非血缘关系HLA全相合造血干细胞移植中供者-受者NK细胞KIR的研究

目的研究自然杀伤细胞免疫球蛋白样受体（KIR）的生物学功能及供者抑制性KIR和受者HLA遗传背景在无关供者全相合造血干细胞移植（HSCT）中的作用。方法采用序列特异性引物聚合酶链反应（SSP—PCR）和序列特异性寡核苷酸探针聚合酶链反应（SSOP—PCR）的方法，对中国造血干细胞捐献者资料库中提供的51对HLA全相合供、受者进行KIR及HLA分型，患者中急性淋巴细胞白血病ALL18例，慢性粒细胞白血病（CML）15例，急性髓系白血病（AML）10例及其他患者8例。结果51对供者一受者均存在2DL1、2DL2／L3、2DIA、3DL2和3DL3，基因频率均为1，96．7％的个体表达KIR3DL1。供-受者中21．57％KIR完全相同；78．43％KIR不完全相同，而KIR不相同又分为受者KIR基因型包含供者KIR为25．49％，供者KIR基因型包含受者KIR为27．45％。74.62％的供者KIR2DL1无受者C2配体，5．91％的供者KIR2DL2／L3无受者C1配体，19．74％的供者KIR3DLl无受者Bw4配体，54．91％的供者KIR3DL2无受者A3、A11配体。结论KIR独特型和HLA-Ⅰ类抗原是独立遗传的。供者KIR2DL1和KIR3DL2是主要引起NK细胞异源活性的受体，供、受者的KIR／HLA不匹配可能在全相合的无关供者异基因HSCT中起着十分重要的作用。KIR受体-配体模型可以更好地提示HSCT的预后。     

异基因反应性NK细胞在供者淋巴细胞输注治疗单倍体相合造血干细胞移植后肺癌复发中的作用研究

本研究旨在探讨预处理中并基因反应性NK细胞（allo—reactive NK cells,Allo—NK）在供者淋巴细胞输注（DLI）治疗单倍体相合造血干细胞移植后肺癌复发中的作用。采用免疫磁珠富集F1供鼠（H-2^d/b）脾脏allo—NK,流式细胞术和LDH法检测其异体反应性。建立小鼠单倍体相合造血干细胞移植后肺癌复发模型,采用放射性核素标记法追踪DLI的体内分布。比较不同处理组小鼠的肿瘤大小、淋巴细胞浸润程度,采用RayBio小鼠细胞因子抗体芯片检测血清中22种细胞因子的变化。结果表明：在移植后24—48小时,allo—NK组的回输供者淋巴细胞在受者肺脏、脾脏、肾脏中明显蓄积,浓度最高,时间最长。化疗＋DLI组与化疗＋PBS组的肿瘤体积相比无明显区别,但allo．NK＋DLI组的肿瘤体积显著小于化疗＋DLI组（P〈0．05）和allo—NK＋PBS组（P〈0．01）组,且镜下可观察到肿瘤局部较多的淋巴细胞浸润。allo—NK＋DLI组中MCP-1、IL-17、IL—12和MCP-5较对照组分别升高1．56、1．36、1.20、1．17倍;而IL-10降低42．8％。结论：allo-NK可以延长DLI在宿主体内停留时间,促进炎性因子和Th1类细胞因子分泌,抑制肿瘤生长,提高DLI治疗移植后肺癌复发的疗效。     

杀伤细胞免疫球蛋白样受体与供者特异性人类白细胞抗原抗体在单倍体相合造血干细胞移植供者选择中的研究进展

目前,单倍体相合造血干细胞移植(haplo-HSCT)为治疗血液系统恶性疾病的重要手段之一,其为需要接受异基因造血干细胞移植(allo-HSCT)患者解决了供者缺乏的问题.但是移植排斥、移植物抗宿主病(GVHD)、植入失败(GF)、植入功能不良均为haplo-HSCT相关不良反应.目前,对于影响非人类白细胞抗原(HLA)相合程度的因素,如供者性别、年龄、供者特异性人类白细胞抗原抗体(DSA)及杀伤细胞免疫球蛋白样受体(KIR)匹配程度等,在haplo-HSCT供者选择方面的作用越来越受到研究者的关注.KIR3DL1为自然杀伤(NK)细胞表面重要的抑制性受体之一,在诱导异源反应性NK细胞中发挥重要作用.为了优化haplo-HSCT的供者选择,笔者拟就DSA、KIR匹配程度在haplo-HSCT供者选择中的作用进行综述.     

杀伤细胞免疫球蛋白样受体基因及其配体相合或错配对单倍相合骨髓移植效果的影响

本研究分析杀伤细胞免疫球蛋白样受体（killer cell Ig-like receptor,KIR）及其配体分子相合与否对单倍体相合骨髓移植效果的影响。分析了74例KIR基因及其配体分子HLA-Cw的分布频率和特点,同时比较KIR分子配体缺失与否对单倍相合骨髓移植患者总体生存、无病生存、GVHD发生以及复发等的影响。结果表明：19个KIR基因表型中2DL1、2DL4和3DL2-3在所有个体100%分布,其他高频率分布的还有3DP1（98.6%）、2DP1（98.6%）、3DL1（97.3%）、2DL3（97.3%）;抑制型基因分布频次是活化型的1.37倍;所有单倍体都含有2DL1、KIR3DL2、3DL3和2DL4,其中每个单倍体中均含有2DL2和/或2DL3。HLA-C14个等位基因中Cw7分布频率最高为37.8%;KIR2DL2/2DL3识别配体Group2（HLA-Cw1,3,7,8,13,14）组占43.2%。32例单倍相合骨髓移植中KIR基因错配发生比例为43.8%,其中9/14例为2DL不相合、5/14为2DL2或3DL1不相合;46对单倍相合骨髓移植中HLA-Cw全相合者29例,不相合者14例,HLA-Cw发生完全错配比例为30.4%,全相合比例为63.4%。14例KIR基因不相合和13例KIR基因相合移植病例中,KIR基因相合组生存率高于KIR基因不相合者（p=0.032）;17例KIR分子配体HLA-Cw不相合移植患者的DFS明显高于24例相合者（p=0.024）。按供受者KIR配体是缺失的GVHD矢量组生存率高于非GVHD矢量组（p=0.015）。急性重症GVHD发生与活化型KIR2DS1/2DS2有关,2DS1/2DS2不相合组高发急性重症GVHD同时复发减低,而2DS1/2DS2相合组低GVHD但是复发增多。14例KIR配体不相合髓系白血病患者骨髓移植后1例复发死亡,而12例KIR配体相合组患者有4例复发死亡。结论：单倍体相合骨髓移植的主要特点就是HLA不全相合,KIR基因表型不一致性及其配体缺失也是其主要免疫学特征,供者型KIR配体的缺失与移植效果有密切关系,在单倍相合移植中分析KIR基因及其配体对于供者选择和判断预后有重要意义。     

去甲基化处理对NK-92MI细胞杀伤活力的影响

为观察DNA去甲基化处理对NK细胞系NK-92M1细胞抑制性KIR表达及细胞杀伤活力的影响,探讨抑制性k打基因去甲基化与NK细胞功能之间的可能联系,采用甲基化抑制剂5-氮胞苷处理NK-92MI细胞,采用流式细胞术检测NK-92MI细胞表面KIR3DL1、KIR2DL2／KIR2DL3、KIR2DL1表达及细胞活力,并采用流式细胞仪将NK-92MI细胞分选为KIR3DL1阳性和KIR3DL1阴性细胞群,采用乳酸脱氢酶释放法检测NK-92MI细胞对白血病细胞系K562的杀伤活力。结果显示：应用终浓度为1．0、2．5和5μmol／L的5-氮胞苷作用72小时可以诱导NK-92MI细胞KIR3DL1、KIR2DL2／KIR2DL3、KIR2DL1表达增加,同时NK-92MI对K562细胞的杀伤活力降低,5-氮胞苷在1—5μmol／L范围内不影响NK-92MI的细胞活力,KIR3DL1阳性NK-92MI细胞的杀伤活力较KIR3DL1阴性细胞的杀伤活力低。结论：去甲基化处理通过诱导抑制性KIR表达增加抑制NK-92MI细胞的杀伤活力。     

Roles for HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of effector function

Interactions between killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I ligands regulate the development and response of human natural killer (NK) cells. Natural selection drove an allele-level group A KIR haplotype and the HLA-C1 ligand to unusually high frequency in the Japanese, who provide a particularly informative population for investigating the mechanisms by which KIR and HLA polymorphism influence NK cell repertoire and function. HLA class I ligands increase the frequencies of NK cells expressing cognate KIR, an effect modified by gene dose, KIR polymorphism, and the presence of other cognate ligand-receptor pairs. The five common Japanese KIR3DLI allotypes have distinguishable inhibitory capacity, frequency of cellular expression, and level of cell surface expression as measured by antibody binding. Although KIR haplotypes encoding 3DL1*001 or 3DL1*005, the strongest inhibitors, have no activating KIR, the dominant haplotype encodes a moderate inhibitor, 3DL1*01502, plus functional forms of the activating receptors 2DL4 and 2DS4. In the population, certain combinations of KIR and HLA class I ligand are overrepresented or underrepresented in women, but not men, and thus influence female fitness and survival. These findings show how KIR-HLA interactions shape the genetic and phenotypic KIR repertoires for both individual humans and the population.     

几类恶性血液病病人杀伤免疫球蛋白样受体基因型的表达在高危及标危组的差异分析

本研究探讨几类高危和标危恶性血液病患者杀伤免疫球蛋白样受体（KIR）基因表达的异同。54例恶性血液病患者分为高危组（27例）和低危组（27例），其中急性髓系白血病14例、急性淋巴细胞白血病16例、慢性髓系白血病20例、骨髓增生异常综合征3例、急性混合白血病1例。用序列特异性引物PCR分型技术检测了6个激活性KIR基因（KIR2DS1-S5、3DS1）和6个抑制性KIR基因（KIR2DL1-2DL4、3DL1-3DL2）的表达，其中24例患者用流式细胞仪测定了NK细胞、T细胞表面CD158a（KIR2DL1、2DS1）、CD158b（KIR2DL2、2DL3、2DS2、2DS3）、CD158e（KIR3DL1、3DS1）的表达。结果显示，标危组病人激活性KIR基因的阳性率均高于高危组患者，2DS1（P=0.014）、2DS2（P=0.046）、3DS1（P=0．005）的差异有统计学意义；抑制性KIR基因的表达在两组患者之间的差异无统计学意义（P〉0.05）。在髓系恶性血液病病人中标危组病人KIR激活性基因表达率仍然高于高危组患者，其中2DS1（66.7％vs29.4％．P=0.022）、2DS2（57.6％vs17.6％，P=0.013）和2DS3（33.3％vs5.9％。p=0．039）。高危组患者同时表达两种以上激活性KIR基因的比例显著低于标危组（P=0.035）。高危和标危组患者NK细胞和T细胞表面CD158a、CD158b、CD158e的表达差异无统计学意义（P〉0．05）。结论：几类恶性血液病患者激活性KIR基因表达在高危和标危之间存在差异。     

同种反应性自然杀伤细胞在小鼠主要组织相容性复合物半相合骨髓移植中的作用

目的研究同种反应性自然杀伤(NK)细胞在小鼠主要组织相容性复合物(MHC)半相合骨髓移植(BMT)中对宿主粒细胞和T细胞的清除、造血重建、植入及移植物抗宿主病(GVHD)的影响。方法以(C257BL／6×BALB／c)BCF_1(H-2~(d/b+))为供鼠,BALB／c(H-2~d)为受鼠建立小鼠MHC半相合BMT模型。根据照射剂量及NK细胞处理的不同将受鼠分为8．5 Gy对照组及7,6和5 Gy实验组。 8．5 Gy对照组进一步分为单纯照射组和BMT组,7,6及5 Gy实验组均进一步分为单纯照射组、BMT 组、非同种反应性NK(non-alloNK)细胞组及同种反应性NK(alloNK)细胞组。以外周血白细胞和血小板计数、受鼠型粒细胞及T细胞计数、H-2~(d/b+)细胞表达水平以及病理学检查等指标评价同种反应性 NK细胞的预处理作用。结果8．5 Gy单纯照射组生存期为(6．00±0．82)d,其他组生存期均大于60 d,各组均未观察到GVHD的临床及病理学表现;alloNK细胞组造血重建明显快于其他组(P值均< 0．05);移植后1天各照射剂量alloNK细胞组骨髓及脾细胞中受鼠型H-2~(d+)粒细胞和T细胞明显减少, 与相同照射剂量BMT组及non-alloNK细胞组比较差异有统计学意义(P值均<0．05);7,6和5 Gy alloNK细胞组供鼠H-2~(d/b+)细胞植入率显著高于相同照射剂量BMT和non-alloNK细胞组(P值均< 0．05)。结论alloNK细胞在小鼠MHC半相合BMT中具有清除受鼠体内残存粒细胞和T细胞、提高供鼠细胞植入和促进造血重建的作用,且不引起GVHD。     

人NK细胞表面KIR与HLA I类抗原

KIR受体与HLA相互作用在人类遗传、免疫应答及异基因造血干细胞移植等方面发挥着重要作用.KIR能识别HLA I类抗原;KIR与HLA各自能独立遗传,HLA配型不能准确推断KIR表型;KIR的表达主要与其基因型有关,HLA对KIR表达的调节作用微乎其微,KIR的基因型可以决定其表现型.供受者间KIR受体-HLA配体不相合有利于异基因造血干细胞移植,尤其是单倍体相合造血干细胞移植,KIR受体-配体模型可以更好地提示造血干细胞移植的预后.本文就KIR与HLA在造血干细胞移植中的相互作用、KIR与HLA的遗传学基础、NK细胞表面KIR表达与HLA的关系及KIR和HLA在免疫应答中的作用作一综述.     

杀伤细胞免疫球蛋白样受体对造血干细胞移植的影响

自然杀伤(NK)细胞表面的杀伤细胞免疫球蛋白样受体(KIR),可通过与人类白细胞抗原(HLA)相互作用,影响NK细胞活性,进而影响异基因造血干细胞移植(allo-HSCT)的预后.在去T淋巴细胞allo-HSCT中,KIR/HLA错配的异源反应性NK细胞,在抗急性髓细胞白血病(AML)中发挥了重要作用;而未去T淋巴细胞allo-HSCT中,KIR/HLA错配对患者预后影响迥异.研究者提出,在自体造血干细胞移植(auto-HSCT)中也存在移植物抗白血病(GVL)效应,并且患者存在不同KIR对移植预后影响不同.笔者拟就KIR/HLA错配在造血干细胞移植中作用的研究进展进行综述.     

低剂量氟达拉滨、环磷酰胺联合供者NK细胞作为非清髓性预处理用于小鼠单倍相合造血干细胞移植

本研究探讨低剂量氟达拉滨、环磷酰胺联合供者异体反应性NK细胞(flu cy allo-NK)作为新的非清髓性单倍相合造血干细胞移植(haploidentical HSCT)预处理方案的可行性。利用免疫磁珠富集F1供鼠(H-2d/b)脾脏NK细胞,检测其中Ly49C 、Ly49A 细胞的比例;LDH法检测其异体反应性。建立小鼠单倍相合造血干细胞移植模型,并比较清髓性方案(9 Gy TBI)、各种非清髓性方案(6.5 Gy TBI,flu cy,及flu cy allo-NK)的体内清髓效果、移植后供者嵌合率、GVHD的发生率及严重程度。结果表明:与其他非清髓性预处理方案相比,flu cy allo-NK组不能增加清髓程度,但可显著提高单倍相合移植后的供者嵌合率,移植后21天的嵌合率在骨髓为(28.70±5.90)%,脾脏为(46.40±5.00)%,并持续2个月。与flu cy组相比,flu cy allo-NK组出现的GVHD反应轻微,仅有50%(5/10)受鼠出现体重减轻,flu cy allo-NK组小鼠的肝脏、小肠、肾脏及皮肤的病理切片均未见明显的组织损伤。结论:供者allo-NK具有促进单倍相合供者造血干细胞植入,减轻GVHD强度的作用;低剂量flu cy allo-NK方案为高龄和一般情况差的肿瘤患者开展单倍相合造血干细胞移植提供了新的途径。     

DNA methylation maintains allele-specific KIR gene expression in human natural killer cells

Killer immunoglobulin-like receptors (KIR) bind self-major histocompatibility complex class I molecules, allowing natural killer (NK) cells to recognize aberrant cells that have down-regulated class I. NK cells express variable numbers and combinations of highly homologous clonally restricted KIR genes, but uniformly express KIR2DL4. We show that NK clones express both 2DL4 alleles and either one or both alleles of the clonally restricted KIR 3DL1 and 3DL2 genes. Despite allele-independent expression, 3DL1 alleles differed in the core promoter by only one or two nucleotides. Allele-specific 3DL1 gene expression correlated with promoter and 5' gene DNA hypomethylation in NK cells in vitro and in vivo. The DNA methylase inhibitor, 5-aza-2'-deoxycytidine, induced KIR DNA hypomethylation and heterogeneous expression of multiple KIR genes. Thus, NK cells use DNA methylation to maintain clonally restricted expression of highly homologous KIR genes and alleles.     

供者活化型KIR2DS5在单倍体相合造血干细胞移植预后中的意义

本研究探讨供者杀伤细胞免疫球蛋白样受体（KIR）和受者人类白细胞抗原（HLA）基因型在单倍体相合造血干细胞移植（HSCT）预后中的意义。26例患者接受单倍体相合HSCT,均未进行体外T细胞去除（TCD）。采用序列特异性引物聚合酶链式反应（PCR-SSP）分型技术对供、受者HLA和供者KIR基因型进行检测。根据供者KIR和受者HLA-Bw4组、-C1组、-C2组等位基因分析结果进行供受者KIR/HLA分组。对KIR/HLA不合与相合组、Bw4不合与相合组、C2不合与相合组、Bw4和C2均不合组与至少其一相合组在造血重建、移植物抗宿主病（GVHD）、无病生存（DFS）、移植后感染及移植相关死亡（TRM）等方面的差异进行比较;并就供者活化型KIR对单倍体相合HSCT预后的影响进行评价。结果表明：各组在造血重建、急性或慢性GVHD发生率、DFS、移植后感染及TRM等方面均无显著性差异（p均〉0.05）,但C2不合组发生严重GVHD4例。供者活化型KIR2DS5阳性组2年DFS明显优于阴性组,分别为（85.7±13.2）%、（31.2±12.8）%,p〈0.05〕。结论：供者KIR及受者HLA基因型对单倍体相合HSCT预后有影响,供者携带活化型KIR2DS5可能有助于提高患者的DFS。     

Natural killer cell alloreactivity for leukemia therapy

Donor-versus-recipient natural killer (NK) cell alloreactivity derives from a mismatch between donor NK clones, carrying specific inhibitory receptors for self MHC class I molecules, and MHC class I ligands on recipient cells. When faced with mismatched allogeneic targets, these donor NK clones sense the missing expression of self HLA class I alleles and mediate alloreactions. Transplantation from NK alloreactive haploidentical donors controls acute myeloid leukemia relapse and improves engraftment without causing graft-versus-host disease.     

转录因子E2F1调控KIR3DL1基因表达的分子机制

目的 研究转录因子E2F1对KIR3DL1基因启动子转录活性的影响,明确其调控KIR3DL1基因表达的分子机制.方法 采用PCR方法 从K562细胞基因组DNA中扩增突变型KIR3DL1基因启动子序列,将产物连接到pGL3-Basic荧光素酶载体,构建报告重组子;采用染色质免疫共沉淀方法 检测E2F1与KIR3DL1启动子在细胞内的结合;采用阳离子脂质体SuperFect包裹突变型和野生型KIR3DL1启动子-荧光素酶报告重组子,然后转染K562细胞,染色质免疫共沉淀方法 检测E2F1与重组子在细胞内的结合,双荧光素酶检测试剂盒测定荧光素酶活性;将E2F1真核表达载体与野生型KIR3DL1启动子-荧光素酶报告重组子共转染K562细胞,双荧光素酶检测试剂盒测定荧光素酶活性.结果 成功构建突变型KIR3DL1启动子-荧光素酶报告重组子,测序证实,在K562细胞KIR3DL1启动子区1个潜在的转录因子E2F1结合位点有1个自然发生的点突变(TTTGGCGC→TTCGGCGC);E2F1完全不能与K562细胞中突变型KIR3DL1启动子结合,但能与NK-92MI细胞中没有突变的野生型KIR3DL1启动子结合;突变型KIR3DL1启动子.荧光素酶报告重组子保留了部分与E2F1的结合能力,但其相对荧光素酶活性较野生型降低了50%;共转染E2F1真核表达载体使野生型KIR3DL1启动子-荧光素酶报告重组子的相对荧光素酶活性增加为对照组的2倍以上.结论 转录因子E2F1参与调控KIR3DL1基因转录激活,E2F1结合位点上CpG二核苷酸的数量和甲基化模式可能影响转录因子E2F1与靶序列的结合.     

A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells

Human natural killer (NK) cells express several killer cell immunoglobulin (Ig)-like receptors (KIRs) that inhibit their cytotoxicity upon recognition of human histocompatibility leukocyte antigen (HLA) class I molecules on target cells. Additional members of the KIR family, including some that deliver activation signals, have unknown ligand specificity and function. One such KIR, denoted KIR2DL4, is structurally divergent from other KIRs in the configuration of its two extracellular Ig domains and of its transmembrane and cytoplasmic domains. Here we show that recombinant soluble KIR2DL4 binds to cells expressing HLA-G but not to cells expressing other HLA class I molecules. Unlike other HLA class I-specific KIRs, which are clonally distributed on NK cells, KIR2DL4 is expressed at the surface of all NK cells. Furthermore, functional transfer of KIR2DL4 into the cell line NK-92 resulted in inhibition of lysis of target cells that express HLA-G, but not target cells that express other class I molecules including HLA-E. Therefore, given that HLA-G expression is restricted to fetal trophoblast cells, KIR2DL4 may provide important signals to maternal NK decidual cells that interact with trophoblast cells at the maternal-fetal interface during pregnancy.     

供者自然杀伤细胞减轻GVHD并提高移植成活率的实验研究

本研究观察激活的供、受者NK细胞能否减轻骨髓移植后GVHD和增加移植成活率。以C57BL／6小鼠作为供者，将接受6．5Gy^60COγ射线全身照射的BALB／c受鼠随机分为4组，对照1组（无任何处理）、对照2组（骨髓移植）、实验1组（骨髓移植同时输注激活的受者NK细胞）和实验2组（骨髓移植同时输注激活的供者NK细胞），观察每组小鼠的活存期、嵌合率、GVHD临床和病理改变。结果表明：对照1组全部活存，实验1组比对照2组活存期短（P〈0．05），实验2组比对照2组活存期延长（P〈0.01）。实验1组GVHD临床及病理评分高于对照2组（P〈0.05），实验2组GVHD临床及病理评分明显低于对照2组（P〈0.01）。实验1组和实验2组嵌合率均较对照2组高（P〈0．05），实验2组的嵌合率比实验1组明显增加（P〈0．01）。结论：激活的供者NK细胞能延长实验小鼠的活存期，降低GVHD严重程度，增加移植成活率；而激活的受者NK细胞却缩短实验小鼠的活存期，加重GVHD严重程度。受者NK细胞也增加移植成活率，但比供者NK细胞作用小。     

Granulocyte–colony‐stimulatory factor: a strong inhibitor of natural killer cell function

BACKGROUND: The human cytokine granulocyte–colony stimulatory factor (G‐CSF) has found widespread application in the medical treatment of neutropenia and to mobilize hematopoietic stem cells used for transplantation. So far, the effect of G‐CSF on natural killer (NK) cells has not been fully investigated. STUDY DESIGN AND METHODS: The effect of G‐CSF on the phenotype, cytokine secretion profile, and cytotoxicity of NK cells was assessed. NK cells incubated in vitro in presence of G‐CSF for 48 hours as well as NK cells isolated from peripheral blood of G‐CSF–mobilized stem cell donors (in vivo) were used. RESULTS: In vitro, G‐CSF caused a strongly altered phenotype in NK cells with 49% down regulation of NKp44 frequency. Furthermore, the expression of the activating receptors NKp46 and NKG2D decreased 40 and 64%, respectively. The expression of KIR2DL1 and KIR2DL2 decreased by 46% each. In cytotoxicity assays, the lytic capacity of G‐CSF–exposed NK cells is reduced by up to 68% in vitro and up to 83% in vivo. Accordingly, granzyme B levels of in vivo G‐CSF–exposed NK cells were reduced by up to 87% in comparison to nonstimulated NK cells. Cytokine production of in vitro and in vivo incubated NK cells was strongly decreased for interferon‐γ, tumor necrosis factor‐α, and granulocyte macrophage colony‐stimulating factor as well as interleukin (IL)‐6 and IL‐8. Furthermore, we observed a reduction in proliferation and a positive feedback loop that increased the expression of the G‐CSF receptor. CONCLUSION: G‐CSF was demonstrated to be a strong inhibitor of NK cells activity and may prevent their graft‐versus‐leukemia effect after transplantation.     

Clinical Impact of KIR2DS3 and KIR2DL3 Genes in Neuroblastoma Patients

ObjectiveNeuroblastoma is a common fatal tumor of childhood. Natural killer (NK) cells can exert direct cytotoxicity on tumor cells. The killer immunoglobulin-like receptor (KIR) family of NK cell receptors is involved in activation/inhibition of NK cells. In the KIR gene cluster, six of them (3DS1, 2DS1–5) encode receptors triggering activation, while seven of them (3DL1–3, 2DL1–3, 2DL5) encode receptors triggering inhibition. We aimed to assess the distribution of genetic polymorphisms of KIRs on the clinical course of neuroblastoma and provide guidance on potential therapeutic options.MethodsOur study group included 50 neuroblastoma patients and 100 healthy children as controls. Twenty-eight patients were boys, and twenty-two were girls; median age was 36 months. Fourteen patients had stage 1, 2, 3, or 4S disease, and 36 patients had stage 4 disease. Isolated DNA from the peripheral blood was amplified for sequence-specific oligonucleotide probe analysis of 16 KIR genes. The Fisher''s exact test was used to evaluate the variation of KIR gene distribution.ResultsAll patients had a lower frequency of KIR2DS3 compared to the control group (p = 0.005). Evaluation of individual KIR genes/genotypes in patients with early stages (stage 1, 2, 3, and 4S) versus stage 4 disease revealed that the frequency of KIR2DS3 was increased in early stages (p = 0.023). Inhibitory KIR2DL3 was increased in the patient group compared to controls (p = 0.038). Furthermore, the frequency of KIR2DL3 was higher in stage 4 neuroblastoma patients compared to the patients with early stages (p = 0.023).ConclusionOur data suggest a role for KIR2DS3 and KIR2DL3 in development of neuroblastoma. Thus, modulation of KIR2SD3 and/or KIR2DL3 expression or function might present a novel therapeutic strategy for neuroblastoma.     

免疫球蛋白样抑制性受体KIR基因与HLA-Cw的匹配对NK细胞活性的影响

本研究探索自然杀伤（naturalkiller,rqK）细胞表面杀伤细胞免疫球蛋白样抑制性受体（killerimmunoglobulin—like inhibitionreceptor,KIR）基因与HLA—Cw配体匹配对NK细胞活性的影响。对27名健康人取新鲜外周血20ml,用NK细胞免疫磁珠分选试剂盒负向分选高纯度NK细胞。对30例初治确诊急性髓系白血病（AML）患者取新鲜骨髓液10ml,分离单个核细胞作为靶细胞。流式细胞仪检测NK细胞CD158a,CD158b表达水平。取健康人和患者的外周血各2ml,以PROTRANS法抽提DNA,采用序列特异性引物PCR—SSP分型技术分别检测HLA—Cw、KIR基因。MTT法检测KIR与HLA—Cw基因不同组合的NK细胞对AML患者白血病细胞的杀伤率。结果表明：分选后的NK细胞,纯度为（90．8±6．08）％。NK细胞KIR基因与靶细胞HLA—Cw等位基因的匹配数少则NK细胞的杀伤效应强,0个等位基因匹配的杀伤率为（50．66±8．40）％,1个匹配与2个匹配的杀伤率分别为（38．28±6．71）％,（19．74±4．15）％,（F=20．226,P〈0．001）。NK细胞的KIR表达水平与杀伤作用也有一定的联系（F=16．276,P值〈0．001）。结论：NK细胞对AML白血病细胞的杀伤作用与抑制性KIR／HLA—Cw的匹配数及KIR的表达相关,匹配越少、KIR表达越低,杀伤率越高。