沙利度胺对白血病细胞NKG2D配体表达及NK细胞杀伤敏感性的影响

目的:探究沙利度胺对白血病细胞自然杀伤细胞及活性受体D(natural killer cell group 2 member D,NKG2D)配体表达及自然杀伤细胞(natural killer,NK)杀伤敏感性的影响.方法:取对数生长期白血病细胞株HL-60、K562细胞,以不同浓度沙利度胺作用48 h,并设置未经沙...     

NKG2D ligands expression and NKG2D-mediated cytotoxicity in human laryngeal squamous carcinoma cells

The NKG2D is an activating immunoreceptor expressed by NK cells and CD8⁺ T cells. Engagement of NKG2D by its ligands is critical for both innate and adoptive immunity. While the overexpression of NKG2D ligands on certain tumour cells has previously been demonstrated, little is known about NKG2D ligand expression on human laryngeal tumour cells. In this study, we first verified that the interaction between NKG2D and its ligands was critical for NK cell-based immune response to human laryngeal squamous carcinoma cells Hep-2. This NKG2D-mediated effect was observed by transfecting the recombinant eukaryotic expression vector pEGFP-N1/NKG2D as well as the NKG2D blockade. The mRNA and protein expression of NKG2D ligands, MHC class I-related chain molecules A (MICA) and UL16-binding proteins (ULBPs), in human laryngeal carcinoma cell line Hep-2 and fresh tumour tissues were evaluated. Compared with non-tumour tissues of vocal cords polyps, MICA and ULBP-3 were strongly overexpressed on both the human laryngeal carcinoma cell line Hep-2 and fresh human laryngeal carcinoma tissues. The mechanism and impact of NKG2D ligands overexpression on NK cell-mediated anti-laryngeal cancer immune response would require further investigation.     

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。     

HMBOX1 negatively regulates NK cell functions by suppressing the NKG2D/DAP10 signaling pathway

HMBOX1 is a new member of the homeobox family. Homeobox members have been reported to participate in embryonic development and systemic metabolism, but the function of HMBOX1 remains unclear, especially in the hematopoietic system. Here, we show that HMBOX1 is expressed at a high level in primary human NK cells but is expressed at much lower levels in NK cell lines. Overexpression of HMBOX1 significantly inhibited NK cell activities, including natural cytotoxicity against tumor cells, the level of CD107a (a marker protein for degranulation) and the production of cytolytic proteins (perforin and granzymes). More interestingly, HMBOX1 negatively regulated the expression of NKG2D and the activation of the NKG2D/DAP10 signaling pathway in NK cells. This effect was reversed by knocking down HMBOX1. Taken together, these findings demonstrate that HMBOX1 may act as a negative regulator of NK cell functions via suppressing the NKG2D/DAP10 signaling pathway.     

NKG2D in NK and T Cell-Mediated Immunity

One of the best characterized NK cell receptors is NKG2D, a highly conserved C-type lectin-like membrane glycoprotein expressed on essentially all NK cells, as well as on γδ-TcR⁺ T cells and αβ-TcR⁺ CD8⁺ T cells, in humans and mice. Here we review recent studies implicating NKG2D in T cell and NK cell-mediated immunity to viruses and tumors, and its potential role in autoimmune diseases and allogeneic bone marrow transplantation.     

NKG2D配体的表达直接影响NK细胞对不同发育阶段DC的杀伤

目的:探讨NKG2D配体在不同发育阶段树突状细胞(DC)表面的表达及其对自然杀伤(NK)细胞杀伤活性的影响.方法:用细胞因子(rh IL-4、rhGM-CSF、TNF-α)体外诱导培养单核细胞来源的未成熟树突状细胞(iDC)和成熟树突状细胞(mDC)并鉴定形态和表型,免疫磁珠法分离纯化NK细胞.流式细胞术(FCM)检测iDC和mDC表面NKG2D配体MICA/B、ULBP1-3的表达.用LDH释放法检测NK细胞对iDC和mDC的杀伤活性以及抗NKG2D单克隆抗体(mAb)阻断NK细胞后的杀伤活性.结果:培养的iDC和mDC具有典型的细胞形态和免疫表型特征.iDC表面表达MICA、MICB、ULBP1、ULBP3,表达率分别为(32.39±8.30)%、(17.75±3.40)%、(26.71±6.48)%、(38.37±6.89)%;mDC表面表达MICA 、ULBP3,表达率分别为(7.82±2.67)%、(8.36±2.42)%,比iDC表面相应配体表达率低(P<0.01).各效靶比NK细胞对iDC的杀伤活性均比对mDC的杀伤活性高,差异有统计学意义(P<0.01).抗NKG2D mAb阻断NK细胞后对iDC杀伤活性比阻断前减弱(P<0.05);对mDC的杀伤活性与阻断前相比无统计学意义(P>0.05).结论:NKG2D配体在iDC表面表达高,介导了NK细胞对iDC的杀伤,而对mDC的杀伤无影响,是NK细胞对iDC选择性高杀伤的分子机制之一.     

脂多糖对蜕膜NK细胞表达NKG2D水平的促进作用

为探讨脂多糖(LPS)及其受体TLR4相互作用并影响蜕膜NK细胞的可能机制,在孕E6.5给BALB/c×C57BL/6孕鼠腹腔注射LPS,而E10.5采用流式细胞术检测小鼠蜕膜NKG2D+TGF-β-NK细胞构成比,计算小鼠胚胎吸收率。此外,采用磁珠亲和细胞分选术纯化E10.5小鼠蜕膜NK细胞并培养,用LPS刺激所培养的细胞,并观察刺激后NK细胞表达NKG2D水平的变化。研究发现,在体内和体外实验中LPS刺激均可显著增高BALB/c×C57BL/6孕鼠蜕膜NK细胞NKG2D的表达水平,其中腹腔注射可显著增高胚胎吸收率。在体外细胞培养实验中,预先在培养基中加入抗TLR4抗体,则LPS刺激后细胞NKG2D表达水平无显著升高。这些结果提示,LPS与TLR4相互作用可增强小鼠蜕膜NK细胞NKG2D的表达,而NKG2D的过高表达不利于同种异基因妊娠成功。     

Human NKG2F is expressed and can associate with DAP12

The NKG2 family of C-lectin type molecules is important for regulating the function of natural killer and subpopulations of T cells. NKG2A/B contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and accordingly functions as an inhibitory receptor, whereas NKG2C and -E/H associate with DAP12 via a positively charged residue in their transmembrane domains and function as activation receptors. Each of these molecules is paired with CD94 for expression and recognizes HLA-E as a ligand. NKG2F is an orphan gene within the NKG2 family whose translated product would contain both a positively charged residue in its transmembrane region, an intracellular ITIM-like sequence and an extracellular domain (62 residues) that is truncated relative to other NKG2 molecules. We show that NKG2F is expressed as a protein in NK cells. Expression appears to be confined to intracellular compartments probably due to its inability to associate with CD94. It can however associate with DAP12 thereby providing activation signaling potential. We were unable to demonstrate phosphorylation of the Tyr residue in the ITIM-like motif suggesting that it is a mock ITIM. NKG2F could be a receptor component with an as yet unidentified partner(s), could function to regulate cell activation through competition for DAP12 with other receptors, such as NKG2C and -E/H, or it could simply be a vestigial gene product.     

Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus-associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8⁺ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D-dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.     

NKG2D-DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathway

The immune recognition receptor complex NKG2D-DAP10 on natural killer cells is stimulated by specific ligands carried on virus-infected and malignant cells. Because DAP10 does not have an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail, its ability to trigger killing has been debated. Here we show that a crucial Tyr-Ile-Asn-Met amino acid motif in the cytoplasmic tail of DAP10 couples receptor stimulation to the downstream activation of phosphatidylinositol 3-kinase, Vav1, Rho family GTPases and phospholipase C. Unlike that of ITAM-containing receptors, the activation of NKG2D-DAP10 proceeds independently of Syk family protein tyrosine kinases. Yet the signals initiated by NKG2D-DAP10 are fully capable of inducing killing. Our findings identify a previously unknown mechanism by which receptor complexes that lack ITAM motifs can trigger lymphocyte activation.     

Low expression of CD161 and NKG2D activating NK receptor is associated with impaired NK cell cytotoxicity in metastatic melanoma patients

Natural killer (NK) cells play a role in the innate and adaptive antitumor immune responses. The activity of NK cells is regulated by functionally opposing, activating and inhibitory receptors whose balance ultimately determines whether target cells will be susceptible to NK cell mediated lysis. As melanoma is an immunogenic tumor, the effect of immunomodulating agents is consistently investigated. In this study in 79 metastatic melanoma (MM) patients and 52 controls NK activity, expression of activating NKG2D and CD161 receptors and KIR receptors, CD158a and CD158b, on freshly isolated PBL and NK cells were evaluated. Native NK cell activity of melanoma patients in clinical stage I–III and MM patients was determined against NK sensitive K562, NK resistant Daudi, human melanoma FemX, HeLa and HL 60 target tumor cell lines. In addition, predictive pretherapy immunomodulating effect after 18 h in vitro treatments of PBL of MM patients with rh IL-2, IFN-α (IFN), 13-cis retinoic acid (RA) and combination IFN-α and RA was evaluated with respect to NK cell lyses against K562 and FemX cell lines. In this study we show for the first time that low expression of CD161 and activating NKG2D receptors, without increased expression of KIR receptors CD158a and CD158b, as well as a decrease in the cytotoxic, CD16^bright NK cell subset, is associated with a significant impairment in NK cell activity in MM patients. Furthermore, the predictive pretherapy finding that IL-2, IFN, IFN and RA, unlike RA alone, can enhance NK cell activity of MM patients against FemX melanoma tumor cell line can be of help in the design and development of therapeutic regimens, considering that it has recently been shown that low-dose combination of different immunomodulators represents the most promising approach in the therapy of MM.     

妊娠子宫微环境中uNK细胞NKG2A和NKG2D及其配体表达的研究

目的：研究妊娠子宫微环境中子宫自然杀伤细胞（uNK细胞）NKG2A和NKG2D及其相应配体的表达，探讨NKG2A与NKG2D的不平衡表达在母胎免疫耐受形成中的作用。方法：选择30例孕6-9周的正常妊娠妇女,分离其新鲜蜕膜组织，除去绒毛,分离蜕膜和外周血单个核细胞，采用流式细胞仪测定NK细胞的数量及NKG2A与NKG2D的表达；采用RT-PCR技术检测滋养层组织NKG2A与NKG2D配体人类白细胞抗原-E(HLA-E)、主要组织相容性复合体-Ⅰ类分子相关蛋白A(MICA)mRNA的表达结果：妊娠子宫蜕膜淋巴细胞中NK细胞约占70%,流式细胞分析的结果显示，子宫自然杀伤细胞NKG2A的表达显著高于外周血NK细胞，分别为97.86%±1.75%与33.35%±10.92%（〖AKx-D〗±s），两者差异显著（P<0.05），在滋养层细胞中检测到其配体HLA-E的表达；而与外周血相比，uNK细胞表面NKG2D的表达与之较为相近，分别为93.21%±4.52%与97.80%±1.72%，但两者仍有显著差异（P<0.05）。在滋养层组织未检测到其相应配体MICA mRNA的表达结论：蜕膜中的淋巴细胞主要为NK细胞，其免疫学表型与外周血NK细胞有较大的区别，妊娠期子宫自然杀伤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人类白细胞抗原-E，这可能是维持母胎界面免疫耐受的重要因素。     

IL-2-activated haploidentical NK cells restore NKG2D-mediated NK-cell cytotoxicity in neuroblastoma patients by scavenging of plasma MICA

NK group 2D (NKG2D)-expressing NK cells exhibit cytolytic activity against various tumors after recognition of the cellular ligand MHC class I chain-related gene A (MICA). However, release of soluble MICA (sMICA) compromises NKG2D-dependent NK-cell cytotoxicity leading to tumor escape from immunosurveillance. Although some molecular details of the NKG2D-MICA interaction have been elucidated, its impact for donor NK (dNK) cell-based therapy of solid tumors has not been studied. Within an ongoing phase I/II trial, we used allogeneic IL-2 activated dNK cells after haploidentical stem cell transplantation for immunotherapy of patients with high-risk stage IV neuroblastoma. NKG2D levels on activated dNK cells increased strongly when compared with freshly isolated dNK cells and correlated with enhanced NK-cell cytotoxicity. Most importantly, elevated sMICA levels in patients plasma correlated significantly with impaired dNK-cell-mediated cytotoxicity. This effect could be reversed by high-dose infusion of activated dNK cells, which display high levels of surface NKG2D. Our data suggest that the provided excess of NKG2D leads to clearance of sMICA and preserves cytotoxicity of dNK cells via non-occupied NKG2D. In conclusion, our results identify this tumor immune escape mechanism as a target to improve immunotherapy of neuroblastoma and presumably other tumors.     

Increased TLR responses in dendritic cells lacking the ITAM-containing adapters DAP12 and FcRgamma

The inhibitory effect of DAP12 on macrophages has been revealed by examining myeloid cells from DAP12-deficient mice. In this report, we demonstrate that both DAP12 and the FcepsilonRIgamma-chain (FcRgamma) are required for negative regulation of TLR responses in bone marrow-derived dendritic cells (DC). Loss of both DAP12 and FcRgamma enhanced the pro-inflammatory cytokine production and maturation of DC after TLR stimulation, resulting in a greater percentage of DC that produced IL-12 p40, TNF, and IL-6, and expressed high levels of MHC class II, CD80, and CD86. Whereas DC lacking only DAP12 showed some increased TLR responses, those lacking only FcRgamma had a greater enhancement of maturation and cytokine production, though to a lesser extent than DC lacking both DAP12 and FcRgamma. Additionally, antigen-specific T cell proliferation was enhanced by DAP12(-/-)FcRgamma(-/-) DC relative to wild-type DC after maturation. Similar to DAP12(-/-)FcRgamma(-/-) DC, Syk-deficient DC also had increased inflammatory cytokine production, maturation, and antigen presentation. These results confirm the inhibitory effect of immunoreceptor tyrosine-based activation motif (ITAM) signaling in myeloid cells and show that DC and macrophages differ in their dependence on the ITAM-containing adapters DAP12 and FcRgamma for negative regulation of TLR signaling.     

NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways

INTRODUCTION: Ewing sarcoma (EWS) is a malignant bone-associated sarcoma, with poor prognosis in case of metastasis or relapse. To explore the feasibility of natural killer (NK) cell mediated immunotherapy and to identify molecular mechanisms involved, the susceptibility of EWS to NK cells was investigated. METHODS AND RESULTS: All EWS cell lines tested (n=7) were lysed by purified allogeneic NK cells from healthy donors, and the efficacy of lysis was increased by activating NK cells with interleukin-15 (IL-15). FACS analysis and immunohistochemistry revealed that EWS cell lines as well as primary tumor cells expressed ligands for the activating NK cell receptors NKG2D and DNAM-1. NK cell cytotoxicity to EWS cells critically depended on the combination of NKG2D and DNAM-1 signaling, since blocking either of these receptors abrogated lysis by resting NK cells. Cytokine-activated NK cells more efficiently recognized EWS cells, since only combined, but not single blockade of NKG2D and DNAM-1 by antibodies inhibited lysis of EWS cells. Induction or blockade of HLA class I on EWS cells did not significantly influence lysis. This suggests that predominantly activating, rather than inhibitory signals on EWS cells determined susceptibility to NK cell cytotoxicity. NK cell cytotoxicity to EWS cells and K562 was reduced in EWS patients at diagnosis (n=11) compared to age matched controls, despite normal NK cell numbers and increased expression of NKG2D. The impaired function of these NK cells was restored after activation with IL-15 in vitro. CONCLUSION: These results demonstrate that EWS cells are potentially susceptible to NK cell cytotoxicity due to the expression of activating NK cell receptor ligands. The use of cytokine-activated NK cells rather than resting NK cells in immunotherapy may be instrumental to optimize NK cell reactivity to EWS.