活化性杀伤细胞免疫球蛋白样受体阳性的NK细胞对靶细胞的杀伤作用

目的 研究活化性杀伤细胞免疫球蛋白样受体(KIR)KIR2DS1阳性的自然杀伤(NK)细胞对急性髓系白血病(AML)靶细胞的杀伤作用;并探讨NK细胞KIR及人类白细胞抗原Cw位点(HLA-Cw)与靶细胞HLA-Cw错配对靶细胞的杀伤机制.方法 获取健康供者的外周血,经Dnyal磁珠负选高纯度NK细胞;取初治确诊的AML患者新鲜骨髓液,分离后的白血病细胞作为靶细胞.用抗CD158a和CD158b单克隆抗体封闭NK细胞抑制性KIR(如:KIR2DL1、KIR2DL2和KIR2DL3),并用噻唑蓝(MTT)比色法分别检测抑制性KIR封闭前和封闭后NK细胞对靶细胞的杀伤作用.采用聚合酶链反应和顺序特异性引物(PCR-SSP)基因分型技术分别检测NK细胞及靶细胞的KIR基因和HLA-Cw;根据HLA-Cw将NK细胞和靶细胞分别分为C1组(表达HLA-Cw 01、03、07、08、12、14、16分子)、C2组(表达HLA-Cw02、04、05、06、15、17、18分子)和C1/C2组(既表达C1组的等位基因又表达C2组的等位基因).结果 分选后的NK细胞经流式细胞仪检测,其纯度为(90.8±6.08)%;NK细胞抑制性KIR封闭后与封闭前比较,其对靶细胞的杀伤作用明显增强(t=-3.00,P=0.005);KIR2DS1阳性的NK细胞C1组对靶细胞C2组的杀伤作用高于靶细胞C1及C1/C2组,杀伤率分别为(57.370±1.400)%、(44.190±4.666)%和(36.770±6.560)%(F=11.87,P=0.021);NK细胞抑制性KIR封闭后,其对各组靶细胞的杀伤率更高(F=18.72,P=0.009).结论 NK细胞抑制性KIR封闭后其活性增强,KIR2DS1阳性的NK细胞对靶细胞的杀伤率高于KIR2DS1阴性的NK细胞;NK细胞KIR及HLA-Cw与靶细胞HLA-Cw错配,能够介导KIR2DS1阳性的NK细胞C1组抗原的异源反应性,实现"丢失自我"的识别作用,从而使NK细胞对靶细胞杀伤率增强.     

KIRB单倍型无关供者造血干细胞移植可提高急性髓细胞白血病患者的无复发存活率

急性髓细胞白血病（AML）患者接受无关供者造血干细胞移植（HSCT）后的存活率与移植相关死亡率和复发率密切相关。由供者杀伤细胞免疫球蛋白样受体（killer—cellimmunoglobulin—likereceptor,KIR）和受者HLA共同介导的自然杀伤细胞同种异基因反应性可改善受者存活率、促进植入、减少移植物抗宿主病的发生和降低复发率,关系着AML患者的HSCT能否成功。     

肾移植受者血他克莫司浓度对外周血自然杀伤细胞及其受体的影响

目的 研究肾移植受者血他克莫司(Tac)浓度对外周血自然杀伤(NK)细胞及其受体的影响.方法 将2007年12月至2009年7月间的60例受者纳入研究,术后受者均采用以Tac为基础的免疫抑制方案.根据术后6个月监测到的血Tac浓度将受者分为低浓度组和高浓度组[各为30例,术后6个月时血Tac浓度分别为(6.84±1.72)和(11.88±2.59)μg/L],另以20名健康志愿者作为对照组.术前和术后6个月,采用流式细胞术检测NK细胞及其抑制性受体(CD85j和CD158d)和活化性受体( CD94、NKG2D)的表达情况,采用酶联免疫吸附试验法检测免疫耐受分子分泌型HLA-G5( sH LA-G5)的表达水平.结果 术前低浓度组和高浓度组受者外周血NK细胞绝对值均较对照组显著降低(P＜0.05),术后6个月时低浓度组和高浓度组NK细胞比例及绝对值较对照组均显著降低(P＜0.05),低浓度组NK细胞绝对值显著高于高浓度组(P＜0.05).术前两组间CD85j、CD158d、CD94、NKG2D表达的差异均无统计学意义(P＞0.05);术后6个月时低浓度组和高浓度组CD85j和CD158d的表达较术前升高,CD94和NKG2D的表达下降,而低浓度组CD85j和CD158d的表达显著高于高浓度组(P＜0.05).经Spearman系数统计,CD85j和CD158d与sHLA-G5呈正相关(P＜0.01),NKG2D与sHLA-G5呈负相关(P＜0.01).结论 肾移植受者血Tac浓度与外周血NK细胞数量及其受体的表达具有相关性,低血Tac浓度受者的NK细胞数量及其抑制性受体的表达升高,仍然能有效保护移植肾功能.     

HLA不相合异基因造血干细胞移植后输注供者CD34^＋祖细胞来源的自然杀伤细胞可预防肿瘤复发

动物实验研究表明,NK细胞可以杀伤肿瘤细胞而不引起移植物抗宿主病（graft—versus—host disease,GVHD）,这一独特的生物学效应使HLA不相合造血干细胞移植（hematopoietic stem cell transplantation,HSCT）后输注供者NK细胞有可能成为有效预防肿瘤复发的措施,但前提是要明确在人体中输注供者NK细胞不会引起GVHD以及其他不良反应。     

供者NK细胞及其嵌合状态在异基因造血干细胞移植中的研究进展

目前,异基因造血干细胞移植(allo-HSCT)已广泛应用于造血系统疾病的治疗,但移植术后也存在一系列并发症。NK细胞的运用为改善allo-HSCT受者预后带来希望,供者来源NK细胞通过其细胞膜上的杀伤细胞免疫球蛋白样受体与其配体错配发挥同种异体反应,该过程具有保留移植物抗白血病和减少移植物抗宿主病双重效应。NK细胞是allo-HSCT后受者体内最早重建的免疫细胞群,因此移植后供、受者NK细胞嵌合状态评估对预测疾病预后及指导干预治疗具有重要意义。基于NK细胞嵌合状态的供者NK细胞输注免疫干预疗法可改善疾病预后,在血液系统疾病治疗中表现出良好的应用前景。本文就近年来供者NK细胞及其嵌合状态在allo-HSCT中的研究进展作一综述。     

输注供者自然杀伤细胞对小鼠单倍型相合造血干细胞移植的影响

目的 探讨输注供者自然杀伤(NK)细胞对小鼠单倍型相合造血干细胞移植的影响.方法 选取C57BL/6(H-2b)雄性小鼠为供者、CB6F1(H-2d/b)雌性小鼠为受者.移植前制备供者的骨髓细胞(BMC)、脾细胞(SC)及脾NK细胞,NK细胞经体外培养扩增和激活;所有受者均接受直线加速器X线全身照射(TBI)预处理.TBI后将受者分为4组(每组10只),分别进行单倍型相合造血干细胞移植.单纯TBI组:TBI后不输注细胞,仅作为对照;单纯BMC输注组:输注5×106个BMC;诱发GVHD组:输注5×106个BMC+1.5×107个SC;NK细胞输注组:输注5 x 106个BMC+1.5×107个SC+1×107个NK细胞,并腹腔注射100 ng重组人白细胞介素2(rhIL-2)和1μg rhIL-15,持续7 d.移植后观察各组受者GVHD的发生情况,并对各组受者进行组织病理学、供者细胞嵌合度和免疫功能重建等检测.另取TBI后受者20只,设白血病复发组和白血病治疗组,每组10只.白血病复发组:输注5×106个BMC+1×107个SC+2×106个白血病细胞株EL9611;白血病治疗组:在白血病复发组的基础上再输注1 x 107个NK细胞,并腹腔注射100 ng rhIL-2和1μg rhIL-15,持续7 d.观察两组受者白血病复发情况和移植后100 d的存活率.结果 单纯BMC输注组受者无GVHD发生,NK细胞输注组受者GVHD的评分和组织病理学改变均较诱发GVHD组轻(P<0.05)f诱发GVHD组的免疫功能重建较NK细胞输注组延迟.白血病复发组和白血病治疗组移植后100 d的存活率分别为20%和90%,两组比较,差异有统计学意义(P<0.01).结论 输注激活的供者NK细胞可以减轻小鼠单倍型相合造血干细胞移植后的GVHD,减少白血病复发,促进免疫功能重建.     

杀伤细胞免疫球蛋白样受体在移植中的作用

杀伤细胞免疫球蛋白样受体（killer cell immunog lobulin—like receptor，KIR）是表达于NK细胞和部分T细胞表面的一系列免疫球蛋白样超家族分子，可识别MHC—Ⅰ类分子并与之结合，传导激活或抑制性信号，从而调节NK细胞和部分T细胞活性。研究表明，KIR在病毒感染、肿瘤形成、移植受者免疫状态和自身免疫性疾病中发挥着重要作用。本文就近年来KIR在移植中作用的研究进行回顾。     

供者表达活化性杀伤细胞免疫球蛋白样受体对造血干细胞移植预后的影响

目的 观察供者表达活化性杀伤细胞免疫球蛋白样受体（aKIR）对受者造血干细胞移植（HSCT）预后的影响。方法 1996年至2001年共行亲缘性人类白细胞抗原（HLA）全相合骨髓移植59例，以序列特异性引物多聚酶链反应法（SSP-PCR）检测供者aKIR的表型。分析供者表达aKIR对受者移植后病毒、细菌和真菌感染及出血、复发、存活情况的影响。结果 供者表达aKIR与受者移植后出血、病毒及细菌感染发生的概率无明显相关性；当供者表达KIR3DS1表型时，发生真菌感染概率增高（X^4．804，P=0．028）。供者表达aKIR对受者HSCT后存活率和白血病复发率均无明显影响。结论 亲缘性HLA全相合HSCT中，供者表达aKIR并不能改善受者的移植效果。     

自然杀伤T淋巴细胞与移植物抗宿主病的研究进展

移植物抗宿主病(GVHD)是目前导致异基因造血干细胞移植(allo-HSCT)失败和受者死亡的重要因素,与移植物抗白血病效应(GVL)和受者的生活质量密切相关.GVHD是由供者移植物巾的T淋巴细胞识别宿主抗原引起的免疫反应所导致受者多器官损伤,涉及各种免疫活性细胞、细胞因子和炎症因子[1].     

自然杀伤T淋巴细胞与移植物抗宿主病的研究进展

移植物抗宿主病(GVHD)是目前导致异基因造血干细胞移植(allo-HSCT)失败和受者死亡的重要因素,与移植物抗白血病效应(GVL)和受者的生活质量密切相关.GVHD是由供者移植物巾的T淋巴细胞识别宿主抗原引起的免疫反应所导致受者多器官损伤,涉及各种免疫活性细胞、细胞因子和炎症因子[1].     

The impact of donor natural killer cell alloreactivity on allogeneic hematopoietic transplantation

Although natural killer (NK) cells are triggered to kill by many activating receptors, lysis of autologous cells is blocked by inhibitory receptors (called Killer cell Ig-like receptors or KIRs) which recognize epitopes shared by certain major histocompatibility complex (MHC) class I allele groups (called KIR ligands). As these inhibitory receptors are clonally distributed, they constituted a repertoire containing different allospecificities. Thus, the NK cells in the repertoire are lytic against allogeneic targets that do not express their inhibitory KIR ligands. In hematopoietic human-leukocyte-antigen (HLA)-haplotype mismatched transplantation, donor-vs-recipient alloreactive NK cells improve engraftment, decrease the incidence of leukemia relapse and do not cause Graft-vs-Host disease (GvHD). Pre-transplant molecular high-resolution HLA of recipient and donor, KIR genotyping of the donor and direct assessment of the donor NK repertoire identify donors with the potential for donor-vs-recipient NK cell alloreactivity.     

Analysis of KIR2D receptors on peripheral blood lymphocytes from liver graft recipients

KIR2D receptors are killer cell immunoglobulin-like receptors (KIRs) specific for HLA-C epitopes, that are expressed on NK cells as well as on minor peripheral blood T-cell subsets, and are able to control NK and T cells activity. The present work explores NK, and particularly CD8(+) T cells expressing KIR2D2L1/S1 (CD158a) or KIR2D2L2/3/S2 (CD158b) receptors in liver graft alloresponse. Flow cytometry was used to analyse peripheral blood mononuclear cells stained with anti-CD158a and anti-CD158b antibodies from 110 liver recipients and 46 healthy controls, previous to and along the first month after transplantation. Pre-transplantation data shows that both CD158a and CD158b molecules can be detected on NK and T cells from all patients and controls, but both KIR2D(+)NK cells are significantly under-represented in patients respect to controls (P<0.001), and CD3(+)CD8(+)CD158a(+) cells decreased particularly in patients suffering from acute rejection (4.03+/-1.33 cells/microL) compared with controls (7.8+/-2.4 cells/microL). Following transplantation, KIR2D(+)CD8(+) T-cell repertoires increased through the first month, mainly in recipients with a good graft acceptance. In summary, monitoring of KIR2D(+)CD8(+) T cells, particularly KIR2DL1/S1(+)CD8(+) T cells at pre-transplant, and both KIR2DL1/S1(+) and KIR2DL2/3/S2(+) T-cell subsets at early post-transplant period, could offer useful information for clinical follow-up of liver grafts.     

HLA-Cw4 expression on porcine endothelial cells reduces cytotoxicity and adhesion mediated by CD158a+ human NK cells

Abstract: Background:  Human natural killer (NK) cell-mediated cytotoxicity represents a hurdle in pig-to-human xenotransplantation. It was previously reported that the expression of human major histocompatibility complex class I molecules, including HLA-B27, -Cw3, -E, and -G, partially protects porcine endothelial cells (pEC) from human NK-mediated cytotoxicity and that HLA-G inhibits NK adhesion to pEC. Here, we tested if HLA-Cw4 expression on pEC alone, or concurrently with HLA-Cw3, prevents human NK adhesion and cytotoxicity against pEC via recognition of the killer-cell immunoglobulin-like receptors (KIR) CD158a (KIR2DL1) and CD158b (KIR2DL2/3), respectively.
Methods:  Two pEC lines (2A2 and PEDSV.15) were transfected with HLA-Cw3 and HLA-Cw4. HLA and KIR expression on porcine and human cells were analyzed by flow cytometry. The effect of HLA expression on pEC on human NK-mediated cytotoxicity and adhesion was tested by ⁵¹Cr-release and dynamic adhesion assays, respectively.
Results:  HLA-Cw4 expression on pEC reduced cytotoxicity mediated by CD158a⁺ polyclonal human NK cells by an average of 58%, and by CD158a^bright NK cell clones by 68%, but not by NK cells expressing low levels of CD158. Co-expression of HLA-Cw3 and HLA-Cw4 on pEC did not mediate further protection against NK cytotoxicity. The expression of HLA-Cw4 reduced the adhesion of human NK cells on pEC by a mean of 53%.
Conclusions:  While transgenic expression of HLA-Cw4 on pEC reduces NK cell adhesion and cytotoxicity, co-expression with HLA-Cw3 is not sufficient to completely overcome human NK-mediated cytotoxicity in vitro.     

Natural killer-cell activity after human renal transplantation in relation to killer immunoglobulin-like receptors and human leukocyte antigen mismatch

BACKGROUND: Natural killer (NK) cells use killer immunoglobulin-like receptors (KIR) that bind to self-class I major histocompatibility complex (MHC) molecules to prevent killing of autologous cells. Mismatched allografts, which do not express recipient MHC class I molecules, can therefore be potential targets for NK-cell killing. In our living related-unrelated renal transplantation program, donor-recipient pairs vary in the amount of both HLA and KIR genes they share. This provides us with a unique opportunity to dissect the influence of KIR on NK-cell function after transplantation. METHODS: Recipient NK cells were used in a cytotoxicity assay against donor peripheral blood mononuclear cells 2 days before, on the day of, and 3 days after transplantation. Results were correlated to HLA-KIR compatibility between donor and recipient. RESULTS: NK killing, in a direct ex vivo setting, was demonstrated to be HLA mismatch dependent. Recipient NK antidonor cytotoxicity was unaltered despite having received 2 days' treatment with cyclosporine A before transplantation. However, cytotoxicity increased 3 days after transplantation in 71% of recipients. Recipients exhibiting increased NK cytotoxicity against their donors after transplantation were found to possess more activating KIR genes specific for donor class I MHC molecules than those in whom killing activity did not increase (P<0.04). CONCLUSIONS: NK cells are activated after transplantation despite quadruple immunosuppression, suggesting that recipient NK-cell cytotoxicity against the donor may be a previously unrecognized area of the rejection process, especially in poorly matched donor-recipient pairs where the recipient may not express the correct repertoire of inhibitory receptors to prevent killing of donor cells.     

KIR/HLA ligand incompatibility in kidney transplantation

BACKGROUND: The polymorphic family of killer-cell immunoglobulin-like receptors (KIRs) consists of activating and inhibitory receptors expressed by natural killer (NK) cells and effector T cells that recognize human leukocyte antigen (HLA) class I ligands. It has been suggested that KIR/HLA incompatibility exerts beneficial effects in hematopoietic stem cell transplantation. METHODS: To elucidate whether certain receptor-ligand combinations between recipient KIR and donor HLA antigens lead to enhanced alloreactivity of NK cells associated with acute rejection (aRx) after kidney transplantation, we analyzed the entirety of matches/mismatches between KIR genes and known HLA ligands for aRx patients (n=105) compared to patients with stable renal function (n=119). RESULTS: Whereas HLA-C ligand incompatibility between donor and recipient has no influence on aRx, grafts derived from donors homozygous for HLA-C group 2 alleles seem to demonstrate a better outcome (P=0.052). Additionally, a higher number of inhibitory receptors in the recipient's genotype (P=0.042), a significant higher number of matches for the receptors KIR2DL2/DS2 (P=0.004), as well as a higher number of mismatches for KIR2DL3 (P=0.014) could be observed for patients with stable renal function. CONCLUSION: Our data illustrate that certain KIR/HLA class I ligand combinations between donor and recipient might influence graft short-term outcome after renal transplantation.     

KIR‐Ligand Mismatches Are Associated With Reduced Long‐Term Graft Survival in HLA‐Compatible Kidney Transplantation

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system with the ability to detect HLA class I disparities via killer‐cell immunoglobulin‐like receptors (KIR). To test whether such KIR‐ligand mismatches contribute to the rejection of human solid allografts, we did a retrospective cohort study of 397 HLA‐DR‐compatible kidney transplantations and determined the KIR and HLA genotypes of recipients and the HLA genotypes of donors. In transplantations compatible for HLA‐A, HLA‐B and HLA‐DR (n = 137), in which a role for T cells and HLA antibodies in rejection was minimized, KIR‐ligand mismatches were associated with an approximately 25% reduction in 10‐year death‐censored graft survival (p = 0.043). This effect was comparable to the effect of classical HLA‐A and HLA‐B incompatibility, and in HLA‐A,‐B‐incompatible transplantations (n = 260) no significant additional effect of KIR‐ligand mismatches was observed. Multivariate Cox regression analysis confirmed the effect of KIR‐ligand mismatching as an independent risk factor in HLA‐A,‐B,‐DR‐compatible transplantations (hazard ratio 2.29, range 1.03–5.10, p = 0.043). This finding constitutes the first indication that alloreactive NK cells may thwart the success of HLA‐compatible kidney transplantations, and suggests that suppression of NK‐cell activity can improve the survival of such kidney grafts.     

Impaired natural killer cell lysis in breast cancer patients with high levels of psychological stress is associated with altered expression of killer immunoglobin-like receptors

BACKGROUND: We previously reported that cancer-related psychological stress is associated with reduced natural killer (NK) cell lysis. We hypothesized that reduced NK cell cytotoxicity in patients with increased levels of stress would correlate with alterations in the expression of inhibitory NK cell receptors (killer immunoglobulin-like receptors, or KIRs). The specific aim of this study was to examine KIR expression in patients with high or low levels of psychologic stress and correlate alterations in KIR expression with NK cell function. MATERIALS AND METHODS: Two hundred twenty-seven patients underwent baseline evaluation of cancer-related psychological stress and were randomized to psychosocial intervention versus observation. From this population, two groups were defined based on pretreatment measurements of NK lytic activity, stress levels, and the availability of cryopreserved peripheral blood mononuclear cells (PBMC). Group I (n=9) had low stress by the Impact of Events Scale (IES), and high NK cell lysis at the 50:1 effector: target ratio (NK(50)=52-89%). Group II (n=8) had high stress and low NK(50) (27-52%). Lymphokine activated killer (LAK) activity, antibody dependent cellular cytotoxicity (ADCC), and expression of cytokine receptors, adhesion molecules, and killer immunoglobulin-like receptors (KIRs) were assessed in PBMC. RESULTS: Incubation of PBMC with NK-stimulatory cytokines (IL-2, IL-12, or IL-15) led to significant increases in cytotoxic activity regardless of IES/NK(50) scores. There were no significant group differences in NK cell surface expression of the IL-2 receptor components CD25 and CD122, antibody-dependent lysis of HER2/neu-positive SKBr3 cells treated with an anti-HER2/neu monoclonal antibody, expression of adhesion molecules (CD2, CD11a, CD18) and markers of activation (CD69), or expression of the KIRs CD158a, NKG2a, NKB1, and CD161. However, levels of CD158b were significantly higher in Group I after incubation in media alone or with IL-2, and CD94 expression was significantly lower in Group I after incubation with IL-2. CONCLUSIONS: In this study of a small subset of breast cancer patients chosen from a previous clinical trial of psychosocial intervention for breast cancer, impaired NK lysis in breast cancer patients with high levels of psychological stress was associated with alterations in surface expression of killer immunoglobulin-like receptors. However, immune effectors retained the ability to lyse antibody-coated targets and to initiate lymphokine-activated killer activity, irrespective of stress levels or baseline NK(50).     

Granular cells of the hepatic capillary sinuses--receptors

Pit cells or hepatic natural killer (NK) cells represent an organ-associated NK cell population. They are situated in liver sinusoids and exert high spontaneous cytotoxic activity against tumor cell lines and may act as a primary defense barrier to metastasing tumor cells and to virus infections. Pit cells express two types of receptors on their cell membrane. One type activates NK cell killing (NCR or natural cytotoxicity receptors, as NKp46, NKp44, NKp30, NKG2D) by recognizing ill-defined molecules on target cells. The second type of receptors inhibits the lytic pathway by recognition of self class I MHC molecules and are represented by KIR or killer cell Ig-like receptors, as KIR2D, CD94/NKG2. Pit cells express on their cell membrane and other type of molecules as CD2, CD54, CD11a/CD18 and are CD3 negative.     

A decrease in graft-vs.-host disease without loss of graft-vs.-leukemia reactivity after MHC-matched bone marrow transplantation by selective depletion of donor NK cells in vivo.

It is thought that natural killer cells may play a role in graft-vs.-host reactions after allogeneic bone marrow transplantation, but the use of NK cell-specific reagents has been limited. In this report, an NK allele-specific monoclonal antibody, anti-NK 1.1, was used to study the impact of in vivo donor NK cell depletion on GVH disease, graft-vs.-leukemia (GVL) reactivity and donor T cell chimerism after allogeneic murine BMT. AKR/J (H-2k) recipient mice were preconditioned with suboptimal irradiation (9 Gy = LD50) and transplanted with major histocompatibility complex-matched B10.BR (H-2k) BM cells with or without added spleen cells as a source of T cells. The addition of increasing numbers of spleen cells to the BM inoculum produced GVHD of varying intensities. The beneficial effect of NK depletion on GVHD was dependent on the intensity of the GVH reaction. Donor NK cell depletion had no effect on the survival of mice with severe GVHD after MHC-matched BMT (B10.BR into AKR) or after MHC-mismatched BMT (B10.BR into DBA/2; H-2k into H-2d). However, donor NK depletion increased survival of AKR hosts given sufficient B10.BR splenic T cells to induce mild-to-moderate GVHD. Ex vivo depletion of donor CD8+ T cells also reduced GVH-associated mortality, but the use of both CD8 and NK depletion offered no improvement over either alone, suggesting an interaction between CD8+ and NK 1.1+ cells. In contrast to CD8 depletion, donor NK depletion did not compromise the rapid and complete establishment of donor T cell chimerism nor the ability of chimeras to mount an effective GVL reaction. Thus, elimination of donor NK cells provides an alternate strategy for reducing GVHD without loss of GVL reactivity following MHC-matched allogeneic BMT.