【作者】 吴功强；

【导师】 黄河；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 杀伤细胞的免疫球蛋白样受体(Killer cell immunoglobulin-like receptor,KIR)是主要表达于人NK细胞及部分T细胞表面的一类受体。编码KIR的基因位于人19号染色体(19q13.4),该基因家族包括15个基因和2个假基因。KIR家族基因呈现高度遗传多态性,不同种族间KIR基因型的分布存在着很大的差异。依照KIR基因数量和种类,人类KIR主要分为两种单倍型:A和B。KIR主要通过与靶细胞表面的HLAⅠ类分子结合,传递抑制或活化信号,从而调控NK细胞的功能。在抗感染免疫、肿瘤免疫、清除老化变异细胞、造血干细胞移植以及自身免疫性疾病等领域发挥重要的调节作用。异基因造血干细胞移植(allo-HSCT)是治疗多种恶性病及一些非恶性病的有效方法,尤其在治疗急性白血病方面,可使50%以上患者长期无病生存。但allo-HSCT治疗就像一把双刃剑,在有效发挥造血重建、移植物抗白血病(GVL)等作用的同时,也会出现一些严重的并发症而导致病人死亡。引起病人死亡的主要原因是移植物抗宿主病(GVHD)和移植后复发。因此,如何减少GVHD和防止复发成为异基因造血干细胞移植迫切需要解决的关键问题。动物实验和临床观察提示allo-HSCT后,由KIR-配体不相合引起的NK细胞异源反应性能通过释放多种促造血因子促进骨髓植入和造血重建,通过杀伤受者来源的抗原递呈细胞(APC)从而阻止T细胞介导的GVHD,同时由于活化NK细胞具有强大的抗肿瘤效应,因此还能增强GVL效应,避免移植后复发。这一重大发现提示KIR不相合所引起的异源反应性NK将为从根本上解决骨髓移植中GVHD和GVL的矛盾问题带来新的希望。上述现象最初发现于半相合移植,而后研究提示在HLA相合的亲缘或非亲缘移植中均有发现。但也有研究发现,KIR-配体不相合引起的NK细胞异源反应性与TRM升高及总存活率下降有关,故在这方面的研究仍是热点,仍存在许多争议。目前国内关于KIR的临床研究较少,我们将探讨KIR基因型对异基因造血干细胞移植的影响及抑制性KIR功能修饰后对NK细胞功能的影响,并研究KIR的人群分布情况。研究共分为3章:第1章:在非亲缘、亲缘异基因造血干细胞移植中KIR与HLA基因型对移植结果的影响本研究第1章中分析KIR与HLA基因型对亲缘及非亲缘移植结果的影响。抽提供者与受者外周血或骨髓的DNA,并采用序列特异性引物PCR法(PCR-SSP)对其进行KIR与HLA-C位点基因检测。HLA-C位点分为两组:C1组(基因Cw1、3、7及8)和C2组(基因为Cw2、4、5及6),HLA-B位点分为Bw4与Bw6。KIR2DLl的配体为HLA-C2,KIR2DL2/2DL3的配体为HLA-C1,KIR3DL1的配体为HLA-Bw4。供受者KIR-配体不相合指供者存在上述抑制性基因,而受者缺乏上述一个或一个以上的相应配体。供者KIR可被分为三类基因型AA、AB及BB,研究时分为两组:AA及Bx(AB和BB)。结果显示,在无关供者移植的病人中,KIR不相合与复发风险减少密切相关(p=0.019;HR=0.329,95%CI 0.131-0.830),尤其是在髓系白血病病人,KIR不相合除了与复发的减少相关外(p=0.003;HR=0.193,95%CI 0.066-0.563),还能改善5年总生存(p=0.031;HR=0.423,95%CI 0.194-0.924)及无病生存(p=0.024;HR=0.445,95%CI 0.221-0.897),而在淋系白血病病人,KIR不相合对移植结果没有影响;供者KIR基因型为AA相对于Bx累积复发率更低,分别为12.9%与28.6%(p=0.024),进一步发现供者KIR含有更多的激活性受体基因与移植后复发风险增高密切相关(p=0.005;HR=1.463,95%CI 1.123-1.906);对髓系白血病病人来说,供者KIR基因中含有KIR2DS3,是个重要的预后危险因素:复发风险增加(p=0.003;HR=5.045,95%CI 1.746-14.575),而且5年总生存(p=0.006;HR=3.046,95%CI 1.378-6.732)及无病生存(p=0.003;HR=2.919,95%CI 1.430-5.959)降低。在亲缘移植病人中,病人HLA-C位点为杂合型C1C2,Ⅱ-Ⅳ度aGVHD发生增高(p=0.032;HR=2.685,95%CI1.087-6.635),在亲缘髓系病人中,也有类似的结果(p=0.024;HR=4.178,95%CI1.206-14.475)。在亲缘移植淋系白血病病人中,供者含有激活性受体KIR2DS1基因与Ⅱ-Ⅳ度aGVHD密切相关(p=0.023;HR=11.86,95%CI 1.415-99.432)。第2章:中国华东地区汉族与台湾汉族人群中KIR基因型和单倍型分布情况本研究第2章中随机选择116名华东地区汉族与97台湾汉族无血缘关系的健康志愿者作为研究对象,分析两人群的KIR分布情况,以及对移植的影响。KIR检测方法同第1章,并根据Hsu等的标准进行基因型和单倍型分析。结果显示:这两个人群能检出所有目前已知的17个KIR基因,KIR2DL4、KIR3DL2、KIR3DL和3KIR3DP1基因存在于所有个体中,基因频率均为1.00。除了KIR3DS1与2DS4*003两个基因频率在这两个人群间有显著性差异外,其它KIR基因频率都很类似,在华东地区汉族人群可检测到35种基因型,在台湾人群有29种。以(2,2)和(1,2)两种基因型最为常见,在华东汉族人群中的频率分别为23.6%与19.8%;而在台湾汉族分别为20.6%与24.7%。两个人群均有10种基因型可同时在其它多个人群中检测到,约占所有华东研究个体的65%,台湾个体的71%。华东汉族人群的A单倍型频率为68.1%,台湾汉族A单倍型频率为74.1%,均明显高于B单倍型。每个人群均有4种基因型无法根据Hsu等的标准模式分析出单倍型。第3章:KIR功能修饰对NK细胞的功能影响本研究第3章应用单克隆抗体CD158a和CD158b封闭KIR两个重要的抑制性受体KIR2DL1与KIR2DL3后,对NK细胞的功能影响,进一步去阐明KIR功能修饰后NK细胞实现GVL与GVHD分离的可能机制。应用Rosettesep人NK分选试剂盒分选人外周血NK细胞,使用乳酸脱氢酶释放法检测NK细胞对不同白血病细胞(人肿瘤细胞株NB4、Raji细胞和K-562)及同种异体的成熟或未成熟的DC的杀伤活性,并观察单克隆抗体封闭后,NK细胞杀伤活性的改变。应用CCK-8试剂检测混合淋巴细胞反应中NK细胞对T淋巴细胞增殖的影响。应用ELISA法检测混合淋巴细胞反应中细胞因子TGF-β1的表达水平。结果显示,任何一个抗体封闭KIR后,NK细胞杀伤NB4活性均增强,并与抗体浓度呈现量效关系(p＜0.05),两个封闭抗体联用能增强NK细胞的杀伤活性;对淋系白血病Raji细胞的杀伤活性则改变较小,仅0μg/ml组与20μ/ml组有统计学差异(p＜0.05);NK细胞本身对K-562细胞具有很强的杀伤活性,但经抗体封闭后,这种活性并没有提高。抗体作用后,对NB4细胞克隆形成的抑制率增高,并与封闭抗体浓度呈现量效关系(7.55%±4.43%vs.28.68%±2.31%,p＜0.05)。对同种异体未成熟DC具有一定的杀伤作用,但抗体封闭后,NK细胞的杀伤活性改变较小,各浓度组间没有显著性差异。成熟DC几乎耐受NK细胞的杀伤,但经抗体封闭KIR后,NK细胞对成熟DC的杀伤活性明显增强,并与抗体浓度呈现量效关系(2.20%±1.10%vs.37.59%±5.06%),各组间比较p＜0.05。经抗体封闭后的NK细胞在混合淋巴细胞反应中能明显提高对T细胞的增殖抑制(77.85%±8.31%vs.43.05%±5.95%,p＜0.05),检测上清液中的细胞因子TGF-β1含量增加。综上所述,本研究目前得出结论如下:1)在非亲缘造血干细胞移植后的髓系白血病病人,在GVH方向的KIR-配体不相合与移植后病人5年生存的改善密切相关,而且复发风险也显著降低。2)在非亲缘移植中,供者含有更多的KIR激活性受体基因与病人的复发风险增高密切相关;在髓系白血病病人,供者KIR中含有KIR2DS3是个预后不良的因素。3)在亲缘移植中,病人HLA-C位点为杂合型C1C2与aGVHD发生密切相关;在淋系白血病病人中,供者含有激活性受体KIR2DS1基因也与aGVHD发生密切相关。4)华东地区汉族与台湾汉族人群KIR基因分布表现出中国汉族人群KIR基因遗传多态性的一些共同特点。5)华东地区汉族与台湾汉族人群KIR分布的部分差异可能与“遗传漂移”有关。6)在移植前通过检测KIR基因可以来筛选供者及预测移植风险。7)KIR抑制性受体体外封闭后,能明显提高NK细胞杀伤急性髓系白血病细胞的能力,这在体外论证了,KIR功能修饰后,能提高NK细胞的GVL作用。8)KIR抑制性受体体外封闭后,能明显提高NK细胞杀伤同种异体成熟DC的能力,并通过分泌TGF-β1,抑制T细胞的增殖、活化,可能是阻止了T细胞介导的GVHD发生机理之一。更多还原

【Abstract】 Killer cell immunoglobulin—like receptors(KIRs ) are a family of inhibitory and activating receptors expressed on natural killer(NK) cells and some T lymphocytes. They belong to the Ig superfamily,and are encoded by a compact cluster of genes located on chromosome 19q13.4.To date,at least 15 functional KIR genes and two pseudogenes have been identified.KIR gene family shows a high degree of genetic polymorphism.Significant differences in KIR genotypes exist among different ethnic populations.In general,two kinds of KIR haplotypes are recognized:group A and group B.KIRs interact with HLA I molecules,which transmit inhibitory or activating signal,thereby regulating the function of NK cells.KIRs play an important regulatory role in a variety of processes,including anti-infective immunity,tumor immunity,the clearance of aging cells,autoimmune diseases as well as hematopoietic stem cell transplantation.Allogeneic hematopoietic stem cell transplantation(allo-HSCT) is a successful curative therapy for a variety of hematological malignancies and non-hematologic malignancies.Especially for acute leukemia,more than 50%patients obtain a long-term disease-free survival after allogeneic hematopoietic stem cell transplantation.But allo-HSCT therapy is just a double-edged sword.On one hand,the transplant can effectively establish hematopoietic reconstitution and graft-versus-leukemia(GVL);on the other hand,it can also arouse a series of serious complications and result in patient’s death.There are two major reasons for the patient’s death:graft-versus-host disease (GVHD) and relapse after transplantation.Therefore,how to reduce GVHD and prevent relapse in allogeneic hematopoietic stem cell transplantation is in urgent need and addressing the key issues involved in it is of great importance.Some animal trials and clinical observation identify alloreactive NK cells caused by KIR-ligand mismatch can promote the implantation of bone marrow and hematopoietic reconstitution by releasing a variety of hematopoietic promoting factors and eliminate recipient antigen presenting cells(APC) which have been demonstrated to initiate T cell-mediated aGVHD.Meanwhile,alloreactive NK cells can kill residual leukemia cells,thereby can make patient avoid post-transplant relapse.These major discoveries indicate KIR-ligand mismatch will fundamentally solve the problem of contradiction between GVHD and GVL in bone marrow transplantation and bring new hope.The above-mentioned phenomenon was first discovered in haploidentical hematopoietic stem cell transplant.It was also observed in HLA-match unrelated donor transplantation and sibling transplantation in the following research.However,other studies have arrived at the opposite conclusion,particularly in unrelated donor HSCT (URD-HSCT),and have shown a deleterious effect of KIR-ligand mismatch.For example,KIR-ligand mismatch was associated with increased transplant- related mortality(TRM).In short,it is a hot area for research worldwide,but there are still many controversies over it.At present,KIR is not well studied in China.In order to investigate the effects of KIR genotype on outcome following hematopoietic stem cell transplantation as well as the mechanism.We conducted this study to address the following questions:a) Whether KIR genotype has the same effect on outcome in sibling or unrelated transplantation? b) What is the mechanism? c) How about the distribution of KIR genes in Eastern Chinese Han and Taiwanese Han populations?Section 1:The Effects of KIR and HLA Genotypes on Clinical Outcome Following Unrelated Donor or Sibling Allogeneic Hematopoietic Stem Cell TransplantationIn section 1,the effects of KIR and HLA genotypes on clinical outcome following unrelated-donor and sibling hematopoietic stem cell transplantation were investigated. Patient and donor DNA were prepared from peripheral blood or bone marrow mononuclear cells prior to transplantation.HLA-C loci genes were analyzed using a PCR-SSP kit.HLA-C and HLA-B alleles were segregated into the epitope groups: HLA-C group 1,HLA-C group 2 and HLA-Bw4.KIR genotype analysis was performed by the PCR-SSP technique,using a KIR genotyping PCR-SSP kit.KIR genotyping was performed according to methods described previously.The inhibitory KIRs identified with HLA ligands are as follows: KIR2DL2/KIR2DL3,which bind group 1 HLA-C molecules(HLA-C1) characterized by serine at position 77 and asparagine at position 80,(Cw*01,03,07,08);KIR2DL1, which recognizes group 2 HLA-C molecules(HLA-C2) characterized by asparagine 77 and lysine 80(Cw*02,04,05,06);and KIR3DL1,which recognizes the HLA-Bw4 alleles.KIR-ligand mismatch was defined as the absence of one or more recipient HLA epitopes for the corresponding donor-inhibitory KIRs.Samples containing either one (A/B heterozygotes) or two(B/B homozygotes) group B haplotypes were assigned the genotype designation B/x.Samples that lacked all KIR B loci were assigned the genotype A/A.The results showed that in the setting of URD-HSCT,KIR-ligand mismatch was significantly associated with a decreased leukemic relapse risk(p=0.019;HR=0.329, 95%CI 0.131-0.830),mainly in myeloid disease(p=0.003;HR=0.193,95%CI 0.066-0.563).In myeloid disease,KIR-ligand mismatch also improved 5-year overall survival(OS)(p=0.031;HR=0.423,95%CI 0.194-0.924) and disease-free survival (DFS)(p=0.024;HR=0.445,95%CI 0.221-0.897).In lymphoid disease,KIR-ligand mismatch had not any beneficial effect on outcome.AA in the donor significantly decreased the cumulative incidence of relapse compared with Bx(12.9%vs.28.6%, P=0.024).We further found that more activating KIR genes in the donor resulted in an increased relapse risk(p=0.005;HR=1.463,95%CI 1.123-1.906).Meanwhile,the presence of donor activating KIR2DS3 was an independent factor associated with increased relapse risk(p=0.003;HR=5.045,95%CI 1.746-14.575) and decreased 5-year OS(p=0.006;HR=3.046,95%CI 1.378-6.732) and DFS(p=0.003;HR=2.919,95%CI 1.430-5.959) in myeloid disease.In the setting of sibling HSCT,KIR-ligand mismatch was not any beneficial effect on clinical outcome.In a multivariate analysis,heterozygous C1C2 in the patient resulted in increasedⅡ-ⅣaGVHD(p=0.032;HR=2.685,95%CI 1.087-6.635).This outcome also could be found in myeloid disease(p=0.024;HR=4.178,95%CI 1.206-14.475).The presence of donor activating KIR2DS1 was an independent factor associated with increasedⅡ-ⅣaGVHD(p=0.023;HR=11.86,95%CI 1.415-99.432) in the lymphoid disease.Section 2:Distribution of KIR Genes in Eastern Chinese Han and Taiwanese Han PopulationsIn section 2,we randomly selected 106 healthy unrelated individuals from Eastern Chinese Han and 97 from Taiwanese Han in order to detect the diversity of KIR gene content and the combination of haplotypes.KIR genotype analysis was performed by the PCR-SSP technique and the genotypes and haplotypes were assigned according to standard modal of Hsu et al.The results showed that all 17 KIR genes were observed in the two populations. Framework genes KIR2DL4,KIR3DL2,KIR3DL3 and KIR3DP1 were present in all individuals.The two populations had very similar frequencies for most loci,however, significant differences were noted in the frequencies of KIR3DS1 and KIR2DS4*003.A total of 35 genotypes were identified in the individuals from Eastern Chinese Han and 29 in the Taiwanese Han.Ten genotypes were also shared with other populations. These genotypes contained the most commonly observed genotypes,which were possessed by over 65%of Eastern Chinese Han and 71%of Taiwanese Han individuals. The most common KIR genotypes were(2,2)and(1,2),with frequencies of 23.6%and 19.8%,respectively,in the Eastern Chinese Han population;20.6%and 24.7%, respectively,in the Taiwanese Han population.Our data are consistent with the broad classification of KIR regions into A and B haplotypes,with a predominance of the group A haplotypes in our two populations. Group A outnumbered group B haplotypes with a frequency of 68.1%in the population of Eastern Chinese Han and 74.1%in the Taiwanese Han.Four new KIR genotypes in every population could not be assigned to the haplotypes,according to standard method of Hsu.Section 3:Effects on Activity of Human NK Cells by KIR Functional ModificationIn section 3,the effect of human NK cells on leukemic cells and allogeneic dendritic cells(DC) by blocking the inhibitory receptors KIR2DL1 and KIR2DL2/2DL3 with monoclonal antibody was observed.And we aim to further clarify the possible mechanism about NK cells achieving the separation of GVL and GVHD through KIR functional modification.Human peripheral blood NK cells are isolated by Rosettesep human NK sorting kit. Using lactate dehydrogenase(LDH) release assay is to detect NK cells killing activity against different leukemic cells(human tumor cell lines NB4,K-562 and Raji cells) and against allogeneic mature or immature DC.And to observe the effect on killing activity of NK cells by KIR2DL1 and KIR2DL2/2DL3 blockade,we established NK cell and T lymphocyte reaction system in vitro.The proliferation of T lymphocytes was detected using CCK-8 reagent.The level of TGF-β1 were detected by ELISA method.The results showed that the cytotoxicity of NK cells to NB4 cells was augmented, and the clone-forming capacity of NB4 cells was inhibited remarkably with the increase of concentration of the antibody.Combination with both antibodies can enhance NK cell killing activity.NK cells have a strong ability of killing K-562 cells.However,the cytotoxicity to K-562 cells was not a little of amplification with the inhibitory receptors blockade.The cytotoxicity to Raji cells was not evidently augmented,and statistical difference was observed only between groups of 0μg/ml and 20μg/ml,p＜0.05.The cytotoxicity to immature DC was also not evidently augmented,and statistical difference was not observed.But the cytotoxicity of NK cells to mature DC was augmented remarkably with the increase of concentration of the antibodies (2.20%±1.10%vs.37.59%±5.06%,p＜0.05).In mixed lymphocyte reaction,NK cells by the two antibodies blockade can evidently inhibit T cell proliferation(77.85%±8.31% vs.43.05%±5.95%,p＜0.05).The content of TGF-β1 in the supernatant was increased with the increase of concentration of the antibodies.In summary:1) In the setting of unrelated HSCT for myeloid leukemia,KIR-ligand mismatch was associated with a long-term survival advantage and decreased relapse.2) In the setting of unrelated HSCT,low number of activating KIRs genes in the donor could reduce the risk of relapse;The presence of KIR2DS3 in the donor was an important risk factor for myeloid leukemia.3) In the setting of sibling HSCT,heterozygous C1C2 in the patient was associated increasedⅡ-ⅣaGVHD;The presence of donor activating KIR2DS1 was also an independent factor associated with increasedⅡ-ⅣaGVHD for lymphoid leukemia.4) The KIR gene polymorphisms in Eastern Chinese Han and Taiwanese Han populations were similar to those of other Chinese Han populations.5) Some differences of the distribution of KIR genes in Eastern Chinese Han and Taiwanese Han populations may be related to "genetic drift".6) The study of the distribution of KIR genes will help choose a suitable donor and predict risks of transplantation in the two communities.7) The cytotoxic effects of human NK cells by the inhibitory receptors KIR2DL1 and KIR2DL2/2DL3 blockade against leukemic cells,especially acute myeloid leukemia, were significantly enhanced.This is demonstrated in vitro,alloreactive NK cells by KIR functional modification have a stronger GVL effect;8) Human NK cells by KIR2DL1 and KIR2DL2/2DL3 blockade significantly enhanced the cytotoxic effects against allograft mature DC and also secreted the protein of TGF-β1,which further inhibited T cells proliferation.This might be one of the mechanisms in preventing the T cell-mediated aGVHD.更多还原