表达HLA—G1的K562细胞抵抗外周血NK细胞杀伤作用的研究

目的 研究HLA－G1分子对NK细胞杀伤活性的影响。方法 供助脂质体介导的DNA转染技术,将本室构建的真核南闰pcDNA3-HLA-G1转染人K562细胞;通过G418筛选,获得克隆化细胞株K562－G1;应用RT－PCR及流式细胞术分别在RNA水平和蛋白质水平检测HLA－G1的表达;最后,应用MTT比色法检测转染细胞对不同个体外周血NK细胞杀伤活性的抑制效应。结果 与转染了空质粒的对照组相比,外周血NK细胞对K562－G1的杀伤率降低32.43%（P＜0.01）。结论 靶细胞表达HLA－G2分子可明显抑制NK细胞的杀伤效应。     

HLA-G分子在体外抑制NK细胞杀伤效应的研究

在获得稳定表达HLA G分子的K5 62细胞克隆的基础上 ,观察 4h及 16h不同效靶比例时NK细胞对靶细胞的杀伤效应 ,结果显示 ,NK细胞对转染HLA G基因的K5 62细胞的杀伤作用较未转染细胞明显降低 (P <0 0 1) ,用W6/ 32及BFL 1单克隆抗体阻断HLA G分子 ,可以恢复NK细胞的杀伤活性。用Y9/Z2 70及XA185 /Z2 70单抗特异性阻断抑制性受体CD94/NKG2A ,未观察到明显影响     

Raji细胞逃逸NK细胞免疫杀伤机制的初步探讨

目的 探讨人B细胞淋巴瘤Raji细胞逃逸NK细胞免疫杀伤的机制.方法 以K562细胞作为对照,应用LDH释放法检测不同效靶比时同种异体NK细胞杀伤Raji细胞的活性.并分别用PCR方法和流式细胞仪检测K562和Raji细胞MICA/B、ULBP1-3、HLA基因型和分子的表达情况.效靶比20∶1时用单抗分别阻断K562和Raji细胞表面MICA、MICB、ULBP1、ULBP2、ULBP3和HLA-Ⅰ类分子,观察NK细胞对其杀伤活性的变化.结果 不同效靶比时NK细胞杀伤Raji细胞的活性明显低于杀伤K562细胞的活性,二者之间差异有统计学意义(P<0.05).K562细胞表达MICMB和ULBP1～3基因和分子,不表达HLA-Ⅰ类分子,Raji细胞表达ULBP1～3基因和HLA-Ⅰ类分子,不表达MICA/B和ULBP1～3分子.Raji细胞HLA基因型为A*3、3.B*71、71,Cw3、4.用单抗封闭MICMB和ULBP1～3分子后,NK细胞对K562细胞的杀伤活性明显降低,对Raji细胞的杀伤活性无明显改变.结论 Raji细胞逃逸NK细胞免疫杀伤机制和Raji细胞高表达HLA-Ⅰ类分子,不表达NKG2D的配体MI-CA/B和ULBPI～3有关.     

同种异体NK细胞对不同肿瘤细胞的体外杀伤活性及其机制的初步探讨

为了观察同种异体NK细胞对不同肿瘤细胞的体外杀伤活性,并初步探讨其分子机制。以K562细胞为对照,应用LDH释放法检测不同效靶比时同种异体NK细胞杀伤CNE2、KG1a和U251细胞的活性。应用RT-PCR和流式细胞仪分别检测4种细胞MHCI类链相关分子(MICA/B)和人巨细胞病毒糖蛋白UL16结合蛋白(ULBP1~3)基因和分子的表达情况。效靶比20∶1时用AMO-1、BMO-1、M295、M310和M551单抗分别阻断肿瘤细胞表面MICA、MICB、ULBP1、ULBP2和ULBP3分子,观察NK细胞对其杀伤活性的变化。结果:NK细胞对K562、CNE2和KG1a细胞均有杀伤活性,对U251细胞无杀伤活性。在mRNA水平4种细胞均表达MICA/B和ULBP1~3基因。K562细胞表达MICA/B和ULBP1~3全部分子;KG1a和U251细胞均不表达5种分子;CNE2细胞表达MICA/B和ULBP2,不表达ULBP1和ULBP3。CNE2、KG1a和U251细胞均高表达HLAI分子,而K562细胞不表达。用单抗分别阻断靶细胞表面相应的NKG2D配体分子,NK细胞对KG1a和U251细胞的杀伤活性无变化。NK细胞对K562和CNE2细胞的杀伤活性可部分被封闭。同种异体NK细胞在体外对不同肿瘤细胞的杀伤活性不同,其杀伤机制也不完全相同。     

同种异体NK细胞对人脐静脉内皮细胞ECV304的杀伤活性差异及分子机制探讨

目的 探讨供者KIR分子表达差异对NK细胞杀伤人脐血内皮细胞系ECV304活性的影响,并观察参与杀伤的活化信号通路.方法 RT-PCR及流式细胞仪检测ECV304细胞NKG2D配体MICA/B、ULBP1-3表达,PCR-SSP法行HLA-A、B、Cw分型.自8例健康供者分离外周血NK细胞,流式细胞仪检测KIR2DL1的表达率,LDH释放法测定NK细胞在效靶比20∶1时对ECV304细胞的杀伤活性及anti-KIR2DL1mAb对NK细胞杀伤活性的影响.结果 ECV30细胞在mRNA水平表达MICA/B、ULBP1-3,但在膜蛋白水平均不表达.HLA分型表明,ECV304表达KIR2DL1的配体,而不表达KIR2DL2/3、KIR3DL1的配体.8例健康供者NK细胞KIR2DL1表达率有较大差异,对ECV304细胞的杀伤活性也有不同,双变量相关分析示个体KIR2DL1表达率与NK细胞对ECV304的杀伤率存在负相关(rS=-0.994,P=0.000).anti-KIR2DL1 mAb明显增强NK细胞对ECV304的杀伤活性(t=-4.860,P=0.002).结论 NK细胞对ECV304细胞的杀伤分子机制主要为HLA-KIR信号系统错配,目前已知的NKG2D配体MICMB、ULBP1-3并不参与,这有助于临床活体器官移植时在遗传指导下选择供体.     

两种不同HLA-B分子对NK细胞表面受体表达的研究

目的：探讨两种HLA-B分子(HLA-B51和HLA—B39)对NK细胞表面活化性受体CDl6和抑制性受体KIR3DL1表达的调节。方法：采用流式细胞仪检测NK细胞分别与转染HLA-BSl和HLA-B39分子的K562细胞相互作用后，CD16和KIR3DL1的表达情况。结果：外周血淋巴细胞与K562细胞作用24小时后，CD56 CD16 细胞数、KIR3DL1 细胞数均明显增加。与表达HLA-B39的K562细胞相比，表达HLA-B51的K562细胞与外周血淋巴细胞作用后，CD56 CD16 细胞数、KIR3DL1 细胞数均明显下降。结论：NK细胞杀伤靶细胞时，活化性受体CD16表达上调后会伴有抑制性受体KIR3DL1的上调；HLA-B51分子表达在K562细胞后，表达外源性HLA-B51分子的K562细胞与NK细胞作用，NK细胞表面受体KIR3DL1的表达可下调，同时伴有CD16的表达下调。     

膜结合型HLA-G1～G4分子的克隆表达及对NK细胞杀伤功能的影响

目的 探讨膜结合型HLA-G1～G4异构体的表达及对NK细胞杀伤功能的影响.方法 通过基因克隆及转染,分别建立稳定表达HLA-G1～G4抗原的人绒癌JAR细胞株.采用RTPCR、流式细胞术、Western blot及免疫细胞化学法分析、鉴定转染细胞中HLA-G的mRNA及蛋白表达.通过加载HLA-G高亲和性KIPAQFYIL抗原肽,观察对HLA-G表达的影响.LDH释放法检测HLA-G1～G4表达对NK细胞杀伤活性的影响.结果 RT-PCR、Western blot及免疫细胞化学结果显示,HLA-G1～G4/pVITRO2-mcs重组质粒成功转染HLA-G表达阴性的人绒癌JAR细胞株.FACS分析显示HLA-G1抗原能在JAR-HLA-G1细胞株表面表达,HLA-G2～G4抗原不能有效到达细胞表面.体外杀伤试验发现表达HLA-G1～G4抗原的细胞均能抑制NK细胞的杀伤活性(P＜0.05);加载HLA-G高亲和性KIPAQFYIL抗原肽对HLA-G表达无明显影响,对NK细胞杀伤抑制程度也未见明显改变.结论 HLA-G1～G4能够明显抑制NK细胞的杀伤活性,提示不同膜结合型HLA-G异构体分子均能作为免疫耐受分子,具备免疫调节功能.     

抗HPV16E6核酶对宫颈癌细胞免疫学特性影响的研究

目的 研究特异性抗体HPV16E6核酶的转染对宫颈癌细胞免疫学特性的影响。方法 以脂质体法将抗HPV16E6-ribozyme、空载体质粒分别导入CaSKi细胞,命名为CaSKi、CaSKi-P细胞。流式细胞仪分别检测3种细胞HLA－1、HL2－2、B7－1和B7－2基因的表达,分析CaSKi细胞转染酶后免疫学特性的变化。诱导制备NK、LAK、CD3AK和肿瘤细胞共激活杀伤细胞,检测各种免疫细胞对CaSKi-R、CaSKi-P、CaSKi细胞的杀伤效应。结果 CaSKi和CaSKi-p细胞中HLA－2、B7－1、B7－2表达都很低,两者差异无显著性。CaSKi-R中HLA－2、B7－1、B7－2表达率均明显升高。3种细胞中HLA－1表达率都很高。NK、LAK、CD3AK细胞对CaSKi-R细胞伤率显著高于CaSKi细胞,CaSKi、CaSKi-R分别与rIL－2共激活杀伤细胞（称CASKI和CASKI－R）的样伤活性无明显差别,对CaSKi-R的杀伤活性高于CaSKi细胞。而各种免疫细胞对CaSKi-P细胞的杀伤率与CaSKi细胞差异无显著性。结论 抗HPV16E6－nrbozyme的导入能使宫颈癌CaSKi细胞易于被机体免疫系统识别杀伤,难于免疫逃避,但不能增强其免疫原性。     

血管内皮细胞(ECV304)表达的HLA-G1对NK细胞杀伤活性的抑制作用

目的:证实HLA-G1分子能够抑制NK细胞对同种血管内皮细胞系的杀伤作用。方法:采用脂质体介导的DNA转染技术,以构建的真核表达质粒pcDNA3-HLA-G1转染人脐静脉内皮细胞系ECV304,再用免疫荧光法检测表达的HLA-G1分子。并用MTT比色法检测HLA-G1对NK细胞杀伤活性的影响。结果:ECV304细胞上可稳定表达HLA-G1。NK细胞对空质粒pcDNA3转染的ECV304细胞的杀伤率为(50.6±18.1)%;而对pcDNA3-HLA-G1转染的ECV304细胞的杀伤率为(29.7±11.4)%,两者差异具有显著意义(P<0.01)。结论:HLA-G1分子可明显抑制NK细胞对同种血管内皮细胞的杀伤效应。     

HLA-G3蛋白能够抑制多克隆NK细胞的杀伤功能

目的 研究HLA－G3蛋白对NK细胞杀伤功能的抑制作用。方法 用RT－PCR方法扩增HLA－G3和HLA－G1的cDNA，并将其克隆到真核表达载体pcDNA3中，采用脂质体转染法将其转入K562细胞，用免疫荧光法确定其在细胞表面表达，细胞毒试验研究其对NK细胞杀伤功能的抑制作用。结果 HLA－G3蛋白和HLA－G1表达在细胞表面，以PBL细胞为效应细胞的细胞毒实验表明K562－G1细胞和K562－G3细胞的细胞裂解率都比K562细胞裂解率低。结论 HLA－G3蛋白能够抑制多克隆NK细胞的杀伤功能。     

Possible roles of KIR2DL4 expression on uNK cells in human pregnancy

PROBLEM: To investigate possible roles of the natural killer (NK) cell receptor killer immunoglobulin-like receptor (KIR)2DL4 expressed on uterine NK (uNK) cells during pregnancy, we investigated KIR2DL4 expression on uNK cells isolated from patients with early recurrent spontaneous abortion (RSA) and normal early pregnancy women, and functions of KIR2DL4 was analyzed in vitro. METHODS OF THE STUDY: Semi-quantitative RT-PCR analysis was introduced to detect KIR2DL4 messenger RNA (mRNA) expression on uNK cells. Cytotoxicity and cytokine production as the result of interaction of KIR2DL4 and its ligand human leukocyte antigen (HLA)-G were analyzed in vitro with lactic dehydrogenase releasing method and enzyme-linked immunosorbent assay, respectively. RESULTS: No significant difference in KIR2DL4 mRNA expression was observed, while the KIR2DL4 protein level in isolated uNK cells is much higher in normal controls than that in RSA patients. Data showed that HLA-G transfection could not reverse the lysis of uNK against HLA-G transfected K562 cells but induced cytokine production. Furthermore, we demonstrated that, via KIR2DL4, membrane-bound HLA-G could induce high cytotoxicity and cytokine production in a high cytotoxic, IL-2 dependent human NK cell line NK-92 cells. CONCLUSION: Our data suggest that KIR2DL4 might play a crucial implication for human pregnancy.     

KIR downregulation by IL‐12/15/18 unleashes human NK cells from KIR/HLA‐I inhibition and enhances killing of tumor cells

To exploit autologous NK cells for cancer immunotherapy, it is highly relevant to circumvent killer cell immunoglobulin‐like receptor (KIR)‐mediated self‐inhibition of human NK cells by HLA‐I–expressing tumor cells. Here, we show that stimulation of NK cells with IL‐12/15/18 for two days led to downregulation of surface expression of the inhibitory KIR2DL2/L3, KIR2DL1 and KIR3DL1 receptors on peripheral blood NK cells. Downregulation of KIR expression was attributed to decreased KIR mRNA levels which could be re‐induced already 3 days after re‐culture in IL‐2. Reduced KIR2DL2/L3 expression on IL‐12/15/18–activated NK cells resulted in less inhibition upon antibody‐mediated KIR engagement and increased CD16‐dependent cytotoxicity in redirected lysis assays. Most importantly, downregulated KIR2DL2/L3 expression enabled enhanced cytotoxicity of IL‐12/15/18–stimulated NK cells against tumor cells expressing cognate HLA‐I molecules. NK cells pre‐activated with IL‐12/15/18 were previously shown to exert potent anti‐tumor activity and memory‐like long‐lived functionality, mediating remission in a subset of acute myeloid leukemia (AML) patients in a clinical trial. Our study reveals a novel mechanism of IL‐12/15/18 in improving the cytotoxicity of NK cells by reducing their sensitivity to inhibition by self–HLA‐I due to decreased KIR expression, highlighting the potency of IL‐12/15/18–activated NK cells for anti‐tumor immunotherapy protocols.     

Possible KIR-driven genetic pressure on the genesis and maintenance of specific HLA-A,B haplotypes as functional genetic blocks

The HLA genomic structure underlines the permanence of fixed haplotypes transmitted in blocks as allelic combinations. One of the most discussed concerns is how and why such a strong linkage between HLA alleles has been maintained for so long. We hypothesized a possible KIR-driven pressure in the genesis of specific HLA-A,B haplotypes. Certain HLA-A and -B molecules are ligands for the same KIR receptors through the Bw4 binding motif spanning residues 77-83 in the α1 domain. We analyzed the HLA-A and -B genomic types of 9897 Caucasian people (3533 newborns and 6364 adults) subdividing them according to the presence/absence of the HLA-B Bw4 serological epitope. For each HLA-B Bw4- and Bw6-cross-reactive group, we evaluated the presence/absence of HLA-A ligands for KIR3DL1 (HLA-A*23, HLA-A*24, HLA-A*32) and KIR3DL2 (HLA-A*03, HLA-A*11). The frequency of HLA-A KIR ligands significantly increased moving from the HLA-B Bw4/Bw4 to the HLA-B Bw4/Bw6 and the HLA-B Bw6/Bw6 groups among both newborns and adults (P<0.0001). Here, we suggest that, when the HLA-B KIR-ligand motif is lacking, the HLA-A KIR-ligand might have a vicarious role in controlling the natural killer cell-mediated innate immune response. Basing upon this compensatory function in the engagement of KIR receptors, we hypothesize that specific HLA-A,B ancestral haplotypes were generated.     

Hematopoietic stem cell transplantation: Improving alloreactive Bw4 donor selection by genotyping codon 86 of KIR3DL1/S1

KIR3DL1 is a natural killer (NK) cell receptor that recognizes the Bw4 epitope of human leukocyte antigen (HLA) class I molecules. Following hematopoietic stem cell transplantation for patients lacking Bw4, KIR3DL1‐expressing NK cells from Bw4‐positive donors can be alloreactive and eliminate tumor cells. However, KIR3DL1 alleles having T instead of C at nucleotide 320 (encoding leucine 86 instead of serine 86) are not expressed on the cell surface. Thus, not all individuals testing positive for KIR3DL1 are optimal donors for Bw4‐negative recipients. Therefore, we developed a method for genotyping codon 86, which was validated by its perfect correlation with NK cell phenotype for 100 donors of diverse KIR3DL1/S1 genotype. We typed 600 donors and found that ～12.2% had the KIR3DL1 gene, but did not express cell‐surface KIR3DL1. By contrast, high‐expressing allotypes were identified when haplotypes from four families with duplicated KIR3DL1/S1 genes were characterized at high resolution. Identifying donors who have KIR3DL1 but lack cell‐surface KIR3DL1 would refine donor selection. With this technique, the number of individuals identified who may not be optimal donors for Bw4‐negative patients increases by threefold, when compared with standard methods. Taken together, we propose that allele typing of killer cell Ig‐like receptor (KIR) polymorphisms should become a standard practice when selecting donors.     

Genetic profile of KIR and HLA in southern Chinese Han population

KIR and their HLA ligands are encoded by two of the most diverse gene families in the human genome. The function of KIR on the NK cell is highly dependent on the normal expression of class I HLA on the target cell. Previous population studies in southern Chinese have been focused on the KIR framework genes and genotypes but little is known about the compound profiles of KIR/HLA. The present study examined 503 unrelated individuals from southern Chinese Han population for the polymorphism of KIR and class I HLA genes. All 16 KIR genes were detected in the study population and the four framework genes KIR3DL2, 3DL3, 3DP1, and 2DL4 were present in all individuals. Thirty unique KIR gene profiles were found reflecting a rather limited number of KIR haplotypes in this population. KIRAA1 was the most common profile observed in 54.7% of the samples. Among the AA1 individuals, 15.6% were homozygous for the deleted KIR2DS4. Haplotype A (74.8%) was more common than haplotype B (25.2%). HLA-C1 was a much more common ligand for 2D KIRs than C2. Bw4-80I, Bw4-80T, and the Bw4-bearing HLA-A alleles were detected at similar frequencies. The matched KIR+HLA pairs 2DL2/3+C1 (98.1%), 3DL1+Bw4 (73.3%), 3DL2+A3/11 (60.0%) were the most common ones whereas 3DS1+Bw4-80I was the least common (9.4%). A total of 193 unique compound profiles of KIR–HLA were identified in 480 informative individuals, 130 of the profiles being detected only once. The study provided a comprehensive analysis of the KIR/HLA profiles in southern Chinese in regards of the presence/absence of KIR genes, HLA ligands, matched KIR+HLA pairs, and KIR/HLA compound profiles. The results could help to better understand the role played by KIR/HLA interaction in associated diseases and clinical transplantation in southern Chinese.     

Isolation of a novel KIR2DL3-specific mAb: comparative analysis of the surface distribution and function of KIR2DL2, KIR2DL3 and KIR2DS2

In recent years an increasing number of sequences coding for new KIRs have been described. However, the limited availability of mAbs with unique KIR specificities has hindered an exhaustive assessment of their actual function, HLA-specificity, expression at the cell surface and distribution in different cell populations. In this study we report the generation of a novel mAb (ECM41) specific for KIR2DL3 molecules. By the use of cell transfectants expressing one or other KIR we show that this reagent allows discrimination of KIR2DL3 from other GL183 mAb-reactive molecules such as KIR2DL2 and KIR2DS2. Moreover we show that this novel mAb can be used to assess the surface expression and distribution of KIR2DL3 in different polyclonal NK populations and in NK cell clones. Along this line, we were able to analyze the HLA class I specificity of NK clones expressing either KIR2DL3 or KIR2DL2, two inhibitory receptors that were so far serologically undistinguishable. Finally, the combined use of GL183 and ECM41 mAbs in redirected killing assays allowed us to investigate the functional outcome of the simultaneous engagement of KIR2DL3 and KIR2DS2 in NK cell clones co-expressing KIRs that display opposite (inhibitory vs activating) function.     

KIR‐HLA‐A and B alleles of the Bw4 epitope against HIV infection in discordant heterosexual couples in Chaco Argentina

Activating and inhibitory killer immunoglobulin‐like receptors (KIR) and their ligands HLA‐Bw4 (loci A and B) were studied by way of establishing whether they can contribute to protection against HIV‐1 infection in highly exposed and persistently seronegative (HESN) patients. Twenty‐three HIV‐1 serodiscordant heterosexual couples, 100 HIV‐1⁺ patients and 200 healthy individuals were included in this retrospective case–control study. HLA typing was performed by means of PCR followed by sequence‐specific oligonucleotide probe reverse hybridization. KIR3DL1 and KIR3DS1 were studied by PCR sequence‐specific primers. The frequency of KIR3DS1(3DS1/3DL1)‐Bw4 combination was significantly higher in HESN patients versus the discordant couples (P = 0·0003) and HIV‐1⁺ patients (P = 0·0001). Conversely, the KIR3DL1/KIR3DL1 homozygosity was significantly decreased in HESN patients versus the discordant couples (P = 0·00003), and HIV‐1⁺ patients (P = 0·00066). The frequency of HLA‐A*32 and HLA‐B*44 was higher in HESN versus their discordant couples (P = 0·009; P = 0·049), and HIV‐1⁺ patients (P = 0·00002; P = 0·0001). This had greater significance in combination with KIR3DS1 (3DS1/3DL1). KIR3DS1(3DS1/3DL1) could have a greater effect on protection against HIV‐1 infection in HESN patients when bound to a specific HLA allele, in this case HLA‐A*32 and HLA‐B*44, both Bw4 alleles. The differences probably arise both in the HLA alleles and in the subtypes of KIR receptors depending on the ethnic group studied.     

Interaction between KIR3DL1 and HLA-B*57 supertype alleles influences the progression of HIV-1 infection in a Zambian population

KIR and HLA loci are both highly polymorphic, and some HLA class 1 products bind and trigger cell-surface receptors specified by KIR genes. We examined whether KIR genes act in concert with HLA-B locus to control HIV-1 infection in a sample of Zambian patients. DNA samples from 88 Zambian patients with HIV-1 were examined. Patients were classified as either slow progressors (SP; n = 54) or rapid progressors (RP; n = 34) to AIDS. All were typed for HLA-B and KIR genes. Our results reveal an association between B*57 supertype (B*57s, which includes B*57 and B*58 alleles) and delayed progression to AIDS (p = 0.0007 by pc = 0.015; OR = 5.25). We also observed an increase incidence of Bw4-I80 in patients with slow progression (p = 0.001 by pc = 0.003, OR = 5). This increase was found to be secondary to B*57s. The presence of both KIR3DL1 and B*57S has a significant effect on progression to AIDS (p = 0.0008; OR = 5.61). B*57s genotypes with another HLA-B allele different from those in the trans position, which also had a specificity different to Bw4-I80 (Bw4-T80 or Bw6), was also greater in the SP than in the RP group (p = 0.00003; OR = 10.11). The presence of the inhibitory allele KIR3DL1 in combination with the HLA-B*57s alleles that contain the Bw4-I80 epitope, has a highly protective effect against progression to AIDS in Zambian patients.