氧化应激可选择性诱导细胞的NKG2D配体的表达

目的 探讨氧化应激与细胞NKG2D配体表达的关系,分析氧化应激对NK细胞功能的影响。方法 加H2O2诱导培养的肿瘤细胞处于氧化应激状态。用RT-PCR、Real-time PCR和流式细胞仪等方法分析细胞多种NKG2D配体的表达。用CCK-8法检测NK92细胞对肿瘤细胞的杀伤活性。结果 氧化应激可诱导肿瘤细胞多种NKG2D配体的表达,不同的肿瘤细胞诱导表达的NKG2D配体不同;NKG2D配体表达上调可有效提高NK细胞的细胞毒活性,此效应可被抗NKG2D抗体所阻断。结论 NKG2D配体可能在机体的免疫应答中发挥正向的调节作用。     

NKG2D及其配体研究进展

NKG2D是较为独特的NK细胞活化性受体，其配体具有多样性，因而其识别机制较独特；其表达范围不仅局限于NK细胞，还在T细胞、巨噬细胞、树突状细胞中有表达，功能上，不仅有直接刺激作用，还能作为协同刺激分子传递第二信号。NKG2D及其配体的研究对抗肿瘤免疫、抗感染免疫、自身免疫病的认识具有重要意义。本文对NKG2D及其配体的研究进展作一综述。     

NKG2D及其配体研究进展

NKG2D是较为独特的NK细胞活化性受体 ,其配体具有多样性 ,因而其识别机制较独特 ;其表达范围不仅局限于NK细胞 ,还在T细胞、巨噬细胞、树突状细胞中有表达 ,功能上 ,不仅有直接刺激作用 ,还能作为协同刺激分子传递第二信号。NKG2D及其配体的研究对抗肿瘤免疫、抗感染免疫、自身免疫病的认识具有重要意义。本文对NKG2D及其配体的研究进展作一综述。     

活化性受体NKG2D及其配体的研究进展

NK细胞是肌体免疫系统至关重要的组成部分,其表达多种活化性和抑制性细胞表面受体。NKG2D是较为独特的活化性受体,属C型凝集素家族跨膜蛋白,分布较广,NK细胞、T细胞和其他免疫细胞都可以产生,其配体具有多样性,MHCⅠ类相关分子（MIC）是人类NKG2D识别的配体之一,应激性表达在一些肿瘤细胞或病原体感染细胞的表面。NKG2D既能直接活化NK细胞,又能以协同刺激的方式促进CD8^＋αβT细胞的活化,在抗肿瘤免疫和病毒感染等方面发挥重要作用。     

结核杆菌耐热抗原对人外周血淋巴细胞NKG2A/NKG2D的影响

目的：以结核杆菌耐热抗原为刺激剂，观察人外岗血淋巴细胞NKG2A受体和NKG2D受体表达量的变化情况。方法：用结核杆菌耐热抗原刺激人外周血淋巴细胞，用流式细胞技术检测NKG2A受体和NKG2D受体的变化。使用RT-PCR和ELISA检测PBMCs中IFN-γ的表达情况。结果：在IL-2和抗原联合刺激下，NKG2A受体的表达量在第12天大幅度上升，NKG2D受体的表达量始终变化不大。NKG2A受体的表达在T细胞、NK细胞、αβT细胞和γδT细胞表面都有不同程度的增加。结论：NKG2A/NKG2D比例的上升有可能会对免疫系统造成一些影响，其中IFN-γ起着不可忽视的作用。     

NKG2D真核表达载体的构建及其对YT细胞生物学功能的影响

目的：建立NK细胞受体NKG2D真核表达载体，通过转染NK细胞系YT，初步探讨NKG2D分子对YT细胞系杀伤功能的增强作用。方法：用RT-PCR方法从NK-92细胞中调取NKG2D基因片段，克隆到pGEM-TEasy载体并对克隆的DNA片段进行序列分析。用限制内切酶EcoRⅠ和BamHⅠ消化pGEM-T Easy／NKG2D重组质粒，分离NKG2D片段，并插入真核表达质粒pEGFP-N1的相应限制酶位点，酶谱分析鉴定重组表达载体pEGFP-N1／NKG2D。然后经脂质体介导转染CHO细胞和YT细胞。应用荧光显微镜观测、Western blot方法和免疫组化染色对转染细胞内pEGFP-NilNKG2D的表达进行鉴定，MTF方法观察YT细胞对肿瘤细胞的杀伤功能。结果：RT-PCR扩增获得650bp基因片段，经DNA序列分析证明所获得的DNA序列与文献报道的NKG2D序列一致。转染的CHO细胞在荧光显微镜下发出强绿色荧光，Western blot分析显示重组蛋白能特异地与抗人NKG2D单克隆抗体结合；免疫组化检测显示，转染的CHO中有棕色颗粒，证明所构建的NKG2D真核表达载体可以在细胞中表达；转染NKG2D真核表达载体的YT细胞对乳腺癌细胞具有更强的杀伤效果。结论：所获得的表达NKG2D分子的真核表达载体，通过转染YT细胞，初步鉴定所表达NKG2D分子具有生物学功能，可以提高NK细胞对肿瘤细胞的杀伤活性。     

NKG2D配体在鼻咽癌细胞的表达及其对NK细胞杀伤活性的影响

NK细胞对靶细胞的杀伤活性与其细胞表面的受体和靶细胞表面的配体密切相关，NKG2D为NK细胞活化性受体，表达于所有的NK细胞表面，是介导NK细胞识别和溶解肿瘤细胞的主要活化性受体。NKG2D配体为MHCⅠ类链相关基因产物（MICA、MICB）及ULBPS（人巨细胞病毒UL16蛋白的结合蛋白ULBP1、ULBP2、ULBP3），NKG2D的配体在多种肿瘤细胞表达，其在鼻咽癌细胞的表达尚未见报道。本文通过流式细胞仪技术探讨其在鼻咽癌细胞CNE2的表达情况，并进一步分析其在NK细胞杀伤CNE2细胞中的作用。     

NKG2D及其配体在白血病免疫监视中的作用

NKG2D是免疫细胞表面的激活性受体,在机体的抗肿瘤免疫、抗感染免疫以及自身免疫病的发生中发挥重要作用。NKG2D识别肿瘤细胞表面的配体,激活效应细胞,产生有效的抗肿瘤免疫应答,是肿瘤免疫监视机制之一。近来研究发现白血病细胞表达多种NKG2D配体,参与白血病的免疫监视。本文就NKG2D及其配体对白血病免疫监视和免疫逃逸的作用作一综述。     

妊娠期uNK、pNK细胞NKG2A与NKG2D的表达及其生物学意义的研究

目的:研究妊娠期妇女子宫NK细胞(uNK细胞)与外周血NK细胞(pNK细胞)表面NKG2A和NKG2D及其相应配体的表达,探讨uNK细胞表面NKG2A和NKG2D的不平衡表达与母胎界面所形成的免疫耐受关系。方法:采用流式细胞术检测30例孕6～9周的正常妊娠妇女uNK细胞和pNK细胞NKG2A、NKG2D的表达状况;RTPCR技术检测绒毛膜组织HLAE、MICA的表达。结果:子宫NK细胞NKG2A的表达显著高于外周血NK细胞,二者分别为(97.86±1.75)%与(33.35±10.92)%;子宫NK细胞NKG2D的表达水平与外周血NK细胞相近,分别为(93.21±4.52)%与(97.80±1.72)%,滋养层组织仅检测到HLAEmRNA的表达。结论:妊娠期子宫NK细胞表面高表达抑制性受体NKG2A,同时滋养层组织表达相应的配体HLAE,这可能是维持母胎界面免疫耐受的重要因素。     

卵巢肿瘤患者自然杀伤细胞活化性受体NKG2D与其配体MICA表达的初步研究

目的:研究卵巢癌、良性卵巢肿瘤患者外周血NK细胞表面活化性受体NKG2D的表达及局部组织中相应配体MICA的表达情况,并结合临床病理因素分析探讨宿主NK细胞受体NKG2D在抗卵巢癌中的作用及其与肿瘤免疫逃逸的关系。方法:对4 2例卵巢癌、2 3例良性卵巢肿瘤及2 0例正常妇女,采用流式细胞术检测外周血NK细胞NKG2D的表达状况,RT PCR技术检测在上述部分相应组织标本中MICA的表达。结果:恶性、良性卵巢肿瘤患者及正常人外周血NK细胞NKG2D的表达水平分别为( 94 2 3±6 0 2 ) %、( 98 70±0 98) %、( 98 6 1±1 5 9) % ,恶性组与另两组之间比较,差异有统计学意义(P <0 0 5 ) ;相应配体MICA在卵巢癌组织中的表达率较良性卵巢肿瘤中明显增高,差异有统计学意义(P <0 0 1) ;在卵巢癌病人是否绝经、不同组织类型、分化程度、手术分期及是否淋巴转移等各组临床病理情况下,其表达率未见明显差异(P >0 0 5 )。结论:卵巢恶性肿瘤患者外周血NK细胞活性降低,其活化性受体NKG2D表达的下降是NK细胞活性下降的原因之一。NKG2D的配体MICA的基因表达可能与卵巢癌的恶性转化有一定的相关性,卵巢癌的免疫逃逸可能与NKG2D表达下调及其配体MI CA的表达升高有关     

Regulation of ligands for the activating receptor NKG2D

The outcome of an encounter between a cytotoxic cell and a potential target cell depends on the balance of signals from inhibitory and activating receptors. Natural Killer group 2D (NKG2D) has recently emerged as a major activating receptor on T lymphocytes and natural killer cells. In both humans and mice, multiple different genes encode ligands for NKG2D, and these ligands are non-classical major histocompatibility complex class I molecules. The NKG2D-ligand interaction triggers an activating signal in the cell expressing NKG2D and this promotes cytotoxic lysis of the cell expressing the ligand. Most normal tissues do not express ligands for NKG2D, but ligand expression has been documented in tumour and virus-infected cells, leading to lysis of these cells. Tight regulation of ligand expression is important. If there is inappropriate expression in normal tissues, this will favour autoimmune processes, whilst failure to up-regulate the ligands in pathological conditions would favour cancer development or dissemination of intracellular infection.     

Murine cytomegalovirus regulation of NKG2D ligands

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes morbidity risk in immunologically suppressed and immunodeficient patients including congenital infections. Approaches to curb the consequences of HCMV infections are restricted by a lack of complete understanding of viral pathogenesis. The infection of mice with murine cytomegalovirus (MCMV) as a model of HCMV infection has been particularly useful in elucidating the role of innate and adaptive immune response mechanisms. A large number of cytomegalovirus genes modulate the innate and the adaptive host immune response. The products of several MCMV genes are involved in subverting the natural killer (NK) cell response by down-modulating cellular ligands for the NKG2D receptor expressed on NK cells and CD8⁺ T cells. Mutant viruses lacking these immunoevasion genes are attenuated with respect to virus growth in vivo. Given the importance of the NKG2D receptor in controlling both NK- and T cell-mediated immunity, it is of tremendous importance to understand the molecular mechanisms and consequences of viral regulation of the NKG2D ligands.     

NKG2D ligands expression patterns in gut mucosa from patients with coeliac disease

Introduction

The activating MICA/NKG2D interaction is involved in the response of intraepithelial lymphocytes (IELs) in coeliac disease (CD). The aim of this study was to investigate the expression of NKG2D ligands (MICA, MICB), IL-15 and NKG2D receptor in gut mucosa of CD patients, and the correlation with the severity of histological damage.

Patients and methods

Intestinal biopsies from 20 CD patients and five healthy controls were selected. All patients were positive for anti-transglutaminase 2 antibodies and for DQ2 or DQ8. Patients were divided into two groups according to their grade of mucosal impairment: ten each with mild and severe mucosal damage (MMD and SMD, respectively). The expression of proposed genes was determined at mRNA level. MICA expression was also determined by immunohistochemistry.

Results

Overexpression of MICA and MICB was observed in biopsies from coeliac patients compared to healthy controls (P < 0.001). Nevertheless, the expression was considerably higher in the group of patients with MMD (P < 0.0001) than in those with SMD. The levels of NKG2D receptor and IL-15 were also higher in patients than in controls, but no relationship with the severity of the mucosal lesion was found.

Conclusions

Our results suggest that NKG2D ligands may play an important role during the onset of the inflammatory process in the early stages of the development of coeliac disease.     

A dual function of NKG2D ligands in NK-cell activation

NK-cell killing requires both the expression of activating receptor ligands and low MHC class I expression by target cells. Here we demonstrate that the expression of any of the murine ligands for the NK-cell activating receptor NKG2D results in a concomitant reduction in MHC class I expression. We show this both in tumor cell lines and in vivo. NK-cell lysis is enhanced by the decrease in MHC class I expression, suggesting the change is biologically relevant. These results demonstrate that NKG2D ligand expression on target cells not only allows for activating receptor recognition, but also actively reduces expression of the inhibitory ligand, MHC class I, leading to enhanced recognition and killing by NK cells.     

NKG2D ligands: unconventional MHC class I-like molecules exploited by viruses and cancer

Our best teachers in revealing the importance of immune pathways are viruses and cancers that have subverted the most prominent pathways to escape from immune recognition. Viruses and cancer impair antigen presentation by classical MHC class I to escape adaptive immunity. The activating receptor NKG2D and its MHC class I-like ligands are other recently defined innate and adaptive immune pathways exploited by viruses and cancer. This review discusses recent advances in the understanding of how NKG2D, expressed on innate immune cells including natural killer cells, gammadelta+ T cells and macrophages, and adaptive immune cells such as CD8+ T cells, recognize stress-induced, MHC class I-like, self-ligands. Moreover, we describe how viruses and cancer have developed strategies to evade this recognition pathway.     

NKG2D blockade attenuated cardiac allograft vasculopathy in a mouse model of cardiac transplantation

A previous paper has reported that blockade of NKG2D was effective in protecting allograft in murine models of cardiac transplantation, but the mechanism of NKG2D blockade on attenuated cardiac allograft vasculopathy (CAV) was still unknown. In our current study, we found that wild‐type recipients treated with anti‐NKG2D monoclonal antibody (mAb) plus cytotoxic T lymphocyte antigen (CTLA)‐4‐immunoglobulin (I)g showed prolonged allograft survivals (>90 days, P < 0·001) significantly and attenuated CAV. These in‐vivo results correlated with reduced alloantibody production, low expression of interleukin (IL)‐17 and IL‐6, while infiltration of regulatory T cells increased. IL‐6 administration induced shorter allograft survival and higher CAV grade in CTLA‐4–Ig plus anti‐NKG2D mAb‐treated recipients, whereas IL‐17 had no significant effect on allograft survival and CAV grade in CTLA‐4–Ig plus anti‐NKG2D mAb‐treated recipients. Furthermore, the prolonged allograft survival induced by NKG2D blockade was abrogated partially with depletion of regulatory T cells. In conclusion, blockade of NKG2D combined with CTLA‐4–Ig attenuated CAV and this effect was associated with lower alloantibody production, inhibited IL‐6 expression and enhanced expansion of regulatory T cells.