下注射;
3)小鼠肝脾淋巴细胞分离和计数;
4)DMAHAS荧光释放法检测CTL杀伤活性;
5)荷瘤乳鼠血清肿瘤细胞特异性抗体检测;
6)荷瘤小鼠肝脾淋巴细胞流式细胞术免疫表型分析;
4、荷瘤小鼠NKT细胞活化及功能的研究
1)人肝癌细胞HCCLM3小鼠肝内原位移植;
2)低剂量CsA(10 mg/kg)小鼠皮下注射;
3)分离小鼠肝脾淋巴细胞并计数;
4)乳酸脱氢酶释放法检测NKT细胞杀伤活性;
5)肝脾淋巴细胞NKT活化与CD1d表达分析;
6)血清及肝脏组织IL-4和IFN-γELISA定量检测;
7)体外免疫细胞特异性清除;
8)LDH释放法测定CTL活性。
研究结果
第一部分
DMAHAS体外标记细胞成像
DMAHAS可在30分钟内快速标记活细胞或多聚甲醛固定的细胞,最适工作浓度为5μM。DMAHAS定位于细胞质,当受到紫外或长波长光(800nm)激发时,可自发地发射蓝色荧光。
DMAHAS体外标记细胞与细胞毒性分析
采用工作浓度5μM的DMAHAS标记HepG2细胞培养24小时进行MTT、NR、CB和PI流式细胞术检测表明DMAHAS对标记细胞无显著影响(P＞0.05)。
DMAHAS标记肿瘤细胞体内示踪和荧光成像
DMAHAS标记的人肝癌HepG2和HCCLM3细胞可在受体鼠肝内存活4周以上,移植肿瘤细胞随时间延长逐渐减少,在移植后六周受体鼠肝内未检测到肿瘤细胞或荧光信号。而且检测过程中未见DMAHAS荧光信号的衰减。
乳鼠肝脏移植瘤组织病理分析
人肝癌HCCLM3细胞原位移植于乳鼠肝内后,第8天左右肝内正常组织出现淋巴细胞的募集,移植瘤周围出现淋巴细胞浸润,无明显的炎性细胞如中性粒细胞或巨噬细胞介入。部分肿瘤移植物仍可存活4周以上,未见明显的肝内转移。低剂量CsA处理可有效减少淋巴细胞浸润,延长肿瘤细胞存活期至5周以上。
第二部分
荷瘤小鼠肝脾淋巴细胞变化
荷瘤乳鼠肝脏淋巴细胞数量于移植后的10天和16天增加(P＜0.05),同时脾脏淋巴细胞数量减少,同样的结果可见于移植后4天、8天和10天的成鼠。并且荷瘤小鼠脾脏或肝脏淋巴细胞未见明显凋亡。
荷瘤小鼠肝脾淋巴细胞免疫分析
移植后第16天,乳鼠肝脏CD4~+T细胞绝对计数和百分比显著增加(P＜0.01),NK和CD8~+T细胞数量绝对计数在肝脏内增加,而在脾脏中减少(P＜0.05)。成鼠于移植后2天即见CD8~+T细胞在肝内的增加与脾内的减少。
荷瘤小鼠脾脏淋巴细胞杀伤功能检测
荷瘤16天的乳鼠脾脏淋巴细胞在10:1,20:1和50:1效靶比时对HCCLM3细胞的杀伤显著增加(P＜0.05)。
低剂量CsA对移植瘤存活及其引发的免疫反应的影响
低剂量CsA处理可显著地减少乳鼠肝内淋巴细胞侵润,延长肿瘤移植物存活期至5周以上。然而其对移植物在成鼠肝内的存活无明显影响。除对CD4~+和CD8~+细胞有抑制效果外,低剂量CsA可有效地抑制NKT细胞的活化。此外,低剂量CsA处理可有效抑制荷瘤乳鼠脾脏效应细胞的功能。
第三部分
HCCLM3异种移植物诱导的肝脏NKT细胞活化
HCCLM3移植于3日龄乳鼠肝内后,其肝脏NKT细胞NK1.1分子在移植后5天和10天表达丢失。16天时表达增加并伴随NKT细胞活性增强。成鼠在移植后2天和4天可见NK1.1分子的大量丢失,并于10天时恢复至正常水平。脾脏NK/NKT细胞表面NK1.1分子的表达在移植前后无显著变化。
荷瘤小鼠肝脏和脾脏淋巴细胞CD1d表达
乳鼠在移植后10天时肝脏NK1.1~+细胞表面CD1d的表达开始增加,随后CD1d的表达上调可见于移植后16天和28天肝脏CD3~+T和NK1.1~+细胞。类似的结果可见于移植后4天和10天时的成鼠肝脾淋巴细胞。
荷瘤小鼠血清IL-4和IFN-γ检测
血清取自荷瘤10天和16天的乳鼠或4天和8天的成鼠。IFN-γ的水平在移植后的乳鼠和成鼠血清中均有增加(P＜0.05),而IL-4水平的升高仅见于移植后10天的乳鼠和4天的成鼠血清(P＜0.05)。
低剂量CsA对NKT细胞活化和功能的影响
低剂量CsA处理可显著抑制荷瘤乳鼠肝脏NKT细胞的功能(P＜0.05)及移植2天时的成鼠NKT细胞活化。此外低剂量CsA可显著抑制受体鼠血清IL-4和IFN-γ的分泌,而并未显著下调肝脾T细胞或NK1.1~+T细胞表面CD1d的表达。
荷瘤乳鼠淋巴细胞体外免疫清除CTL活性分析
体外特异性清除荷瘤乳鼠淋巴细胞中的CD3或CD8细胞,可显著降低CTL细胞的杀伤活性(P＜0.05);而特异性清除CD4或NK1.1~+细胞却无明显影响。
研究结论
1.首次应用新型双光子荧光染料DMAHAS标记人肝癌细胞及荧光成像研究,并初次证实其对标记细胞的活力、总蛋白合成和细胞周期与凋亡无显著影响。体内研究表明DMAHAS可作为一种安全可靠的探针用于肿瘤细胞示踪和荧光成像研究。
2.初步证实乳鼠肝脏较成鼠肝脏更适宜于异种肿瘤细胞的存活与生长。
3.淋巴细胞在荷瘤小鼠脾脏中的减少及肝脏中的增加暗示脾脏淋巴细胞以某种方式参与了肝脏异种肿瘤移植物的免疫排斥反应。
4.乳鼠和成鼠肝内发生了对肿瘤移植物不同的细胞免疫排斥反应。参与乳鼠肿瘤延迟排斥反应的免疫细胞主要是CD4~+T、NK1.1~+细胞及CD8~+T细胞,而参与成鼠肿瘤急性排斥反应的免疫细胞主要是CD8~+T细胞、NK1.1~+细胞及大量的炎性细胞,如中性粒细胞或巨噬细胞。
5.肝脏NKT细胞在异种肿瘤的排斥反应早期活化、功能及活性增强,表明其在排斥反应的早期起着重要作用。
6.CD1d分子在荷瘤小鼠肝脏淋巴细胞不同时期表达的增加,表明其参与了异种肿瘤抗原的免疫呈递过程及异种移植物的免疫清除。
7.低剂量CsA处理可显著地抑制乳鼠的免疫排斥反应,延长肿瘤移植物存活期,然而其对成鼠肝内肿瘤移植物存活无明显影响。
8.除对CD4~+和CD8~+T细胞功能有抑制作用外,低剂量CsA可有效抑制肝脏NKT细胞的活化及功能,而且这种抑制方式可能不依赖于CD1d。
Background and objective
Hepatocellular carcinoma(HCC),the third most common cause of cancer-related death worldwide,is the most frequent hepatic tumor with poor prognosis.Tumor xenograft models are widely used for human tumor biology and chemotherapy analysis, but they may be insufficient for studying tumor immunotherapy because most of them are established in immunodeficient hosts.Studies to elucidate the immune mechanisms of tumor-host interactions in immunocompetent tumor xenograft models will benefit our understanding of tumor immunotherapy.Previous studies have demonstrated that tumor xenografts can grow in some immunologically privileged sites,such as the anterior chamber of the eye,the hamster cheek pouch,or under the kidney capsule of mice or rats with limited cyclosporine A treatment.The potential barriers to successful xenograft survival are believed to be T cell mediated xenorejection and tissue microenvironments.The importance of T cells in xenorejection can be manifested by the growth of xenografts in mutant nude mice.However,the interaction between tumor xenografts and local microenvironments where the xenografts grow have received little extent attentions.
As a common site for human HCC transplants,the liver is accepted as a complex immune or tolerance organ in recent years.It contains abundant innate immune cells, such as natural killer(NK),natural killer T(NKT) cells,kupffer cells and dendritic cells(DCs).Besides,classic CD4~+ T and CD8~+ T cells in the liver are present at a reversed ratio other than they are in the peripheral blood.All these innate and adaptive immune cells residing in liver dominate the local tumor surveillance,but their role in tumor xenograft survival has been inadequately explored.
In this study we attempt to establish a human-mouse HCC model by transplanting human HCC cells to the liver of immunocompetent newborn mice.We based on three reasons:first,the elevated expression of alpha-fetaprotein(AFP) in the liver of newborn mice(at day 0～15 after birth) can suppress the anti-tumor immunity;second, newborn mice can generate weak immune rejection because of their incomplete thymus and peripheral immune organs;the third,as a immune privilege organ,the liver is apt to induce immune tolerance.In our previous experiments,we transplanted human HepG2 cells into the liver of immunocompetent newborn KM mice for the first time.We demonstrated that human HepG2 cells survived longer than those in adults.However,following the immune systems of newborn mice maturing,the immune xenorejection enhanced,and tumor cells began necrosis or apoptosis.The tumor cells encountered immune rejection in the liver of newborn mice,which immune cell participated in that process? Transplanted tumor cells were cleared from the liver,how the immune microenvironment of liver changed? In particular the aboundent NKT cells,how their activation and function changed? Whether the fate of different cells in the liver of newborn mice is same or not? No lymphocyte infiltration was detected in tumor tissues of CsA treated newborn mice,how the effect of CsA on cell-mediated xenorejection?
Trying to solve these problems,we transplanted different human HCC lines (HepG2 and HCCLM3) to immunocompetent newborn mice.We intended to examine the fate of transplanted human tumor cells,the cell-mediated immune rejection,and the changes of activation and function of NKT cells.For better understanding this,we adopted low-dose CsA treated newborn and adult mice as controls.This study provides new information on tumor xenograft models in immature animals and throws new insight into NKT-mediated tumor xenorejection.Moreover,it adds to our understanding of the immunosuppressive effects of CsA.
Methods
1.In vitro cell imaging and Cytotoxicity analysis by DMAHAS
1) HepG2 cells labeled with DMAHAS and cell imaging;
2) Fluorescence release analysis of DMAHAS labeled cell;
3) Cell viability was determined using MTT and neutral red assays;
4) Total protein content of cells was measured by Coomassie brilliant blue assay;
5) Cell proliferation and apoptosis were analyzed using the propidium iodide(PI) flow cytometric assay;
2.Orthotopic implantation of human HCC cells in young and adult mice
1) In vitro culture of tumor cells and labeled by DMAHAS;
2) Mice were treated with lose-CsA(10mg/kg) and sera CsA was measured;
3) Orthotopic implantation of human HCC cells in the liver of mice;
4) In vitro frozen section analysis and fluorescence imaging of tumor xenografts;
5) Conventional HE staining and pathological examination of tumor xenografts:
3.Different cellular rejection occurred in young and adult mice was analyzed
1) Orthotopic implantation of human HCC cells in the liver of mice;
2) Mice treated with lose-CsA(10mg/kg) through subcutaneously injection;
3) Isolation of hepatic or splenic lymphocytes and counting;
4) Method based on DMAHAS fluorescence release for cytotoxicity analysis;
5) Specific antibodies toward minor cells were measured in the serum from mice;
6) Phenotypic analysis of hepatic and splenic lymphocytes from mice by FCM;
4.Analysis of the activation and function of NKT from tumor-bearing mice
1) Orthotopic implantation of human HCC cells in the liver of mice;
2) Mice treated with lose-CsA(10mg/kg) through subcutaneously injection;
3) Isolation of hepatic or splenic lymphocytes and counting;
4) NKT activity analysis by LDH release assay;
5) Analysis of NKT activation and CD1d expression of hepatic/splenic lymphocytes;
6) Levels of IL-4 and IFN-γwere measured by ELISA using a quantitative kit;
7) In vitro immune cells depleted by spcific antibodies;
8) Cytotoxicity analysis by LDH release assay.
Results
The first part
Cell imaging in vitro using DMAHAS
We found that both live and fixed cells took up DMAHAS rapidly within 30min. The final recommended working concentration is 5μM.DMAHAS exhibited strong one photon induced blue fluorescence and stable cytosolic localization.
Cytotoxicity of DMAHAS toward HepG2 cells
No significant cytotoxicity was determined following 48h incubation of HepG2 cells with DMAHAS at a working concentration of 5μM.According to MTT,NR,CB and FCM assay.
Tumor tracking in vivo using DMAHAS and fluorescence imaging
DMAHAS labeled HepG2 cells could survive in recipient mice for more than 4 weeks and the same results were obtained with mice receiving HCCLM3 xenografts. Smaller tumor xenografts were observed as survival time increased,and tumor cells tended to aggregated.At six weeks after transplantation,no tumor cells or fluorescence were detected in the liver of recipient mice.At any given time,no significant decline in fluorescence intensity was observed.
Pathological examination of tumor xenografts in newborn mice
Lymphocyte infiltration appeared in and around the xenografts at day 8 after implantation of human HCCLM3 cells in the liver of newborn mice,no macrophages or neutrophils was visible,and the recruitment of lymphocytes appeared in the normal liver tissue near the tumor xenografts.Some of them still survived for more than four weeks,and no evidence of distant metastasis was observed.Low-dose CsA treatments significantly reduced the lymphocytes infiltration and prolonged the survival of xenografts in young mice to five weeks.
The second part
Number changes of hepatic and splenic lymphocytes in mice bearing tumor cells
In young mice,the absolute number of lymphocytes increased in the liver,but decreased in the spleen at days 10 and 16 postimplantation(P＜0.05).Similar results were found in adult mice at days 4,8 and 10 postimplantation.No significant apoptosis of splenic or hepatic lymphocytes was detected in young mice.
Immune analysis of hepatic and splenic lymphocytes in mice bearing tumor cells
In young mice,the absolute number and proportion of hepatic CD4~+ T cells was significantly increased at day 16 postimplantation(P＜0.01);the absolute number of NK cells and CD8~+ T cells increased in the liver,but decreased in the spleen(P＜0.05). In adult mice,the proportion of CD8~+ cells increased in the liver,but decreased in the spleen at day 2 after transplantation.
CTL responses induced by HCCLM3 xenografts in young mice
Splenic lymphocytes from tumor-bearing young mice showed higher cytotoxicity against HCCLM3 cells at the ratio of 10:1,20:1 and 50:1(P＜0.05).
Effects of low-dose CsA on the survival of xenografts and immune response
Low-dose CsA treatments significantly reduced the lymphocytes infiltration and prolonged the survival of xenografts in young mice to five weeks posttransplantation. However,CsA had no obvious effect on the survival of xenografts in adults.Besides the inhibiting effects on CD4~+ and CD8~+ cells as seen in data from young and adult mice,low-dose CsA treatment efficiently suppressed the activation of hepatic NKT cells at day 2 after implantation in adult mice without the loss of NK1.1 expression on NKT cells.Moreover,low-dose CsA treatment efficiently suppressed the splenic CTL activity in tumor-bearing young mice.
The third part
Hepatic NKT cell activation in mice induced by HCCLM3 xenografts
After HCCLM3 cells were transplanted into the livers of 3-day-old mice,the hepatic NKT cells lost expression of NK1.1 at days 5 and 10.NK1.1 molecules reappeared at day 16,and elevated NKT-like activity was detected.In adult mice,a loss of NK1.1 expression was detected in hepatic NK/NKT cells at days 2 and 4 posttransplantation.Then the NK1.1 marker was reexpressed and returned to normal levels at day 10 posttransplantation.There were no significant changes in NK1.1 expression on splenic NK/NKT cells after transplantation.
CD1d expression on lymphocytes from the liver and spleen of tumor-bearing mice
In young mice,the CD1d expression first increased on hepatic NK1.1~+ cells at day 10 after transplantation.Then up-regulation of CD1d expression was detected on hepatic CD3~+ T cells and NK1.1~+ cells at days 16 and 28 after transplantation.Similar results were detected in hepatic or splenic lymphocytes from adults at day 4 and 10 posttransplantation.
Secretions of IL-4 and IFN-γin tumor-bearing young and adult mice
Sera were collected from the retro-orbital veins at days 10 and 16 in young mice and at days 4 and 10 in adult mice after transplanted with human HCCLM3 cells. Elevated secretion of IL-4 was detected at day 10 in young mice and at day 4 in adult mice(P＜0.05),and elevated secretion of IFN-γat any given time in young and adult mice(P＜0.05),compared with control groups.
Effects of low-dose CsA treatment on the activation and function of NKT cells
low-dose CsA treatment efficiently suppressed the activity of NKT cells from tumor-bearing young mice,and the activation of hepatic NKT cells in adult mice at day 2 after implantation.Moreover,low-dose CsA treatment inhibited the secretions of IL-4 and IFN-γin recipient mice(P＜0.05).No down-regulation of CD1d expression on hepatic or splenic T cells and NK1.1~+ cells was detected in mice with low-dose CsA treatment.
CTL analysis after in vitro deletion of specific lymphocytes
In vitro deletion of CD3 and CD8 cells efficiently reduced the cytotoxicity against HCCLM3 cells(P＜0.05),however,deletion of CD4 and NK1.1 cells showed no evident effect.
Conclusion
1.DMAHAS was used to label human HCC cells for fluorescence imaging for the first time.It was demonstrated that DMAHAS had no evident effect on the cell viability,synthesis of total protein or DNA,as well as cell apoptosis.In vivo study indicated that DMAHAS was a reliable probe for tumor cell tracking and imaging.
2.It was demonstrated that the liver of newborn mice was more suitable for the survival of tumor xenografts than that of adults.
3.Lymphocytes significantly increased in the liver,but decreased in the spleen after tumor implantation without enhanced cell apoptosis,suggesting that splenic lymphocytes may participate in the tumor xenorejection.
4.cell-mediated xenorejection in the liver of young mice was different from that in adult liver.In young mice the delayed cellular rejection was mediated mainly by CD4~+T cells,CD8~+T cells and NK1.1~+cells.In contrast,in adult mice the acute cellular rejection was mediated mainly by CD8~+T cells,NK1.1~+cells,macrophages or neutrophils.
5.The rapid activation of hepatic NKT cells with enhanced activity and function, suggested that NKT cells acted as an important role in the xenorejection.
6.Increased expression of CD1d on the surface of hepatic or splenic lymphocytes, implying that CD1d molecules participate in the tumor xeno-antigens presenting and the subsequent tumor clearance.
7.Low-dose CsA treatments significantly prolonged the survival of xenografts in young mice by inhibiting the cell-mediated rejection.However,it had no obvious effect on the survival of tumor xenografts in adults.
8.Besides the inhibiting effects on CD4~+ and CD8~+ cells,low-dose CsA treatment efficiently suppressed the activation and function of hepatic NKT cells,maybe through a CD1d-independent manner.
原发性肝细胞癌(HCC)是世界三大癌症相关死亡之一,是最常见的预后较差的肝脏恶性肿瘤。目前研究者建立了各种肿瘤异种移植模型用于人肝癌生物学和化疗研究,但由于其采用的模型动物主要是免疫缺陷鼠或诱导免疫耐受鼠,致使以此模型进行肿瘤免疫治疗的研究受到限制。因而构建免疫力正常的异种肿瘤移植模型,研究肿瘤与宿主的相互作用将有助于我们更好地理解肿瘤的免疫治疗。然而早先的研究证实肿瘤异种移植物仅可以生长于一些免疫豁免位点,如大鼠的颊囊、眼前房,或环孢霉素A处理的小鼠或大鼠的肾囊下生长。异种肿瘤移植物在免疫力正常宿主体内成功存活的主要障碍被认为是T细胞介导的免疫排斥及组织微环境。T细胞介导的异种免疫排斥的重要性已在裸鼠模型中得到证实,而异种肿瘤移植物与组织微环境的关系研究较少。
肝脏作为人肝癌细胞移植的常见位点,近年被公认为一个复杂的免疫与耐受器官。它富含天然免疫细胞,如NK、NKT、枯否细胞和树突状细胞,尤其是NKT细胞,近年被称为免疫系统的瑞士军刀,其活化后可激活各类免疫细胞参与多种免疫反应。此外,经典CD4~+T和CD8~+T细胞在肝脏中的比例也与外周血相反。所有这些天然免疫和获得性免疫细胞决定着肝脏微环境的肿瘤监视,但它们在肿瘤异种移植物存活中所扮演的角色却知之甚少。
本研究尝试在免疫力正常的乳鼠肝脏内移植入人的肝癌细胞来建立人肝癌—小鼠动物研究模型。其理论依据为:①乳鼠(约出生后0～15d)肝内高表达的甲胎蛋白(alpha-fetaprotein,AFP)具有抑制机体抗肿瘤免疫的生物学特性;②乳鼠胸腺尚未发育成熟,外周免疫系统尚未完善,免疫排斥较弱;③选择“免疫特赦器官”—肝脏作为荷瘤器官,易于诱导免疫耐受。在前期的实验中,我们首次将人肝癌细胞HepG2细胞原位移植到具正常免疫力的昆明种乳鼠肝内,构建了人肝癌HepG2移植性小鼠模型。虽然我们证实在免疫力正常的乳鼠肝内移植人HepG2细胞较成鼠成功率高,移植瘤细胞存活时间较长,但是随着乳鼠发育免疫系统的逐渐完善,肝内异种免疫排斥反应增强,肿瘤细胞在移植后7～9天开始坏死。肿瘤细胞在乳鼠肝内遭遇免疫排斥,何种免疫细胞参与了这一过程?肿瘤细胞在肝内被清除,肝内的免疫微环境发生了怎样的变化?尤其是数量丰富的NKT细胞的活化和功能有着怎样的改变?分化程度不同的异种肿瘤细胞,在乳鼠肝内的命运是否一样?CsA免疫抑制的荷瘤乳鼠肿瘤组织病理切片中并未发现淋巴细胞的浸润和肿瘤细胞的坏死,其对细胞介导的异种免疫排斥有着怎样的影响?
围绕这些问题,本研究将生长特性不同的人肝癌细胞株(HepG2和HCCLM3)移植到免疫力正常的乳鼠体内,探讨人肝癌细胞在乳鼠肝内的存活情况、肝内细胞介导的免疫排斥反应、NKT细胞活化及功能的改变。为更好地阐明这一点,我们同时采用成鼠及低剂量CsA处理的乳鼠和成鼠做移植受体,进行对比研究。本研究为HCC异种模型的建立提供了一种新的尝试,为NKT细胞介导的肿瘤异种排斥呈现了一个新的视角。而且它有助于更好地了解CsA的免疫抑制机制,无论是在临床还是动物模型的研究中。
研究方法
1、DMAHAS体外标记细胞与细胞毒性分析
1)DMAHAS标记人肝癌HepG2细胞并荧光成像;
2)DMAHAS标记细胞荧光释放分析;
3)MTT和中性红法检测DMAHAS处理HepG2细胞的活力;
4)考马斯亮蓝法测定细胞总蛋白;
5)PI流式细胞术检测细胞周期与凋亡;
2、乳鼠和成鼠人肝癌原位移植
1)肿瘤细胞培养与体外DMAHAS标记;
2)低剂量CsA(10 mg/kg)处理小鼠及其血清浓度检测;
3)人肝癌细胞小鼠肝内原位移植;
4)异种移植物组织冰冻切片分析及荧光成像;
5)异种移植物组织常规HE染色及病理分析;
3、荷瘤乳鼠及成鼠不同细胞免疫排斥机理的研究
1)人肝癌细胞HCCLM3小鼠肝内原位移植;
2)低剂量CsA(10 mg/kg)小鼠皮下注射;
3)小鼠肝脾淋巴细胞分离和计数;
4)DMAHAS荧光释放法检测CTL杀伤活性;
5)荷瘤乳鼠血清肿瘤细胞特异性抗体检测;
6)荷瘤小鼠肝脾淋巴细胞流式细胞术免疫表型分析;
4、荷瘤小鼠NKT细胞活化及功能的研究
1)人肝癌细胞HCCLM3小鼠肝内原位移植;
2)低剂量CsA(10 mg/kg)小鼠皮下注射;
3)分离小鼠肝脾淋巴细胞并计数;
4)乳酸脱氢酶释放法检测NKT细胞杀伤活性;
5)肝脾淋巴细胞NKT活化与CD1d表达分析;
6)血清及肝脏组织IL-4和IFN-γELISA定量检测;
7)体外免疫细胞特异性清除;
8)LDH释放法测定CTL活性。
研究结果
第一部分
DMAHAS体外标记细胞成像
DMAHAS可在30分钟内快速标记活细胞或多聚甲醛固定的细胞,最适工作浓度为5μM。DMAHAS定位于细胞质,当受到紫外或长波长光(800nm)激发时,可自发地发射蓝色荧光。
DMAHAS体外标记细胞与细胞毒性分析
采用工作浓度5μM的DMAHAS标记HepG2细胞培养24小时进行MTT、NR、CB和PI流式细胞术检测表明DMAHAS对标记细胞无显著影响(P＞0.05)。
DMAHAS标记肿瘤细胞体内示踪和荧光成像
DMAHAS标记的人肝癌HepG2和HCCLM3细胞可在受体鼠肝内存活4周以上,移植肿瘤细胞随时间延长逐渐减少,在移植后六周受体鼠肝内未检测到肿瘤细胞或荧光信号。而且检测过程中未见DMAHAS荧光信号的衰减。
乳鼠肝脏移植瘤组织病理分析
人肝癌HCCLM3细胞原位移植于乳鼠肝内后,第8天左右肝内正常组织出现淋巴细胞的募集,移植瘤周围出现淋巴细胞浸润,无明显的炎性细胞如中性粒细胞或巨噬细胞介入。部分肿瘤移植物仍可存活4周以上,未见明显的肝内转移。低剂量CsA处理可有效减少淋巴细胞浸润,延长肿瘤细胞存活期至5周以上。
第二部分
荷瘤小鼠肝脾淋巴细胞变化
荷瘤乳鼠肝脏淋巴细胞数量于移植后的10天和16天增加(P＜0.05),同时脾脏淋巴细胞数量减少,同样的结果可见于移植后4天、8天和10天的成鼠。并且荷瘤小鼠脾脏或肝脏淋巴细胞未见明显凋亡。
荷瘤小鼠肝脾淋巴细胞免疫分析
移植后第16天,乳鼠肝脏CD4~+T细胞绝对计数和百分比显著增加(P＜0.01),NK和CD8~+T细胞数量绝对计数在肝脏内增加,而在脾脏中减少(P＜0.05)。成鼠于移植后2天即见CD8~+T细胞在肝内的增加与脾内的减少。
荷瘤小鼠脾脏淋巴细胞杀伤功能检测
荷瘤16天的乳鼠脾脏淋巴细胞在10:1,20:1和50:1效靶比时对HCCLM3细胞的杀伤显著增加(P＜0.05)。
低剂量CsA对移植瘤存活及其引发的免疫反应的影响
低剂量CsA处理可显著地减少乳鼠肝内淋巴细胞侵润,延长肿瘤移植物存活期至5周以上。然而其对移植物在成鼠肝内的存活无明显影响。除对CD4~+和CD8~+细胞有抑制效果外,低剂量CsA可有效地抑制NKT细胞的活化。此外,低剂量CsA处理可有效抑制荷瘤乳鼠脾脏效应细胞的功能。
第三部分
HCCLM3异种移植物诱导的肝脏NKT细胞活化
HCCLM3移植于3日龄乳鼠肝内后,其肝脏NKT细胞NK1.1分子在移植后5天和10天表达丢失。16天时表达增加并伴随NKT细胞活性增强。成鼠在移植后2天和4天可见NK1.1分子的大量丢失,并于10天时恢复至正常水平。脾脏NK/NKT细胞表面NK1.1分子的表达在移植前后无显著变化。
荷瘤小鼠肝脏和脾脏淋巴细胞CD1d表达
乳鼠在移植后10天时肝脏NK1.1~+细胞表面CD1d的表达开始增加,随后CD1d的表达上调可见于移植后16天和28天肝脏CD3~+T和NK1.1~+细胞。类似的结果可见于移植后4天和10天时的成鼠肝脾淋巴细胞。
荷瘤小鼠血清IL-4和IFN-γ检测
血清取自荷瘤10天和16天的乳鼠或4天和8天的成鼠。IFN-γ的水平在移植后的乳鼠和成鼠血清中均有增加(P＜0.05),而IL-4水平的升高仅见于移植后10天的乳鼠和4天的成鼠血清(P＜0.05)。
低剂量CsA对NKT细胞活化和功能的影响
低剂量CsA处理可显著抑制荷瘤乳鼠肝脏NKT细胞的功能(P＜0.05)及移植2天时的成鼠NKT细胞活化。此外低剂量CsA可显著抑制受体鼠血清IL-4和IFN-γ的分泌,而并未显著下调肝脾T细胞或NK1.1~+T细胞表面CD1d的表达。
荷瘤乳鼠淋巴细胞体外免疫清除CTL活性分析
体外特异性清除荷瘤乳鼠淋巴细胞中的CD3或CD8细胞,可显著降低CTL细胞的杀伤活性(P＜0.05);而特异性清除CD4或NK1.1~+细胞却无明显影响。
研究结论
1.首次应用新型双光子荧光染料DMAHAS标记人肝癌细胞及荧光成像研究,并初次证实其对标记细胞的活力、总蛋白合成和细胞周期与凋亡无显著影响。体内研究表明DMAHAS可作为一种安全可靠的探针用于肿瘤细胞示踪和荧光成像研究。
2.初步证实乳鼠肝脏较成鼠肝脏更适宜于异种肿瘤细胞的存活与生长。
3.淋巴细胞在荷瘤小鼠脾脏中的减少及肝脏中的增加暗示脾脏淋巴细胞以某种方式参与了肝脏异种肿瘤移植物的免疫排斥反应。
4.乳鼠和成鼠肝内发生了对肿瘤移植物不同的细胞免疫排斥反应。参与乳鼠肿瘤延迟排斥反应的免疫细胞主要是CD4~+T、NK1.1~+细胞及CD8~+T细胞,而参与成鼠肿瘤急性排斥反应的免疫细胞主要是CD8~+T细胞、NK1.1~+细胞及大量的炎性细胞,如中性粒细胞或巨噬细胞。
5.肝脏NKT细胞在异种肿瘤的排斥反应早期活化、功能及活性增强,表明其在排斥反应的早期起着重要作用。
6.CD1d分子在荷瘤小鼠肝脏淋巴细胞不同时期表达的增加,表明其参与了异种肿瘤抗原的免疫呈递过程及异种移植物的免疫清除。
7.低剂量CsA处理可显著地抑制乳鼠的免疫排斥反应,延长肿瘤移植物存活期,然而其对成鼠肝内肿瘤移植物存活无明显影响。
8.除对CD4~+和CD8~+T细胞功能有抑制作用外,低剂量CsA可有效抑制肝脏NKT细胞的活化及功能,而且这种抑制方式可能不依赖于CD1d。
Background and objective
Hepatocellular carcinoma(HCC),the third most common cause of cancer-related death worldwide,is the most frequent hepatic tumor with poor prognosis.Tumor xenograft models are widely used for human tumor biology and chemotherapy analysis, but they may be insufficient for studying tumor immunotherapy because most of them are established in immunodeficient hosts.Studies to elucidate the immune mechanisms of tumor-host interactions in immunocompetent tumor xenograft models will benefit our understanding of tumor immunotherapy.Previous studies have demonstrated that tumor xenografts can grow in some immunologically privileged sites,such as the anterior chamber of the eye,the hamster cheek pouch,or under the kidney capsule of mice or rats with limited cyclosporine A treatment.The potential barriers to successful xenograft survival are believed to be T cell mediated xenorejection and tissue microenvironments.The importance of T cells in xenorejection can be manifested by the growth of xenografts in mutant nude mice.However,the interaction between tumor xenografts and local microenvironments where the xenografts grow have received little extent attentions.
As a common site for human HCC transplants,the liver is accepted as a complex immune or tolerance organ in recent years.It contains abundant innate immune cells, such as natural killer(NK),natural killer T(NKT) cells,kupffer cells and dendritic cells(DCs).Besides,classic CD4~+ T and CD8~+ T cells in the liver are present at a reversed ratio other than they are in the peripheral blood.All these innate and adaptive immune cells residing in liver dominate the local tumor surveillance,but their role in tumor xenograft survival has been inadequately explored.
In this study we attempt to establish a human-mouse HCC model by transplanting human HCC cells to the liver of immunocompetent newborn mice.We based on three reasons:first,the elevated expression of alpha-fetaprotein(AFP) in the liver of newborn mice(at day 0～15 after birth) can suppress the anti-tumor immunity;second, newborn mice can generate weak immune rejection because of their incomplete thymus and peripheral immune organs;the third,as a immune privilege organ,the liver is apt to induce immune tolerance.In our previous experiments,we transplanted human HepG2 cells into the liver of immunocompetent newborn KM mice for the first time.We demonstrated that human HepG2 cells survived longer than those in adults.However,following the immune systems of newborn mice maturing,the immune xenorejection enhanced,and tumor cells began necrosis or apoptosis.The tumor cells encountered immune rejection in the liver of newborn mice,which immune cell participated in that process? Transplanted tumor cells were cleared from the liver,how the immune microenvironment of liver changed? In particular the aboundent NKT cells,how their activation and function changed? Whether the fate of different cells in the liver of newborn mice is same or not? No lymphocyte infiltration was detected in tumor tissues of CsA treated newborn mice,how the effect of CsA on cell-mediated xenorejection?
Trying to solve these problems,we transplanted different human HCC lines (HepG2 and HCCLM3) to immunocompetent newborn mice.We intended to examine the fate of transplanted human tumor cells,the cell-mediated immune rejection,and the changes of activation and function of NKT cells.For better understanding this,we adopted low-dose CsA treated newborn and adult mice as controls.This study provides new information on tumor xenograft models in immature animals and throws new insight into NKT-mediated tumor xenorejection.Moreover,it adds to our understanding of the immunosuppressive effects of CsA.
Methods
1.In vitro cell imaging and Cytotoxicity analysis by DMAHAS
1) HepG2 cells labeled with DMAHAS and cell imaging;
2) Fluorescence release analysis of DMAHAS labeled cell;
3) Cell viability was determined using MTT and neutral red assays;
4) Total protein content of cells was measured by Coomassie brilliant blue assay;
5) Cell proliferation and apoptosis were analyzed using the propidium iodide(PI) flow cytometric assay;
2.Orthotopic implantation of human HCC cells in young and adult mice
1) In vitro culture of tumor cells and labeled by DMAHAS;
2) Mice were treated with lose-CsA(10mg/kg) and sera CsA was measured;
3) Orthotopic implantation of human HCC cells in the liver of mice;
4) In vitro frozen section analysis and fluorescence imaging of tumor xenografts;
5) Conventional HE staining and pathological examination of tumor xenografts:
3.Different cellular rejection occurred in young and adult mice was analyzed
1) Orthotopic implantation of human HCC cells in the liver of mice;
2) Mice treated with lose-CsA(10mg/kg) through subcutaneously injection;
3) Isolation of hepatic or splenic lymphocytes and counting;
4) Method based on DMAHAS fluorescence release for cytotoxicity analysis;
5) Specific antibodies toward minor cells were measured in the serum from mice;
6) Phenotypic analysis of hepatic and splenic lymphocytes from mice by FCM;
4.Analysis of the activation and function of NKT from tumor-bearing mice
1) Orthotopic implantation of human HCC cells in the liver of mice;
2) Mice treated with lose-CsA(10mg/kg) through subcutaneously injection;
3) Isolation of hepatic or splenic lymphocytes and counting;
4) NKT activity analysis by LDH release assay;
5) Analysis of NKT activation and CD1d expression of hepatic/splenic lymphocytes;
6) Levels of IL-4 and IFN-γwere measured by ELISA using a quantitative kit;
7) In vitro immune cells depleted by spcific antibodies;
8) Cytotoxicity analysis by LDH release assay.
Results
The first part
Cell imaging in vitro using DMAHAS
We found that both live and fixed cells took up DMAHAS rapidly within 30min. The final recommended working concentration is 5μM.DMAHAS exhibited strong one photon induced blue fluorescence and stable cytosolic localization.
Cytotoxicity of DMAHAS toward HepG2 cells
No significant cytotoxicity was determined following 48h incubation of HepG2 cells with DMAHAS at a working concentration of 5μM.According to MTT,NR,CB and FCM assay.
Tumor tracking in vivo using DMAHAS and fluorescence imaging
DMAHAS labeled HepG2 cells could survive in recipient mice for more than 4 weeks and the same results were obtained with mice receiving HCCLM3 xenografts. Smaller tumor xenografts were observed as survival time increased,and tumor cells tended to aggregated.At six weeks after transplantation,no tumor cells or fluorescence were detected in the liver of recipient mice.At any given time,no significant decline in fluorescence intensity was observed.
Pathological examination of tumor xenografts in newborn mice
Lymphocyte infiltration appeared in and around the xenografts at day 8 after implantation of human HCCLM3 cells in the liver of newborn mice,no macrophages or neutrophils was visible,and the recruitment of lymphocytes appeared in the normal liver tissue near the tumor xenografts.Some of them still survived for more than four weeks,and no evidence of distant metastasis was observed.Low-dose CsA treatments significantly reduced the lymphocytes infiltration and prolonged the survival of xenografts in young mice to five weeks.
The second part
Number changes of hepatic and splenic lymphocytes in mice bearing tumor cells
In young mice,the absolute number of lymphocytes increased in the liver,but decreased in the spleen at days 10 and 16 postimplantation(P＜0.05).Similar results were found in adult mice at days 4,8 and 10 postimplantation.No significant apoptosis of splenic or hepatic lymphocytes was detected in young mice.
Immune analysis of hepatic and splenic lymphocytes in mice bearing tumor cells
In young mice,the absolute number and proportion of hepatic CD4~+ T cells was significantly increased at day 16 postimplantation(P＜0.01);the absolute number of NK cells and CD8~+ T cells increased in the liver,but decreased in the spleen(P＜0.05). In adult mice,the proportion of CD8~+ cells increased in the liver,but decreased in the spleen at day 2 after transplantation.
CTL responses induced by HCCLM3 xenografts in young mice
Splenic lymphocytes from tumor-bearing young mice showed higher cytotoxicity against HCCLM3 cells at the ratio of 10:1,20:1 and 50:1(P＜0.05).
Effects of low-dose CsA on the survival of xenografts and immune response
Low-dose CsA treatments significantly reduced the lymphocytes infiltration and prolonged the survival of xenografts in young mice to five weeks posttransplantation. However,CsA had no obvious effect on the survival of xenografts in adults.Besides the inhibiting effects on CD4~+ and CD8~+ cells as seen in data from young and adult mice,low-dose CsA treatment efficiently suppressed the activation of hepatic NKT cells at day 2 after implantation in adult mice without the loss of NK1.1 expression on NKT cells.Moreover,low-dose CsA treatment efficiently suppressed the splenic CTL activity in tumor-bearing young mice.
The third part
Hepatic NKT cell activation in mice induced by HCCLM3 xenografts
After HCCLM3 cells were transplanted into the livers of 3-day-old mice,the hepatic NKT cells lost expression of NK1.1 at days 5 and 10.NK1.1 molecules reappeared at day 16,and elevated NKT-like activity was detected.In adult mice,a loss of NK1.1 expression was detected in hepatic NK/NKT cells at days 2 and 4 posttransplantation.Then the NK1.1 marker was reexpressed and returned to normal levels at day 10 posttransplantation.There were no significant changes in NK1.1 expression on splenic NK/NKT cells after transplantation.
CD1d expression on lymphocytes from the liver and spleen of tumor-bearing mice
In young mice,the CD1d expression first increased on hepatic NK1.1~+ cells at day 10 after transplantation.Then up-regulation of CD1d expression was detected on hepatic CD3~+ T cells and NK1.1~+ cells at days 16 and 28 after transplantation.Similar results were detected in hepatic or splenic lymphocytes from adults at day 4 and 10 posttransplantation.
Secretions of IL-4 and IFN-γin tumor-bearing young and adult mice
Sera were collected from the retro-orbital veins at days 10 and 16 in young mice and at days 4 and 10 in adult mice after transplanted with human HCCLM3 cells. Elevated secretion of IL-4 was detected at day 10 in young mice and at day 4 in adult mice(P＜0.05),and elevated secretion of IFN-γat any given time in young and adult mice(P＜0.05),compared with control groups.
Effects of low-dose CsA treatment on the activation and function of NKT cells
low-dose CsA treatment efficiently suppressed the activity of NKT cells from tumor-bearing young mice,and the activation of hepatic NKT cells in adult mice at day 2 after implantation.Moreover,low-dose CsA treatment inhibited the secretions of IL-4 and IFN-γin recipient mice(P＜0.05).No down-regulation of CD1d expression on hepatic or splenic T cells and NK1.1~+ cells was detected in mice with low-dose CsA treatment.
CTL analysis after in vitro deletion of specific lymphocytes
In vitro deletion of CD3 and CD8 cells efficiently reduced the cytotoxicity against HCCLM3 cells(P＜0.05),however,deletion of CD4 and NK1.1 cells showed no evident effect.
Conclusion
1.DMAHAS was used to label human HCC cells for fluorescence imaging for the first time.It was demonstrated that DMAHAS had no evident effect on the cell viability,synthesis of total protein or DNA,as well as cell apoptosis.In vivo study indicated that DMAHAS was a reliable probe for tumor cell tracking and imaging.
2.It was demonstrated that the liver of newborn mice was more suitable for the survival of tumor xenografts than that of adults.
3.Lymphocytes significantly increased in the liver,but decreased in the spleen after tumor implantation without enhanced cell apoptosis,suggesting that splenic lymphocytes may participate in the tumor xenorejection.
4.cell-mediated xenorejection in the liver of young mice was different from that in adult liver.In young mice the delayed cellular rejection was mediated mainly by CD4~+T cells,CD8~+T cells and NK1.1~+cells.In contrast,in adult mice the acute cellular rejection was mediated mainly by CD8~+T cells,NK1.1~+cells,macrophages or neutrophils.
5.The rapid activation of hepatic NKT cells with enhanced activity and function, suggested that NKT cells acted as an important role in the xenorejection.
6.Increased expression of CD1d on the surface of hepatic or splenic lymphocytes, implying that CD1d molecules participate in the tumor xeno-antigens presenting and the subsequent tumor clearance.
7.Low-dose CsA treatments significantly prolonged the survival of xenografts in young mice by inhibiting the cell-mediated rejection.However,it had no obvious effect on the survival of tumor xenografts in adults.
8.Besides the inhibiting effects on CD4~+ and CD8~+ cells,low-dose CsA treatment efficiently suppressed the activation and function of hepatic NKT cells,maybe through a CD1d-independent manner.
引文
1.刘福英,王秀芳,冯旭.可移植性肿瘤动物模型的复制应用及问题.医学动物防治,2000;16:482-5.
2.Editorial:Human tumour xenografts.Br Med J 1974;16(5941):366-7.
3.Jacobson HI,Bennett JA,Mizejewski GJ.Inhibition of estrogen-dependent breast cancer growth by a reaction product of alpha-fetoprotein and estradiol.Cancer Res 1990;50:415.
4.Kaartinen M,Eray M,Lehtonen E,Matoso-Ferreira A,Tienari J.Implanted solid human tumours grow under the renal capsule of cyclosporin-immunosuppressed rats.Scand J Immunol 1994;39:618.
5..Rygaard J,Povlsen CO.Heterotransplantation of a human malignant tumor to 'nude' mice.Acta Path Microbiol Scand 1969;77:758-60.
6.汤钊猷,马曾层,应越英,等.裸鼠人体肝癌移植模型的研究.模型的建立及其血清学与形态学特征.上海第一医学院学报,1982;9:21-251.
7.胡卫,陈涛.鼠移植性肝癌模型研究进展.河南肿瘤学杂志,2003;16:463-6.
8.Murphy JB.Factors of resistance to heteroplastic tissue-grafting:studies in tisses specificity Ⅲ.J Exp Med 1914;19:513-522.
9.Shirai Y.Transplanting rat sarcoma in adult heterogenous animals.Jap Med World 1921;1:14-5.
10.Murphy JB,Sturm E.Conditions determining the transplantability of tissues in the brain.J Exp Med 1923;38:183-97.
11.Mandel TE,Dillon H,Koulmanda M.The effect of a depleting anti-CD4 monoclonal antibody on T cells and fetal pig islet xenograft survival in various strains of mice.Transpl Immunol 1995;3:265-72.
12.Simeonovic CJ.Xenogeneic islet transplantation.Xenotransplantation 1999;6:1-5.
13.Yi S,Feng X,Hawthorne WJ et al.Cells initiate pancreatic islet xenograft rejection via an interferon-gamma-dependent recruitment of macrophages and natural killer cells.Transplantation 2002;73:437-46.
14.Zhao Y,Swenson K,Wekerle T,Rodriguez-Barbosa JI,Arn JS,Sykes M.The critical role of mouse CD4+ cells in the rejection of highly disparate xenogeneic pig thymus grafts. Xenotransplantation 2000; 7: 129-37.
15. Friedman T, Smith RN, Colvin RB, Iacomini J. A critical role for human CD4+ T-cells in rejection of porcine islet cell xenografts. Diabetes 1999; 48: 2340-8.
16. Lechler RI, Lombardi G, Batchelor JR, Reinsmoen N, Bach FH. The molecular basis of alloreactivity. Immunol Today 1990; 11: 83.
17. Brouard S, Gagne K, Blancho G, and Soulillou JP. T Cell Response in Xenorecognition and Xenografts: A Review. Human Immunology 1999; 60: 455-68.
18. Ricordi C, Lacy PE. Renal subcapsular xenotransplantation of purified porcine islets. Transplantation 1987; 44: 721.
19. Korsgren O, Sandier S, Landstrom AS, Jansson K. Andersson A. Large-scale production of foetal porcine pancreatic islet like cell clusters. An expeimental tool for studies of islet cell differentiation and xenotransplantation. Transplantation 1988; 45: 509.
20. Sandier S, Andersson A, Schnell A, et al. Tissue culture of human foetal pancreas. Development and function of B-cells in vitro and transplantation of explants to nude mice. Diabetes 1985; 34: 1113.
21. Simeonovic CJ, Ceredig R, Wilson JD. Effect of GK1.5 monoclonal antibody dosage on survival of pig proislet xenografts in CD41 T cell-depleted mice. Transplantation 1990; 49: 849.
22. Morris CF, Simenovic CJ, Fung MC, Wilson JD, Hapel AJ. Intragraft expression of cytokine transcripts during pig proislet xenograft rejection and tolerance in mice. J Immunol 1995; 154: 2470.
23. Zhan Y, Brady JL, Irawaty W, Thomas HE, Kay TW, Lew AM. Activated macrophages require T cells for xenograft rejection under the kidney capsule. Immunol Cell Biol. 2003; 81: 451-8.
24. Benda B, Karlsson-Parra A, Ridderstad A, Korsgren O. Xenograft rejection of porcine islet- like cell clusters in immunoglobulin- or Fc-receptor gamma-deficient mice. Transplantation 1996; 62:1207.
25. Medbury HJ, Hibbins M, Lehnert AM, et al. The cytokine and histological response in islet xenograft rejection is dependent upon species combination. Transplantation 1997; 64: 1307.
26. Simeonovic CJ, Townsend MJ, Wilson JD, et al. Eosinophils are not required for the rejection of neovascularized foetal pig proislet xenografts in mice. J Immunol 1997; 158: 2490.
27. Backes MG, Lund RD, Lagenaur CF, Kunz HW, Gill TJ. Cellular events associated with peripherally induced rejection of mature neural xenografts placed into neonatal rat brains. J Comp Neurol 1990; 295: 428.
28. Finsen BR, Sorensen T, Castellano B, et al. Leukocyte infiltration and glial reactions in xenografts of mouse brain tissue undergoing rejection in the adult rat brain. A light and electron microscopical immunocytochemical study. Immunocytochemical characterization of the cellular immune response to intracerebral xenografts of brain tissue. J Neuroimmunol 1991;32:159.
29. Mason DW, Charlton HM, Jones A, Lavy CB, Puklavec M. Simmonds SJ. The fate of allogeneic and xenogeneic neuronal tissue transplanted into the third ventricle of rodents. Neuroscience 1986; 19:685.
30. Wood M, Sloan D, Wood K, Charlton H. Indefinite survival of neural xenografts induced with anti-CD4 monoclonal antibodies. Neuroscience 1996; 70: 775.
31. Honey CR, Clarke DJ, Dallman MJ, Charlton HM. Human neural graft function in rats treated with anti-interleukin II receptor antibody. Neuroreport 1990; 1: 247.
32. Pedersen EB, Zimmer J, Finsen B. Triple immunosuppression protects murine intracerebral, hippocampal xenografts in adult rat hosts: effects on cellular infiltration, major histocompatibility complex antigen induction and blood-brain barrier leakage. Neuroscience 1997;78:685.
33. Smyth MJ, Kershaw MH, Trapani JA. Xenospecific cytotoxic T lymphocytes: potent lysis in vitro and in vivo. Transplantation 1997; 63: 1171-8.
34. Abe M, Cheng J, Qi J, et al. Elimination of porcine hemopoietic cells by macrophages in mice. J Immunol 2002; 168: 621-8.
35. Fox A, Mountford J, Braakhuis A, Harrison LC. Innate and adaptive immune responses to nonvascular xenografts: evidence that macrophages are direct effectors of xenograft rejection. J Immunol 2001; 166: 2133-40.
36. Loudovaris T, Mandel TE, Charlton B. CD 4+ T cell mediated destruction of xenografts within cell-impermeable membranes in the absence of CD8+ T cells and B cells. Transplantation 1996; 61: 1678-84.
37. Zhan Y, Brady JL, Sutherland RM, Lew AM. Without CD4 help, CD8 rejection of pig xenografts requires CD28 costimulation but not perforin killing. J Immunol 2001; 167: 6279-85.
38. Wu G, Korsgren O, Zhang J, Song Z, van Rooijen N, Tibell A. Pig islet xenograft rejection is markedly delayed in macrophagedepleted mice: a study in streptozotocin diabetic animals. Xenotransplantation 2000; 7: 214-20.
39. Bancroft GJ, Schreiber RD, Unanue ER. Natural immunity: a T-cell- independent pathway of macrophage activation, defined in the scid mouse. Immunol Rev 1991; 124: 5-24.
40. Salazar-Mather TP, Orange JS, Biron CA. Early murine cytomegalovirus (MCMV) infection induces liver natural killer (NK) cell inflammation and protection through macrophage inflammatory protein lalpha (MIP-1 alpha) -dependent pathways. J Exp Med 1998; 187: 1-14.
41. Terpstra W, Leenen PJ, van den Bos C et al. Facilitated engraftment of human hematopoietic cells in severe combined immunodeficient mice following a single injection of C12MDP liposomes. Leukemia 1997; 11: 1049-54.
42. Hancock WW, Miyatake T, Koyamada N, et al. Effects of leflunomide and deoxyspergualin in the guinea pig-rat cardiac model of delayed xenograft rejection: suppression of B cell and C-C chemokine responses but not induction of macrophage lectin. Transplantation (in press).
43. Hancock WW. Immunopathology of discordant xenograft rejection. In Xenotransplantation: The Transplantation of Organs and Tissues Between Species, D.K.C. Cooper, E. Kemp, J.L. Platt, D.J.G. White, editors. Berlin, Springer, 1997, pp. 303-313.
44. Hancock WW. Chemokines and the pathogenesis of T cell-dependent immune responses. Am J Pathol 1996; 148:681.
45. Benbaruch A, Xu LL, Young PR, Bengali K, Oppenheim JJ, Wang JM. Monocyte chemotactic protein-3 (MCP3) interacts with multiple leukocyte receptors: C-C CKR1, a receptor for macrophage inflammatory protein-1 alpha/Rantes, is also a functional receptor for MCP3. J Biol Chem 1995; 270: 22123.
46. Horuk R, Colby TJ, Darbonne WC, Schall TJ, Neote K. The human erythrocyte inflammatory peptide (chemokine) receptor: biochemical characterization, solubilization, and development of a binding assay for the soluble receptor. Biochemistry 1993; 32: 5733.
47. Colotta F, Sciacca FL, Sironi M, Luini W, Rabiet MJ, Mantovani A. Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin. Am J Pathol 1994;144:975.
48. Medzhitov R, Janeway CA. Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 1997; 9: 4.
49. Saton M, Kawakami K, Osawa T, Toyoshima S. Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages. J Biochem.(Tokyo) 1992; 111 :331.
50. Candinas D, Koyamada N, Miyatake T, et al. T cell-independence of macrophage and NK cell infiltration, cytokine production and endothelial activation during delayed xenograft rejection. Transplantation 1996; 62:1920.
51. Millan MT, Geczy C, Stuhlmeier K, Goodman DJ, Ferran C, Bach FH. Human monocytes activate porcine endothelial cells, resulting in increased E-selectin, interleukin-8, monocyte chemotactic protein-1, and plasminogen activator inhibitor-type 1 expression. Transplantation 1997; 63:421.
52. Goodman D, von Albertini M, Willson A, Millan M, Bach FH. Direct activation of porcine endothelial cells by human natural killer cells. Transplantation 1996; 61: 763.
53. Kirchhof N, Shibata S, Wijkstrom M, et al. Reversal of diabetes in non-immunosuppressed rhesus macaques by intraportal porcine islet xenografts precedes acute cellular rejection. Xenotransplantation 2004; 11: 396-407.
54. Buhler L, Deng S, O'Neil J, et al. Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade. Xenotransplantation 2002; 9: 3-13.
55. Rijkelijkhuizen JK, Haanstra KG, Wubben J, et al. T-cell-specific immunosuppression results in more than 53 days survival of porcine islets of Langerhans in the monkey. Transplantation 2003;76:1359-68.
56. Tseng YL, Dor FJ, Kuwaki K, et al. Bone marrow transplantation from alphal,3- galactosyl- transferase gene-knockout pigs in baboons. Xenotransplantation 2004;11 :361-70.
57. Cosimi AB, Sachs DH. Mixed chimerism and transplantation tolerance. Transplantation 2004; 77: 943-6.
58. Wood KJ. Is B cell tolerance essential for transplantation tolerance? Transplantation 2005; 79: S40-2.
59. Sykes M, Shimizu I, Kawahara T. Mixed hematopoietic chimerism for the simultaneous induction of T and B cell tolerance. Transplantation 2005; 79: S28-9.
60. Matsuda JL, Mallevaey T, Scott-Browne J, Gapin L.CD1d-restricted iNKT cells, the 'Swiss- Army knife' of the immune system. Curr Opin Immunol 2008; 20: 358-68.
61. Matsuda JL, Naidenko OV, Gapin L, et al. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J Exp Med 2000; 192: 741-54.
62. Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu Rev Immunol 2007; 25: 297-336.
63. Stewart TJ, Smyth MJ, Fernando GJ, Frazer IH, Leggatt GR. Inhibition of early tumor growth requires J18-positive (natural killer T) cells. Cancer Res 2003; 63: 3058-60.
64. Shimizu K, Goto A, Fukui M, Taniguchi M, Fujii S. Tumor cells loaded with α-galactosyl- ceramide induce innate NKT and NK cell-dependent resistance to tumor implantation in mice. J Immunol 2007; 178: 2853-61.
65. Soulard V, Roland J, Sellier C, et al. Primary infection of C57BL/6 mice with Plasmodium yoelii induces a heterogeneous response of NKT cells. Infect Immun 2007; 75: 2511-22.
66. Emoto M, Yoshizawa I, Emoto Y, et al. Reversible NK1.1 surface expression on invariant liver natural killer T cells during Listeria monocytogenes infection. Microbes Infect 2007; 9: 1511-20.
67. Oh K, Kim S, Park SH, et al. Direct regulatory role of NKT cells in allogeneic graft survival is dependent on the quantitative strength of antigenicity. J Immunol 2005; 174: 2030-6.
68. Yang SH, Jin JZ, Lee SH, et al. Role of NKT cells in allogeneic islet graft survival. Clin Immunol 2007; 124: 258-66.
69. Ramirez-Victorio F, Beilke JN, Gill RG. Both innate and adaptive major histocompatibility complex class I-dependent immunity impair long-term islet xenograft survival. Transplant Proc 2008; 40: 557-8.
70. Ikehara Y, Yasunami Y, Kodama S, et al. CD4(+) V alpha 14 natural killer T cells are essential for acceptance of rat islet xenografts in mice. J Clin Invest 2000; 105: 1761-7.
71. Park SH, Weiss A, Benlagha K, Kyin T, Teyton L, Bendelac A. The mouse CD1drestricted repertoire is dominated by a few autoreacitive T cell receptor families. J Exp Med 2001; 193: 893-904.
72. Cardell S, Tangri S, Chan S, Kronenberg M, Benoist C, Mathis D. CD1-restricted CD4+ T cells in MHC class II-deficient mice. J Exp Med 1995; 182: 993-1004.
73. KronenbergM, Engel I. On the road: p rogress in finding the unique pathway of invariant NKT cell differentiation. Curr Opin Immunol 2007; 19: 186 - 93.
74. Geissmann F, Cameron TO, Sidobre S, et al. Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids. PloS Biol 2005; 3: e113.
75. Benlagha K, Kyin T, Beavis A, Teyton L, Bendelac A. A thymic precursor to the NKT cell lineage. Science 2002; 296: 553-55.
76. McNab FW, Berzins SP, Pellicci DG, et al. The influence of CD1d in postselection NKT cell maturation and homeostasis. J. Immunol 2005; 175: 3762-68.
77. Brigl M, Brenner MB. CD1: antigen presentation and T cell function. Annu Rev Immunol 2004; 22: 817-90.
78. Brossay L, Jullien D, Cardell S, et al. Mouse CD1 is mainly expressed on hemopoietic-derived cells. J Immunol 1997; 159: 1216-24.
79. Park SH, Roark JH, Bendelac A. Tissue-specific recognition of mouse CD1 molecules. J Immunol 1998; 160: 3128-34.
80. Durante-Mangoni E, Wang R, Shaulov A, et al. Hepatic CDld expression in hepatitis C virus infection and recognition by resident proinflammatory CD Id-reactive T cells. J Immunol 2004; 173:2159-66.
81. Shimizu K, Goto A, Fukui M, Taniguchi M, Fujii S. Tumor cells loaded with α-galactosyl- ceramide induce innate NKT and NK cell-dependent resistance to tumor implantation in mice. J Immunol, 2007, 178: 2853-61.
82. Nieda M, Okai M, Tazbirkova A, et al. Therapeutic activation of V alpha 24+V beta 11+NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity. Blood 2004; 103: 383-9.
83. Giaccone G, Punt CJ, Ando Y, et al. A phase I study of the natural killer T-cell ligand alpha- galactosylceramide (KRN7000) in patients with solid tumors. Clin Cancer Res 2002; 8:3702-9.
84. Munz C, Steinman RM, Fujii S. Dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity. J Exp Med 2005; 202: 203-7.
85. Duthie MS , Kahn M , White M , et al. Both CD1d antigen presentation and interleukin-12 are required to activate natural killer T cells during trypanosoma cruzi infection. Infect Immun 2005; 73: 1890-4.
86. Wesley JD, Robbins SH, Sidobre S, et al. Cutting edge: IFN{gamma} signaling to macrophages is required for optimal V{alpha}14i NK TV NK cell crosstalk . J Immunol 2005; 174:3864-8.
87. Bezbradica J S, Stanic AK, Matsuki N, et al. Distinct roles of dendritic cells and B cells in Va14Ja18 natural T cell activation i n vivo. J Immunol 2005; 174: 4696 - 4705.
88. Van Kaer L. NKT cells: T lymphocytes with innate effector functions. Curr Opin Immunol 2007; 19:354-64.
89. Kojo S, Seino K, HaradaM, et al. Induction of regulatory properties in dendritic cells by Vα14NKT cells. J Immunol 2005; 175: 3648 - 55.
90. KronenbergM. Toward an understanding of NKT cell biology: progress and paradoxes. Annu Rev Imm unol 2005; 23: 877 -90.
91. Cui J , Sh in T, Kawano T, et al. Requirement fo r V alpha 14 NKT cells in IL-12 mediated rejection of tumours. Science 1997; 278: 1623-6.
92. Apostolou I, et al. Murine natural killer (sic) cells contribute to the granulomatous reaction caused by mycobacterial cell walls. Proc Nat Acad Sci USA 1999; 96: 5141-6.
93. Emoto M, Emoto Y, Buchwalow IB, Kaufmann SHE. Induction of IFN-g producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette-Guerin. Eur J Immunol 1999;29: 650-9.
94. Cooper AM, et al. Disseminated tuberculosis in IFN-g gene disrupted mice. J Exp Med 1993;178:2243-7.
95. Flynn JL, et al. An essential role for IFN-g in resistance to Mycobacterium tuberculosis infection. J Exp Med 1993; 178: 2249-54.
96. Flesch IEA, Wandersee A, Kaufmann SHE. IL-4 secretion by CD4+ NK1+ T cells induces monocyte chemoattractant protein-1 in early Listeriosis. J Immunol 1997; 159: 7-10.
97. Kadena T, et al. TCRab+CD4-CD8-T cells differentiate extrathymically in an Lckin dependent manner and participate in early response against Listeriai monocytogenese infection through interferon-g production. J Immunol 1997; 91: 511-9.
98. Pied S, et al. Liver CD4-CD8- NK1.1+ TCRab intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite stage in vitro. J Immunol 2000; 164: 1463-9.
99. Seki S, et al. Antimetastatic effect of NK1+ T cells on experimental haematogenous tumour metastases in the liver and lungs of mice. J Immunol 1997; 92: 561-6.
100. Smyth MJ, et al. Differential tumour surveillance by natural killer (NK) and NKT cells. J Exp Med 2000; 191: 661-8.
101. Nicol A, et al. Human invariant Va24+ natural killer T cells activated by a-galactosylceramide (KRN7000) have cytotoxic antitumour activity through mechanisms distinct from T cells and natural killer cells. J Immunol 2000; 99: 229-34.
102. Nakamura E, et al. Involvement of NK1+CD4-CD8- ab T cells and endogenous IL-4 in non- MHC-restricted rejection of embryonal carcinoma in genetically resistant mice. J Immunol 1997; 158:5338-48.
103. Hayakawa Y, Takeda K, Yagita H, et al.Critical contribution of IFN-gamma and NK cells, but not perforin-mediated cytotoxicity, to anti-meta-static effect of alpha-galactosylceramide. Eur J Immunol 2001; 31: 1720-7.
104. Albertsson PA, Basse PH, Hokland M, et al. NK cells and the tumour microenvironment: implications for NK-cell function and anti-tumour activity. Trends Immunol 2003; 24: 603-9.
105. Margalit M, Shibolet O, Klein A, et al. Suppression of hepatocellular carcinoma by transplantation of ex-vivo immune-modulated NKT lymphocytes. Int J Cancer 2005; 115:443-9.
106. Renukaradhya GJ, Sriram V, Du W, Gervay-Hague J, Van Kaer L, Brutkiewicz RR. Inhibition of antitumor immunity by invariant natural killer T cells in a T-cell lymphoma model in vivo. Int J Cancer 2006; 118: 3045-53.
107. Terabe M, Matsui S, Noben-Trauth N, et al. NKT cell mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nat Immunol 2000; 1: 515-20.
108. Crowe NY, Coquet JM, Berzins SP, et al. Differential antitumor immunity mediated by NKT cell subsets in vivo. J Exp Med 2005; 202: 1279-88.
109. Wang YF, Goldschneider I, Rourke JO, et al. Blood mononuclear cells induce regulatory NKT thymocytes in anterior chamber2associated immune deviation. Leukoc Bio 2001; 69: 7412-61.
110. Seino K, Fukao K, Muramoto K, et al. Requirement for natural killer T (NKT) cells in the induction of allograft tolerance. Proc Nat Acad Sci USA 2001; 98: 2577-81.
111. Oh K, Kim S ,Park SH, et al. Direct regulatory role of NKT cells in allogeneic graft survival is dependent on the quantitative strength of antigenicity. J Immunol 2005; 174: 2030-6.
112. Harharaguch IK, Takahash IT, Matsumoto A, et al.Host residual invariant NKT cells attenuate graft-versus-host immunity. J Immuno 2005; 175: 1320-28.
113. Toyofuku A, Yasunami Y, NabeyamaK, et al. Natural killer T-cells participate in rejection of islet allografts in the liver of mice. Diabetes 2006; 55: 34-9.
114. Galante NZ, Ozaki KS, Cenedeze MA, et al. Frequency of Valpha24+Vbetal 1+ NKT cells in peripheral blood of human kidney transplantation recipients. Int Immunopharmacol 2005; 5: 53-8.
115. Jones ND, Turvey SE,Van Maurik A, et al. Differential susceptibility of heart, skin, and islet allografts to T cell-mediated rejection. J Immunol 2001; 166: 2824-30.
116. La Cava A, Van Kaer L, Shi FD. CD4+CD25+ Tregs and NKT cells: regulators regulating regulators. Trends Immunol 2006; 27: 322-7.
117. Sonoda KH, Exley M, Snapper S, Balk SP, Stein-Streilein J. CD1-reactive natural killer Tcells are required for development of systemic tolerance through an immune-privileged site. J Exp Med 1999; 190:1215-26.
118. Liu R, La Cava A, Bai X-F, et al. Cooperation of invariant NKT cells and CD4+CD25+ regulatory T cells in the prevention of autoimmune myasthenia. J Immunol 2005; 175: 7898-904.
119. Ly D, Mi QS, Hussain S, Delovitch TL. Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells. J Immunol 2006; 177: 3695-704.
120. Palmer JL, Tulley JM, Kovacs EJ, Gamelli RL, Taniguchi M, Faunce DE. Injury-induced suppression of effector T cell immunity requires CD 1d-positive APCs and CD1d-restricted NKT cells. J Immunol 2006; 177: 92-9.
121.Naumov YN, Bahjat KS, Gausling R, et al. Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets. Proc Natl Acad Sci USA 2001; 98:13838-43.
122. Kojo S, Seino K-i, Harada M, et al. Induction of regulatory properties in dendritic cells by Va14 NKT cells. J Immunol 2005; 175: 3648-55.
123. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001; 94: 153-6.
124. Wada Y, Nakashima O, Kutami R, Yamamoto O, Kojiro M. Clinicopathological study on hepatocellular carcinoma with lymphocytic infiltration. Hepatology 1998; 27: 407-14.
125. Greten TF, Manns MP, Korangy F. Immunotherapy of HCC. Rev Recent Clin Trials 2008; 3: 31-9.
126. Stell A, Biserni A, Delia Torre S, et al. Cancer modeling: Modern imaging applications in the generation of novel animal model systems to study cancer progression and therapy. Int J Biochem Cell Biol 2007; 39: 1288-96.
127. Gao X, Cui Y, Levenson RM, Chung LW, Nie S . In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol 2004; 22: 959-60.
128. Voura EB, Jaiswal JK, Mattoussi H, Simon SM. Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy. Nat Med 2004; 10: 993-8.
129. Rao J, Dragulescu-Andrasi A, Yao H. Fluorescence imaging in vivo: recent advances. Curr Opin Biotechnol 2007; 18: 17-25.
130. Kovar JL, Simpson MA, Schutz-Geschwender A, Olive DM. A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models. Anal Biochem 2007; 367: 1-12.
131. Terai T, Nagano T. Fluorescent probes for bioimaging applications. Curr Opin Chem Biol. 2008; 12:515-21.
132. Ben N G Giepmans, S R Adams, M H Ellisan. The fluorescent toolbox for assessing protein location and function. Science 2006; 312: 217-23.
133. Carreon JR, Stewart KM, Mahon KP Jr, Shin S, Kelley SO. Cyanine dye conjugates as probes for live cell imaging. Bioorg Med Chem Lett 2007; 17: 5182-5.
134. Yan Y. Synthesis and nonlinear optical properties of newsymmetrically substituted stilbenes. J Solid State Chem 2003; 172: 364-9.
135. Li Y, Tang Y, Ye L, et al. Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis- related genes through cDNA microarray.J Cancer Res Clin Oncol 2003;129:43-51.
136.Taitz A,Petruzzelli G,Pak AS,et al.Immune parameters of mice bearing human head and neck cancer.Cancer Immunol Immunother 1995;40:283-91.
137.何维主编.医学免疫学.北京:人民卫生出版社,2005.
138.Hara M,Chosa E,Onitsuka T.The spleen's role in transplantation immunology.Transpl Immunol 2008;18:324-9.
139.Chandra AP,Ou-Yang L,Wong JK,et al.Association between islet xenograft rejection mediated by activated macrophages and upregulated chemokines.Zhong Nan Da Xue Xue Bao Yi Xue Ban 2007;32:26-35.
140.Kajiwara T,Tomita Y,Okano S.Effects of cyclosporin A on the activation of natural killer T cells induced by alpha-galactosylceramide.Transplantation 2007;83:184-92.
141.Zigmond E,Shalev Z,Pappo O,et al.NKT lymphocyte polarization determined by microenvironment signaling:a role for CD8+lymphocytes and beta-glycosphingolipids.J Autoimmun 2008;31:188-95.
142.Emoto M,Yoshizawa I,Emoto Y,et al.Rapid development of a gamma interferon-secreting glycolipid/CD1d-specific Va14t NK1.1~+T-cell subset after bacterial infection.Infect Immun 2006;74:5903-13.
143.Crowe NY,Uidrich AP,Kyparissoudis K,et al.Glycolipid antigen drives rapid expansion and sustained cytokine production by NK T cells.J Immunol 2003;171:4020-7.
144.Wilson MT,Johansson C,Olivares-Villagomez D,et al.The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion.Proc Nat Acad Sci USA 2003;100:10913-8.
2.Editorial:Human tumour xenografts.Br Med J 1974;16(5941):366-7.
3.Jacobson HI,Bennett JA,Mizejewski GJ.Inhibition of estrogen-dependent breast cancer growth by a reaction product of alpha-fetoprotein and estradiol.Cancer Res 1990;50:415.
4.Kaartinen M,Eray M,Lehtonen E,Matoso-Ferreira A,Tienari J.Implanted solid human tumours grow under the renal capsule of cyclosporin-immunosuppressed rats.Scand J Immunol 1994;39:618.
5..Rygaard J,Povlsen CO.Heterotransplantation of a human malignant tumor to 'nude' mice.Acta Path Microbiol Scand 1969;77:758-60.
6.汤钊猷,马曾层,应越英,等.裸鼠人体肝癌移植模型的研究.模型的建立及其血清学与形态学特征.上海第一医学院学报,1982;9:21-251.
7.胡卫,陈涛.鼠移植性肝癌模型研究进展.河南肿瘤学杂志,2003;16:463-6.
8.Murphy JB.Factors of resistance to heteroplastic tissue-grafting:studies in tisses specificity Ⅲ.J Exp Med 1914;19:513-522.
9.Shirai Y.Transplanting rat sarcoma in adult heterogenous animals.Jap Med World 1921;1:14-5.
10.Murphy JB,Sturm E.Conditions determining the transplantability of tissues in the brain.J Exp Med 1923;38:183-97.
11.Mandel TE,Dillon H,Koulmanda M.The effect of a depleting anti-CD4 monoclonal antibody on T cells and fetal pig islet xenograft survival in various strains of mice.Transpl Immunol 1995;3:265-72.
12.Simeonovic CJ.Xenogeneic islet transplantation.Xenotransplantation 1999;6:1-5.
13.Yi S,Feng X,Hawthorne WJ et al.Cells initiate pancreatic islet xenograft rejection via an interferon-gamma-dependent recruitment of macrophages and natural killer cells.Transplantation 2002;73:437-46.
14.Zhao Y,Swenson K,Wekerle T,Rodriguez-Barbosa JI,Arn JS,Sykes M.The critical role of mouse CD4+ cells in the rejection of highly disparate xenogeneic pig thymus grafts. Xenotransplantation 2000; 7: 129-37.
15. Friedman T, Smith RN, Colvin RB, Iacomini J. A critical role for human CD4+ T-cells in rejection of porcine islet cell xenografts. Diabetes 1999; 48: 2340-8.
16. Lechler RI, Lombardi G, Batchelor JR, Reinsmoen N, Bach FH. The molecular basis of alloreactivity. Immunol Today 1990; 11: 83.
17. Brouard S, Gagne K, Blancho G, and Soulillou JP. T Cell Response in Xenorecognition and Xenografts: A Review. Human Immunology 1999; 60: 455-68.
18. Ricordi C, Lacy PE. Renal subcapsular xenotransplantation of purified porcine islets. Transplantation 1987; 44: 721.
19. Korsgren O, Sandier S, Landstrom AS, Jansson K. Andersson A. Large-scale production of foetal porcine pancreatic islet like cell clusters. An expeimental tool for studies of islet cell differentiation and xenotransplantation. Transplantation 1988; 45: 509.
20. Sandier S, Andersson A, Schnell A, et al. Tissue culture of human foetal pancreas. Development and function of B-cells in vitro and transplantation of explants to nude mice. Diabetes 1985; 34: 1113.
21. Simeonovic CJ, Ceredig R, Wilson JD. Effect of GK1.5 monoclonal antibody dosage on survival of pig proislet xenografts in CD41 T cell-depleted mice. Transplantation 1990; 49: 849.
22. Morris CF, Simenovic CJ, Fung MC, Wilson JD, Hapel AJ. Intragraft expression of cytokine transcripts during pig proislet xenograft rejection and tolerance in mice. J Immunol 1995; 154: 2470.
23. Zhan Y, Brady JL, Irawaty W, Thomas HE, Kay TW, Lew AM. Activated macrophages require T cells for xenograft rejection under the kidney capsule. Immunol Cell Biol. 2003; 81: 451-8.
24. Benda B, Karlsson-Parra A, Ridderstad A, Korsgren O. Xenograft rejection of porcine islet- like cell clusters in immunoglobulin- or Fc-receptor gamma-deficient mice. Transplantation 1996; 62:1207.
25. Medbury HJ, Hibbins M, Lehnert AM, et al. The cytokine and histological response in islet xenograft rejection is dependent upon species combination. Transplantation 1997; 64: 1307.
26. Simeonovic CJ, Townsend MJ, Wilson JD, et al. Eosinophils are not required for the rejection of neovascularized foetal pig proislet xenografts in mice. J Immunol 1997; 158: 2490.
27. Backes MG, Lund RD, Lagenaur CF, Kunz HW, Gill TJ. Cellular events associated with peripherally induced rejection of mature neural xenografts placed into neonatal rat brains. J Comp Neurol 1990; 295: 428.
28. Finsen BR, Sorensen T, Castellano B, et al. Leukocyte infiltration and glial reactions in xenografts of mouse brain tissue undergoing rejection in the adult rat brain. A light and electron microscopical immunocytochemical study. Immunocytochemical characterization of the cellular immune response to intracerebral xenografts of brain tissue. J Neuroimmunol 1991;32:159.
29. Mason DW, Charlton HM, Jones A, Lavy CB, Puklavec M. Simmonds SJ. The fate of allogeneic and xenogeneic neuronal tissue transplanted into the third ventricle of rodents. Neuroscience 1986; 19:685.
30. Wood M, Sloan D, Wood K, Charlton H. Indefinite survival of neural xenografts induced with anti-CD4 monoclonal antibodies. Neuroscience 1996; 70: 775.
31. Honey CR, Clarke DJ, Dallman MJ, Charlton HM. Human neural graft function in rats treated with anti-interleukin II receptor antibody. Neuroreport 1990; 1: 247.
32. Pedersen EB, Zimmer J, Finsen B. Triple immunosuppression protects murine intracerebral, hippocampal xenografts in adult rat hosts: effects on cellular infiltration, major histocompatibility complex antigen induction and blood-brain barrier leakage. Neuroscience 1997;78:685.
33. Smyth MJ, Kershaw MH, Trapani JA. Xenospecific cytotoxic T lymphocytes: potent lysis in vitro and in vivo. Transplantation 1997; 63: 1171-8.
34. Abe M, Cheng J, Qi J, et al. Elimination of porcine hemopoietic cells by macrophages in mice. J Immunol 2002; 168: 621-8.
35. Fox A, Mountford J, Braakhuis A, Harrison LC. Innate and adaptive immune responses to nonvascular xenografts: evidence that macrophages are direct effectors of xenograft rejection. J Immunol 2001; 166: 2133-40.
36. Loudovaris T, Mandel TE, Charlton B. CD 4+ T cell mediated destruction of xenografts within cell-impermeable membranes in the absence of CD8+ T cells and B cells. Transplantation 1996; 61: 1678-84.
37. Zhan Y, Brady JL, Sutherland RM, Lew AM. Without CD4 help, CD8 rejection of pig xenografts requires CD28 costimulation but not perforin killing. J Immunol 2001; 167: 6279-85.
38. Wu G, Korsgren O, Zhang J, Song Z, van Rooijen N, Tibell A. Pig islet xenograft rejection is markedly delayed in macrophagedepleted mice: a study in streptozotocin diabetic animals. Xenotransplantation 2000; 7: 214-20.
39. Bancroft GJ, Schreiber RD, Unanue ER. Natural immunity: a T-cell- independent pathway of macrophage activation, defined in the scid mouse. Immunol Rev 1991; 124: 5-24.
40. Salazar-Mather TP, Orange JS, Biron CA. Early murine cytomegalovirus (MCMV) infection induces liver natural killer (NK) cell inflammation and protection through macrophage inflammatory protein lalpha (MIP-1 alpha) -dependent pathways. J Exp Med 1998; 187: 1-14.
41. Terpstra W, Leenen PJ, van den Bos C et al. Facilitated engraftment of human hematopoietic cells in severe combined immunodeficient mice following a single injection of C12MDP liposomes. Leukemia 1997; 11: 1049-54.
42. Hancock WW, Miyatake T, Koyamada N, et al. Effects of leflunomide and deoxyspergualin in the guinea pig-rat cardiac model of delayed xenograft rejection: suppression of B cell and C-C chemokine responses but not induction of macrophage lectin. Transplantation (in press).
43. Hancock WW. Immunopathology of discordant xenograft rejection. In Xenotransplantation: The Transplantation of Organs and Tissues Between Species, D.K.C. Cooper, E. Kemp, J.L. Platt, D.J.G. White, editors. Berlin, Springer, 1997, pp. 303-313.
44. Hancock WW. Chemokines and the pathogenesis of T cell-dependent immune responses. Am J Pathol 1996; 148:681.
45. Benbaruch A, Xu LL, Young PR, Bengali K, Oppenheim JJ, Wang JM. Monocyte chemotactic protein-3 (MCP3) interacts with multiple leukocyte receptors: C-C CKR1, a receptor for macrophage inflammatory protein-1 alpha/Rantes, is also a functional receptor for MCP3. J Biol Chem 1995; 270: 22123.
46. Horuk R, Colby TJ, Darbonne WC, Schall TJ, Neote K. The human erythrocyte inflammatory peptide (chemokine) receptor: biochemical characterization, solubilization, and development of a binding assay for the soluble receptor. Biochemistry 1993; 32: 5733.
47. Colotta F, Sciacca FL, Sironi M, Luini W, Rabiet MJ, Mantovani A. Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin. Am J Pathol 1994;144:975.
48. Medzhitov R, Janeway CA. Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 1997; 9: 4.
49. Saton M, Kawakami K, Osawa T, Toyoshima S. Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages. J Biochem.(Tokyo) 1992; 111 :331.
50. Candinas D, Koyamada N, Miyatake T, et al. T cell-independence of macrophage and NK cell infiltration, cytokine production and endothelial activation during delayed xenograft rejection. Transplantation 1996; 62:1920.
51. Millan MT, Geczy C, Stuhlmeier K, Goodman DJ, Ferran C, Bach FH. Human monocytes activate porcine endothelial cells, resulting in increased E-selectin, interleukin-8, monocyte chemotactic protein-1, and plasminogen activator inhibitor-type 1 expression. Transplantation 1997; 63:421.
52. Goodman D, von Albertini M, Willson A, Millan M, Bach FH. Direct activation of porcine endothelial cells by human natural killer cells. Transplantation 1996; 61: 763.
53. Kirchhof N, Shibata S, Wijkstrom M, et al. Reversal of diabetes in non-immunosuppressed rhesus macaques by intraportal porcine islet xenografts precedes acute cellular rejection. Xenotransplantation 2004; 11: 396-407.
54. Buhler L, Deng S, O'Neil J, et al. Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade. Xenotransplantation 2002; 9: 3-13.
55. Rijkelijkhuizen JK, Haanstra KG, Wubben J, et al. T-cell-specific immunosuppression results in more than 53 days survival of porcine islets of Langerhans in the monkey. Transplantation 2003;76:1359-68.
56. Tseng YL, Dor FJ, Kuwaki K, et al. Bone marrow transplantation from alphal,3- galactosyl- transferase gene-knockout pigs in baboons. Xenotransplantation 2004;11 :361-70.
57. Cosimi AB, Sachs DH. Mixed chimerism and transplantation tolerance. Transplantation 2004; 77: 943-6.
58. Wood KJ. Is B cell tolerance essential for transplantation tolerance? Transplantation 2005; 79: S40-2.
59. Sykes M, Shimizu I, Kawahara T. Mixed hematopoietic chimerism for the simultaneous induction of T and B cell tolerance. Transplantation 2005; 79: S28-9.
60. Matsuda JL, Mallevaey T, Scott-Browne J, Gapin L.CD1d-restricted iNKT cells, the 'Swiss- Army knife' of the immune system. Curr Opin Immunol 2008; 20: 358-68.
61. Matsuda JL, Naidenko OV, Gapin L, et al. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J Exp Med 2000; 192: 741-54.
62. Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu Rev Immunol 2007; 25: 297-336.
63. Stewart TJ, Smyth MJ, Fernando GJ, Frazer IH, Leggatt GR. Inhibition of early tumor growth requires J18-positive (natural killer T) cells. Cancer Res 2003; 63: 3058-60.
64. Shimizu K, Goto A, Fukui M, Taniguchi M, Fujii S. Tumor cells loaded with α-galactosyl- ceramide induce innate NKT and NK cell-dependent resistance to tumor implantation in mice. J Immunol 2007; 178: 2853-61.
65. Soulard V, Roland J, Sellier C, et al. Primary infection of C57BL/6 mice with Plasmodium yoelii induces a heterogeneous response of NKT cells. Infect Immun 2007; 75: 2511-22.
66. Emoto M, Yoshizawa I, Emoto Y, et al. Reversible NK1.1 surface expression on invariant liver natural killer T cells during Listeria monocytogenes infection. Microbes Infect 2007; 9: 1511-20.
67. Oh K, Kim S, Park SH, et al. Direct regulatory role of NKT cells in allogeneic graft survival is dependent on the quantitative strength of antigenicity. J Immunol 2005; 174: 2030-6.
68. Yang SH, Jin JZ, Lee SH, et al. Role of NKT cells in allogeneic islet graft survival. Clin Immunol 2007; 124: 258-66.
69. Ramirez-Victorio F, Beilke JN, Gill RG. Both innate and adaptive major histocompatibility complex class I-dependent immunity impair long-term islet xenograft survival. Transplant Proc 2008; 40: 557-8.
70. Ikehara Y, Yasunami Y, Kodama S, et al. CD4(+) V alpha 14 natural killer T cells are essential for acceptance of rat islet xenografts in mice. J Clin Invest 2000; 105: 1761-7.
71. Park SH, Weiss A, Benlagha K, Kyin T, Teyton L, Bendelac A. The mouse CD1drestricted repertoire is dominated by a few autoreacitive T cell receptor families. J Exp Med 2001; 193: 893-904.
72. Cardell S, Tangri S, Chan S, Kronenberg M, Benoist C, Mathis D. CD1-restricted CD4+ T cells in MHC class II-deficient mice. J Exp Med 1995; 182: 993-1004.
73. KronenbergM, Engel I. On the road: p rogress in finding the unique pathway of invariant NKT cell differentiation. Curr Opin Immunol 2007; 19: 186 - 93.
74. Geissmann F, Cameron TO, Sidobre S, et al. Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids. PloS Biol 2005; 3: e113.
75. Benlagha K, Kyin T, Beavis A, Teyton L, Bendelac A. A thymic precursor to the NKT cell lineage. Science 2002; 296: 553-55.
76. McNab FW, Berzins SP, Pellicci DG, et al. The influence of CD1d in postselection NKT cell maturation and homeostasis. J. Immunol 2005; 175: 3762-68.
77. Brigl M, Brenner MB. CD1: antigen presentation and T cell function. Annu Rev Immunol 2004; 22: 817-90.
78. Brossay L, Jullien D, Cardell S, et al. Mouse CD1 is mainly expressed on hemopoietic-derived cells. J Immunol 1997; 159: 1216-24.
79. Park SH, Roark JH, Bendelac A. Tissue-specific recognition of mouse CD1 molecules. J Immunol 1998; 160: 3128-34.
80. Durante-Mangoni E, Wang R, Shaulov A, et al. Hepatic CDld expression in hepatitis C virus infection and recognition by resident proinflammatory CD Id-reactive T cells. J Immunol 2004; 173:2159-66.
81. Shimizu K, Goto A, Fukui M, Taniguchi M, Fujii S. Tumor cells loaded with α-galactosyl- ceramide induce innate NKT and NK cell-dependent resistance to tumor implantation in mice. J Immunol, 2007, 178: 2853-61.
82. Nieda M, Okai M, Tazbirkova A, et al. Therapeutic activation of V alpha 24+V beta 11+NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity. Blood 2004; 103: 383-9.
83. Giaccone G, Punt CJ, Ando Y, et al. A phase I study of the natural killer T-cell ligand alpha- galactosylceramide (KRN7000) in patients with solid tumors. Clin Cancer Res 2002; 8:3702-9.
84. Munz C, Steinman RM, Fujii S. Dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity. J Exp Med 2005; 202: 203-7.
85. Duthie MS , Kahn M , White M , et al. Both CD1d antigen presentation and interleukin-12 are required to activate natural killer T cells during trypanosoma cruzi infection. Infect Immun 2005; 73: 1890-4.
86. Wesley JD, Robbins SH, Sidobre S, et al. Cutting edge: IFN{gamma} signaling to macrophages is required for optimal V{alpha}14i NK TV NK cell crosstalk . J Immunol 2005; 174:3864-8.
87. Bezbradica J S, Stanic AK, Matsuki N, et al. Distinct roles of dendritic cells and B cells in Va14Ja18 natural T cell activation i n vivo. J Immunol 2005; 174: 4696 - 4705.
88. Van Kaer L. NKT cells: T lymphocytes with innate effector functions. Curr Opin Immunol 2007; 19:354-64.
89. Kojo S, Seino K, HaradaM, et al. Induction of regulatory properties in dendritic cells by Vα14NKT cells. J Immunol 2005; 175: 3648 - 55.
90. KronenbergM. Toward an understanding of NKT cell biology: progress and paradoxes. Annu Rev Imm unol 2005; 23: 877 -90.
91. Cui J , Sh in T, Kawano T, et al. Requirement fo r V alpha 14 NKT cells in IL-12 mediated rejection of tumours. Science 1997; 278: 1623-6.
92. Apostolou I, et al. Murine natural killer (sic) cells contribute to the granulomatous reaction caused by mycobacterial cell walls. Proc Nat Acad Sci USA 1999; 96: 5141-6.
93. Emoto M, Emoto Y, Buchwalow IB, Kaufmann SHE. Induction of IFN-g producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette-Guerin. Eur J Immunol 1999;29: 650-9.
94. Cooper AM, et al. Disseminated tuberculosis in IFN-g gene disrupted mice. J Exp Med 1993;178:2243-7.
95. Flynn JL, et al. An essential role for IFN-g in resistance to Mycobacterium tuberculosis infection. J Exp Med 1993; 178: 2249-54.
96. Flesch IEA, Wandersee A, Kaufmann SHE. IL-4 secretion by CD4+ NK1+ T cells induces monocyte chemoattractant protein-1 in early Listeriosis. J Immunol 1997; 159: 7-10.
97. Kadena T, et al. TCRab+CD4-CD8-T cells differentiate extrathymically in an Lckin dependent manner and participate in early response against Listeriai monocytogenese infection through interferon-g production. J Immunol 1997; 91: 511-9.
98. Pied S, et al. Liver CD4-CD8- NK1.1+ TCRab intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite stage in vitro. J Immunol 2000; 164: 1463-9.
99. Seki S, et al. Antimetastatic effect of NK1+ T cells on experimental haematogenous tumour metastases in the liver and lungs of mice. J Immunol 1997; 92: 561-6.
100. Smyth MJ, et al. Differential tumour surveillance by natural killer (NK) and NKT cells. J Exp Med 2000; 191: 661-8.
101. Nicol A, et al. Human invariant Va24+ natural killer T cells activated by a-galactosylceramide (KRN7000) have cytotoxic antitumour activity through mechanisms distinct from T cells and natural killer cells. J Immunol 2000; 99: 229-34.
102. Nakamura E, et al. Involvement of NK1+CD4-CD8- ab T cells and endogenous IL-4 in non- MHC-restricted rejection of embryonal carcinoma in genetically resistant mice. J Immunol 1997; 158:5338-48.
103. Hayakawa Y, Takeda K, Yagita H, et al.Critical contribution of IFN-gamma and NK cells, but not perforin-mediated cytotoxicity, to anti-meta-static effect of alpha-galactosylceramide. Eur J Immunol 2001; 31: 1720-7.
104. Albertsson PA, Basse PH, Hokland M, et al. NK cells and the tumour microenvironment: implications for NK-cell function and anti-tumour activity. Trends Immunol 2003; 24: 603-9.
105. Margalit M, Shibolet O, Klein A, et al. Suppression of hepatocellular carcinoma by transplantation of ex-vivo immune-modulated NKT lymphocytes. Int J Cancer 2005; 115:443-9.
106. Renukaradhya GJ, Sriram V, Du W, Gervay-Hague J, Van Kaer L, Brutkiewicz RR. Inhibition of antitumor immunity by invariant natural killer T cells in a T-cell lymphoma model in vivo. Int J Cancer 2006; 118: 3045-53.
107. Terabe M, Matsui S, Noben-Trauth N, et al. NKT cell mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nat Immunol 2000; 1: 515-20.
108. Crowe NY, Coquet JM, Berzins SP, et al. Differential antitumor immunity mediated by NKT cell subsets in vivo. J Exp Med 2005; 202: 1279-88.
109. Wang YF, Goldschneider I, Rourke JO, et al. Blood mononuclear cells induce regulatory NKT thymocytes in anterior chamber2associated immune deviation. Leukoc Bio 2001; 69: 7412-61.
110. Seino K, Fukao K, Muramoto K, et al. Requirement for natural killer T (NKT) cells in the induction of allograft tolerance. Proc Nat Acad Sci USA 2001; 98: 2577-81.
111. Oh K, Kim S ,Park SH, et al. Direct regulatory role of NKT cells in allogeneic graft survival is dependent on the quantitative strength of antigenicity. J Immunol 2005; 174: 2030-6.
112. Harharaguch IK, Takahash IT, Matsumoto A, et al.Host residual invariant NKT cells attenuate graft-versus-host immunity. J Immuno 2005; 175: 1320-28.
113. Toyofuku A, Yasunami Y, NabeyamaK, et al. Natural killer T-cells participate in rejection of islet allografts in the liver of mice. Diabetes 2006; 55: 34-9.
114. Galante NZ, Ozaki KS, Cenedeze MA, et al. Frequency of Valpha24+Vbetal 1+ NKT cells in peripheral blood of human kidney transplantation recipients. Int Immunopharmacol 2005; 5: 53-8.
115. Jones ND, Turvey SE,Van Maurik A, et al. Differential susceptibility of heart, skin, and islet allografts to T cell-mediated rejection. J Immunol 2001; 166: 2824-30.
116. La Cava A, Van Kaer L, Shi FD. CD4+CD25+ Tregs and NKT cells: regulators regulating regulators. Trends Immunol 2006; 27: 322-7.
117. Sonoda KH, Exley M, Snapper S, Balk SP, Stein-Streilein J. CD1-reactive natural killer Tcells are required for development of systemic tolerance through an immune-privileged site. J Exp Med 1999; 190:1215-26.
118. Liu R, La Cava A, Bai X-F, et al. Cooperation of invariant NKT cells and CD4+CD25+ regulatory T cells in the prevention of autoimmune myasthenia. J Immunol 2005; 175: 7898-904.
119. Ly D, Mi QS, Hussain S, Delovitch TL. Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells. J Immunol 2006; 177: 3695-704.
120. Palmer JL, Tulley JM, Kovacs EJ, Gamelli RL, Taniguchi M, Faunce DE. Injury-induced suppression of effector T cell immunity requires CD 1d-positive APCs and CD1d-restricted NKT cells. J Immunol 2006; 177: 92-9.
121.Naumov YN, Bahjat KS, Gausling R, et al. Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets. Proc Natl Acad Sci USA 2001; 98:13838-43.
122. Kojo S, Seino K-i, Harada M, et al. Induction of regulatory properties in dendritic cells by Va14 NKT cells. J Immunol 2005; 175: 3648-55.
123. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001; 94: 153-6.
124. Wada Y, Nakashima O, Kutami R, Yamamoto O, Kojiro M. Clinicopathological study on hepatocellular carcinoma with lymphocytic infiltration. Hepatology 1998; 27: 407-14.
125. Greten TF, Manns MP, Korangy F. Immunotherapy of HCC. Rev Recent Clin Trials 2008; 3: 31-9.
126. Stell A, Biserni A, Delia Torre S, et al. Cancer modeling: Modern imaging applications in the generation of novel animal model systems to study cancer progression and therapy. Int J Biochem Cell Biol 2007; 39: 1288-96.
127. Gao X, Cui Y, Levenson RM, Chung LW, Nie S . In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol 2004; 22: 959-60.
128. Voura EB, Jaiswal JK, Mattoussi H, Simon SM. Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy. Nat Med 2004; 10: 993-8.
129. Rao J, Dragulescu-Andrasi A, Yao H. Fluorescence imaging in vivo: recent advances. Curr Opin Biotechnol 2007; 18: 17-25.
130. Kovar JL, Simpson MA, Schutz-Geschwender A, Olive DM. A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models. Anal Biochem 2007; 367: 1-12.
131. Terai T, Nagano T. Fluorescent probes for bioimaging applications. Curr Opin Chem Biol. 2008; 12:515-21.
132. Ben N G Giepmans, S R Adams, M H Ellisan. The fluorescent toolbox for assessing protein location and function. Science 2006; 312: 217-23.
133. Carreon JR, Stewart KM, Mahon KP Jr, Shin S, Kelley SO. Cyanine dye conjugates as probes for live cell imaging. Bioorg Med Chem Lett 2007; 17: 5182-5.
134. Yan Y. Synthesis and nonlinear optical properties of newsymmetrically substituted stilbenes. J Solid State Chem 2003; 172: 364-9.
135. Li Y, Tang Y, Ye L, et al. Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis- related genes through cDNA microarray.J Cancer Res Clin Oncol 2003;129:43-51.
136.Taitz A,Petruzzelli G,Pak AS,et al.Immune parameters of mice bearing human head and neck cancer.Cancer Immunol Immunother 1995;40:283-91.
137.何维主编.医学免疫学.北京:人民卫生出版社,2005.
138.Hara M,Chosa E,Onitsuka T.The spleen's role in transplantation immunology.Transpl Immunol 2008;18:324-9.
139.Chandra AP,Ou-Yang L,Wong JK,et al.Association between islet xenograft rejection mediated by activated macrophages and upregulated chemokines.Zhong Nan Da Xue Xue Bao Yi Xue Ban 2007;32:26-35.
140.Kajiwara T,Tomita Y,Okano S.Effects of cyclosporin A on the activation of natural killer T cells induced by alpha-galactosylceramide.Transplantation 2007;83:184-92.
141.Zigmond E,Shalev Z,Pappo O,et al.NKT lymphocyte polarization determined by microenvironment signaling:a role for CD8+lymphocytes and beta-glycosphingolipids.J Autoimmun 2008;31:188-95.
142.Emoto M,Yoshizawa I,Emoto Y,et al.Rapid development of a gamma interferon-secreting glycolipid/CD1d-specific Va14t NK1.1~+T-cell subset after bacterial infection.Infect Immun 2006;74:5903-13.
143.Crowe NY,Uidrich AP,Kyparissoudis K,et al.Glycolipid antigen drives rapid expansion and sustained cytokine production by NK T cells.J Immunol 2003;171:4020-7.
144.Wilson MT,Johansson C,Olivares-Villagomez D,et al.The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion.Proc Nat Acad Sci USA 2003;100:10913-8.