Perforin- and Fas-dependent mechanisms of natural killer cell-mediated rejection of incompatible bone marrow cell grafts

Natural killer (NK) cells eliminate target cells infected with intracellular pathogens and tumor cells by employing the granule exocytosis and death receptor pathways. They also mediate the acute rejection of incompatible bone marrow cell (BMC) grafts. However, the cytotoxic mechanisms employed during acute BMC graft rejection are obscure. Throughout these studies, BMC graft rejection was compared between two inbred strains of mice: 129 mice, which apparently use perforin- and Fas-dependent cytotoxicity, and C57BL/6 (B6) mice, which are able to exploit perforin- and/or Fas-independent mechanisms. Using perforin-knockout (PKO) mice, we have determined that the granule exocytosis pathway can play a major role in NK cell-mediated rejection of allogeneic and MHC class I-deficient BMC, depending upon the genetic background of the recipient and the environmental housing conditions. Although the granule exocytosis pathway seems to be the most potent cytolytic mechanism of NK cell-mediated rejection, alternative perforin-independent mechanisms, such as death receptor-induced apoptosis, also exist. By preventing both perforin- and Fas-mediated interactions concurrently, we observed that 129 mice were impaired in mediating MHC class I-deficient BMC rejection, while B6 mice maintained strong rejection capacities. The administration of neutralizing TNF antibodies to B6PKO mice before challenging with Fas and MHC class I double-deficient BMC still did not reverse rejection. Thus, our studies reveal the relative importance of perforin-, Fas-, and TNF-based cytotoxicity in NK cell-mediated rejection of incompatible BMC.     

The immunobiology of natural killer cells and bone marrow allograft rejection.

Natural killer (NK) cells mediate the acute rejection of bone marrow cell (BMC) allografts, but not solid tissue grafts, in lethally irradiated mice. However, the mechanisms underlying this capability for rejecting BMC remain unclear. NK cells express (1) inhibitory receptors specific for major histocompatibility complex (MHC) class I molecules and (2) activating receptors with diverse specificities. Inhibitory NK receptors confer to NK cells the ability to discriminate between MHC class I-positive and -negative target cells and are therefore involved in the control of NK cell tolerance to self, as well as in the elimination of cells that have downregulation of MHC class I molecules. Preclinical studies in mice have provided good evidence that subsets of NK cells that bear different combinations of both inhibitory and activating Ly49 receptors can interact with each other and target specific BMC rejection, as well as NK cell responses toward tumor cells. Recent clinical studies have also shown that the use of killer cell immunoglobulin-like receptor ligand incompatibility in patients with leukemia who received hematopoietic stem cell transplants correlated not only with the elimination of graft rejection, but also with eradication of tumor and prevention of graft-versus-host disease; this offers a significant advantage for survival. In this review, we attempt to bring together literature regarding the biology of NK cells and discuss the current issues in bone marrow transplantation and the potential clinical role of NK cell alloreactivity in the efficacy of this procedure for immunotherapy of cancer and infectious states.     

Inhibition of natural killer cell-mediated bone marrow graft rejection by allogeneic major histocompatibility complex class I,but not class II molecules

The role of major histocompatibility complex (MHC) class I and class II molecules in natural killer (NK) cell-mediated rejection of allogeneic, semi-syngeneic and MHC-matched bone marrow grafts was investigated. The use of β₂-microglobulin (β₂m) -/- and β₂m +/- mice as bone marrow donors to MHC-mismatched recipients allowed an analysis of whether the presence of semi-syngeneic and allogeneic MHC class I gene products would be triggering, protective or neutral, in relation to NK cell-mediated rejection. Loss of β₂m did not allow H-2^b bone marrow cells to escape from NK cell-mediated rejection in allogeneic (BALB/c) or semi-allogeneic (H-2D^d transgenic C57BL/6) mice. On the contrary, it led to stronger rejection, as reflected by the inability of a larger bone marrow cell inoculum to overcome rejection by the H-2-mismatched recipients. In H-2-matched recipients, loss of β₂m in the graft led to a switch from engraftment to rejection. At the recipient level, loss of β₂m led to loss of the capability to reject H-2-matched β₂m-deficient as well as allogeneic grafts. When MHC class II-deficient mice were used as donors, the response was the same as that against donors of normal MHC phenotype: allogeneic and semi-syngeneic grafts were rejected by NK cells, while syngeneic grafts were accepted. These data suggest a model in which allogeneic class I molecules on the target cell offer partial protection, while certain syngeneic class I molecules give full protection from NK cell-mediated rejection of bone marrow cells. There was no evidence for a role of MHC class II molecules in this system.     

Inhibition of natural killer cell-mediated lysis of tumor cells by normal and regenerating bone marrow

The natural killer (NK) cells which can lyse certain tumor cells during brief incubation in vitro have also been postulated to be the cells responsible for natural resistance to transplanted hemopoietic cells in vivo. To test this hypothesis, we have now measured: 1) the ability of bone marrow cells to compete with tumor cells as targets for spleen NK cells and 2) the effect of a brief incubation with spleen cells on the hemopoietic grafting potential of bone marrow cells. Firstly, when CBA/J mouse spleen cells were incubated with 51Cr-labelled YAC tumor cells together with DBA/2 mouse bone marrow cells, tumor cell lysis was reduced compared with incubation of spleen cells with tumor cells alone. Tumor cell lysis was even less when post-irradiation regenerating bone marrow was used. Secondly, C57B1/6 mouse bone marrow cells incubated with an excess of DBA/2 mouse spleen cells showed a reduced ability to produce hemopoietic spleen colonies in irradiated 129/J mice, whereas incubation with either thymus cells or fewer spleen cells produced no such effect. The results show that, when incubated with spleen cells under the conditions of a standard NK cell assay, regenerating bone marrow cells competitively inhibit the killing of YAC tumor cells and bone marrow progenitor cells are rendered ineffective in their hemopoietic colony-forming potential (CFU-s). These findings suggest that certain hemopoietic progenitor cells and YAC tumor cells can both serve as targets for NK cells, consistent with the view that the spontaneous cytolysis of tumor cells in vitro and natural resistance to bone marrow transplantation in vivo are mediated by cells of a common lineage.     

Inhibitory NKG2A and activating NKG2D and NKG2C natural killer cell receptor genes: susceptibility for rheumatoid arthritis

The inhibitory (NKG2A) and activating (NKG2D and NKG2C) natural killer (NK) cell receptors are expressed on a subset of NK and T cells. They regulate the innate and adaptive immune systems related to cytotoxicity and cytokine production that are involved in the pathogenesis of rheumatoid arthritis (RA). The role of inhibitory and activating NK cell receptor genes might contribute to chronic inflammation and destruction of bone and cartilage in RA. Therefore, we investigated the association of the NKG2A, NKG2C, and NKG2D genotypes with RA. NKG2A (KLRC1) NKG2C (KLRC2), and NKG2D (KLRK1, D12S249E) genes were genotyped in 210 unrelated patients with RA and 298 controls using a polymerase chain reaction-restriction fragment length polymorphism. We further investigated the relationships between the genotypes of each single nucleotide polymorphism and the presence of rheumatoid factor (RF), antinuclear antibody (ANA), and bony erosions in RA patients. The major NKG2A c.338-90*A/*A, NKG2C102*Ser/*Ser, and NKG2D72*Ala/*Ala genotypes in RA were significantly associated compared with controls [P = 0.013, odds ratio (OR) = 0.6, 95% confidence interval (CI) = 0.44-0.91; P < 0.0001, OR = 2.1, 95% CI = 1.44-2.96; and P = 0.019, OR = 0.6, 95% CI = 0.45-0.93, respectively]. The minor NKG2A c.338-90*G/*G, NKG2C102*Phe/*Phe, and NKG2D72*Thr/*Thr genotypes showed a risk of RA (P = 0.010, OR = 2.0, 95% CI = 1.17-3.54; P < 0.0001, OR = 0.2, 95% CI = 0.12-0.48; and P = 0.032, OR = 2.3, 95% CI = 1.05-5.01, respectively) compared with controls. No significance was observed between the inhibitory (NKG2A) or activating (NKG2C and NKG2D) receptor genotypes and the presence of RF, ANA, or bony erosions in RA.     

Natural killer cell-mediated lysis of dorsal root ganglia neurons via RAE1/NKG2D interactions

Natural killer cells have been reported to be able to kill various transformed and virus-infected target cells. It was recently observed that NK cells also could kill syngeneic dorsal root ganglia (DRG) neurons by a perforin-dependent mechanism. We demonstrate here that this phenomenon does not reflect a general ability of NK cells to kill neurons in culture. While DRG neurons of the peripheral nervous system were readily killed, ventral spinal cord neurons and hippocampal neurons of the central nervous system (CNS) were resistant to lysis. The resistance to NK cell-mediated lysis of the latter neurons was not related to protection by MHC class I molecules, since similar beta(2)-microglobulin(-/-) neurons were equally resistant to lysis. While exploring other possible molecular mechanisms for the selective triggering of lysis of DRG neurons, we observed that the retinoic acid early inducible gene-1 (RAE-1), the product of which is a ligand for the NK cell-activating receptor NKG2D, was expressed at high levels in the DRG neurons. In contrast, RAE-1 was expressed only at very low levels in the resistant CNS-derived neurons. Blocking NK cells withanti-NKG2D antibodies inhibited NK cell-mediated killing of the DRG neurons. Thus, we demonstrate that NK cell-mediated lysis of DRG neurons correlates with the expression of RAE-1 and that this lysis is dependent on activation of NK cells via NKG2D. This observation demonstrates that NK cells can kill non-pathogen-infected or non-transformed syngeneic cells through activation of the NKG2D receptor.     

Human natural killer lymphocytes through the engagement of natural cytotoxicity receptors and NKG2D can trigger self-aggression

NK cell killing of autologous dendritic cells (DC) is mediated by the LFA1-dependent activation of the calcium-calmodulin kinase II, resulting in degranulation and extracellular release of perforin and granzymes. On the other hand, DC killing can also be triggered by the engagement of the phosphatidylinositol 3-kinase upon ligation of the natural cytotoxicity receptors (NCR) NKp30 and NKp46. Furthermore, NK cells can also damage autologous stromal cells (SC) derived from bone marrow (BMSC) or fibroblasts obtained from skin biopsies (SF). Binding of ICAM1, expressed by BMSC or SF, to its receptor, the integrin LFA1 on NK cells, plays a key role in SC/NK interaction. Both types of SC bear the MHC-related molecule MIC-A and the UL16 binding protein ULBP3, which engagement with NKG2D, expressed by NK, is responsible for the delivery of lethal hit. HLA-I molecules do not protect DC or SC from NK cell-mediated injury. Thus, NK cells can be activated upon binding with autologous DC or SC, using different receptor-ligand pairs. On one hand this activation leads to the amplification of the NK arm of innate immune response; on the other hand, the lack of HLA-I-mediated control of such activation may lead to self-reactions elicited by NK cells.     

同种反应性自然杀伤细胞对小鼠单倍体骨髓移植后T细胞重建的影响

目的探讨同种反应性自然杀伤（NK）细胞对小鼠单倍体骨髓移植（BMT）后T细胞重建的可能影响。方法以（C57BL/6×BALB/c）BCF1（H-2d/b）为供鼠,BALB/c（H-2d）为受鼠,鼠龄6～8周,体质量16～20 g。建立小鼠单倍体BMT模型。根据移植物不同将实验分为单纯BMT组、非同种反应性NK（non-alloNK）细胞组、同种反应性NK（allo-NK）细胞组,移植后不同时间点以外周血CD3e的表达、外周血血清白细胞介素4（IL-4）、干扰素（IFN-γ）浓度等指标评价同种反应性NK细胞对T细胞重建的可能影响。结果移植后＋10 d受鼠外周血CD3e的表达allo-NK组较non-alloNK组显著增高,分别为（16.58±5.77）%、（8.64±3.60）%,差异具有统计学意义（P〈0.05）。＋30 d时allo-NK组IL-4的浓度显著低于单纯BMT组,分别为（9.73±2.08）pg/mL、（12.44±1.57）pg/mL,差异具有统计学意义（P〈0.05）。non-alloNK组IFN-γ的浓度显著高于单纯BMT组,分别为（98.78±33.07）pg/mL、（52.15±18.87）pg/mL,差异具有统计学意义（P〈0.05）。结论同种反应性NK细胞用于单倍体BMT小鼠,可能促进T细胞的重建,体内诱导Th1免疫反应的偏移。     

乏氧对人外周血NK细胞NKG2A、NKG2D及CD44表达的影响

目的： 观察乏氧微环境对人外周血自然杀伤细胞(NK)表面自然杀伤细胞2族成员A(NKG2A)、自然杀伤细胞2族成员D(NKG2D)及CD44分子表达的影响,探讨乏氧抑制NK细胞杀伤活性的分子机制。方法: 采用密度梯度离心法分离健康人外周血单个核细胞（PBMC）,贴壁去除单核细胞获得外周血淋巴细胞（PBL）,分别置常氧（21％O2）、乏氧（1％O2）以及有或无人重组白细胞介素2(rhIL-2)（1×106 U/L）刺激条件下培养16 h,流式细胞术（FCM）检测不同 NK细胞亚群 NKG2A、NKG2D以及CD44分子的表达。结果: 常氧条件,人外周血CD3-CD56+NK细胞NKG2A、NKG2D表达的阳性率分别为16.16%和78.45%,乏氧条件下二者表达的阳性率分别为15.16%和71.08%;rhIL-2上调NKG2A和NKG2D的表达,乏氧不影响 rhIL-2对NKG2D、 NKG2A的上调作用;rhIL-2显著上调NK细胞CD44的表达,乏氧抑制CD44的表达（P<0.05）。结论: 乏氧下调外周血NK细胞表面受体NKG2D及CD44的表达,但对NKG2A的表达无显著影响。由此提示,NKG2D及CD44分子可能在乏氧引起的NK细胞杀伤活性抑制中具有重要作用。     

Asymmetric ligand recognition by the activating natural killer cell receptor NKG2D, a symmetric homodimer

Natural killer (NK) cells function through a diverse array of cell-surface natural killer receptors (NCRs). NCRs specific for classical and non-classical MHC class I proteins, expressed in complex patterns of inhibitory and activating isoforms on overlapping, but distinct, subsets of NK cells, play an important role in immunosurveillance against cells that have reduced MHC class I expression as a result of infection or transformation. Another NCR, NKG2D, is an activating NCR first identified on NK cells, but subsequently found on macrophages and a variety of T cell types. NKG2D ligands in rodents include the MHC class I-like proteins RAE-1 and H60 and, in humans, ULBPs and the cell stress-inducible proteins MICA and MICB. NKG2D-MIC and -RAE-1 recognition events have been implicated in anti-viral and -tumor immune responses. Crystallographic analyses of NKG2D-MICA and -RAE-1 complexes reveal an unusual mode of recognition that apparently tolerates a surprising degree of ligand plasticity while generating affinities that are among the strongest TCR- or NCR-ligand affinities, thus, far described.     

Effects of NKG2D haplotypes on the cell-surface expression of NKG2D protein on natural killer and CD8 T cells of peripheral blood among atomic-bomb survivors

NKG2D is a primary activating receptor that triggers cell-mediated cytotoxicity in NK cells against tumor and virus-infected cells. We previously identified the NKG2D haplotypes in the natural killer gene complex region on chromosome 12p. Two major haplotype alleles, LNK1 and HNK1, were closely related to low and high natural cytotoxic activity phenotypes, respectively. Furthermore, the haplotype of HNK1/HNK1 has revealed a decreased risk of cancer compared with LNK1/LNK1. In the present study, using flow cytometry, we evaluated the functional effects of NKG2D haplotypes and five htSNPs in terms of the cell-surface expression of NKG2D protein on NK and CD8 T cells of peripheral blood among 732 atomic-bomb survivors. NKG2D expression on NK cells showed significant increases, in the order of LNK1/LNK1, LNK1/HNK1 and HNK1/HNK1 haplotypes (p for trend=0.003), or with major homozygous, heterozygous, and minor homozygous genotypes for individual htSNPs (p for trend=0.02-0.003). The same trend was observed for NKG2D expression on CD8 T cells. Our findings indicate that the NKG2D haplotypes are associated with the expression levels of NKG2D protein on NK and CD8 T cells, resulting in inter-individual variations in human cytotoxic response.     

Allorecognition by murine natural killer cells: lysis of T-lymphoblasts and rejection of bone-marrow grafts

Summary: Natural killer (NK) cells of inbred mice reject allogeneic bone-marrow cells, and NK cells of F1 hybrid mice can reject parental bone-marrow cells (hybrid resistance). In some cases these patterns of rejection can be mimicked in vitro by utilizing IL-2 cultured NK effector cells and allogeneic or parental T-lymphoblasts as target cells. Lysis of allogeneic parental targets in vitro can be explained on the basis of the missing self hypothesis. Subsets of NK cells that bear non-overlapping MHC class I inhibitory receptors belonging to the Ly49 family lyse allogeneic targets because they do not express self class I molectiles of the NK cell donor. Parental strain targets are lysed because they do not express all of the self class I antigens of the Fl hybrid, and hence fail to deliver inhibitory signals to all subsets of Fl NK cells. The expression of Ly49 receptors on NK cells is regulated by liost MHC to ensure maximal sensitivity to alterations in self class I molecules and to prevent autoreactivity. In many instances, however, the rejection of allogeneic bone marrow cells in vivo cannot be readily explained by the missing self hypothesis. In these instances, it appears that rejection is initiated by class 1 MHC receptors on NK ceils Out recognize allogeneic class I molecules as non-self, and activate rather than inhibit NK cell function.     

Generation of natural killer cells from bone marrow precursors in vitro.

A simple and highly reproducible bone marrow culture system for the generation of cytolytically active NK cells from immature precursors in the bone marrow is described. The NK cells can be generated with various sources of IL-2, including Con A-conditioned medium, supernatants from IL-2-producing cell lines and recombinant IL-2. Neither IL-1, IL-3 nor alpha/beta interferon induced significant cytotoxicity in bone marrow cells. Identification of the cytotoxic cells as NK cells was based on their phenotypic characteristics (aGM1+, Thy 1 +/-, Ly 1-2- X Ia-, RIL-2 +/-, H2+), as well as their spectrum of target specificity. The deliberate addition of peripheral blood mononuclear cells as a source of mature NK cells and elimination of cells expressing markers specific for mature NK cells indicated that the generated NK cells were descendants of precursors of NK cells harboured in the bone marrow and not derived from mature NK cells contaminating the bone marrow preparations. Thus, it was shown that not only functionally active NK cells but also their precursors are highly dependent on IL-2 for differentiation and growth. This culture system should be helpful in studying the origin of NK cells in relation to other cell lineages as well as the regulation of the maturation of NK cells from their precursors.     

白血病患者NK细胞表面NKG2D受体及其配体MICA/B的研究

目的:研究白血病患者NK细胞表面NKG2D受体及其配体MICA/B的表达,探讨白血病细胞逃逸NK细胞杀伤的机制.方法:采用流式细胞术检测NK细胞表面NKG2D受体和骨髓有核细胞表面MICA/B配体.结果:治疗前组和完全缓解组NKG2D受体的表达均较健康组低(P＜0.05);且完全缓解组低于治疗前组(P＜0.05);治疗前组和完全缓解组MICA/B配体的表达均低于增生性贫血组(P＞0.50);完全缓解组与治疗前组比较差异无显著性(P＞0.05).结论:白血病患者体内NKG2D-MICA/B介导的NK细胞功能受抑,这可能导致白血病细胞逃逸NK细胞的细胞毒作用;白血病化疗后完全缓解时其体内NKG2D-MICA/B介导的NK细胞功能仍未恢复,且较治疗前更低.     

Cloning of NKG2-F,a new member of the NKG2 family of human natural killer cell receptor genes

The NKG2 family of genes encodes at least four different type II transmembrane molecules (NKG2-A, NKG2-B, NKG2-C and NKG2-E) which contain a C-lectin domain. These proteins have been shown to be covalently associated with CD94, another C-type lectin member. The heterodimers are involved in natural killer cell-mediated recognition of different HLA-allotypes. Here we describe the cloning of a new NKG2-related gene, termed NKG2-F, localized 25 kb from NKG2-A as well as its relationship with the previously described NKG2-D cDNA. Despite the similarities with the other NKG2 genes, NKG2-F encodes a putative protein which does not contain any lectin domain. However, a conserved 24-amino acid sequence, present in all members of the NKG2 family, suggests that NKG2-F is also able to form heterodimers with CD94.     

The activating receptor NKG2D of natural killer cells promotes resistance against enterovirus‐mediated inflammatory cardiomyopathy

In enterovirus‐induced cardiomyopathy, information regarding the detailed impact of natural killer (NK) cells on the outcome of the disease is limited. We therefore hypothesized that NK cells and certain NK cell receptors determine the different outcome of coxsackievirus B3 (CVB3) myocarditis. Here, we demonstrate in murine models that resistance to chronic CVB3 myocarditis in immunocompetent C57BL/6 mice is characterized by significantly more mature CD11b^high NK cells, the presence of NKG2D on NK cells, and enhanced NKG2D‐dependent cytotoxicity compared to CVB3‐susceptible A.BY/SnJ mice. The highly protective role of NKG2D in myocarditis was further proven by in vivo neutralization of NKG2D as well as in NKG2D‐deficient mice but was shown to be independent of CD8⁺ T‐cell‐dependent immunity. Moreover, the adoptive transfer of immunocompetent C57BL/6 NK cells pre‐ (day ?1) as well as post‐infectionem (day +2) displayed the potential to prevent permissive A.BY/SnJ mice from a progressive outcome of CVB3 myocarditis reflected by significantly improved cardiopathology and heart function. Altogether, our results provide firm evidence for a protective role of NKG2D‐activated NK cells in CVB3 myocarditis leading to an effective virus clearance, thus offering novel therapeutic options in the treatment of virus‐induced myocarditis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Tbet promotes CXCR6 expression in immature natural killer cells and natural killer cell egress from the bone marrow

Tbet-deficient mice have reduced natural killer (NK) cells in blood and spleen, but increased NK cells in bone marrow and lymph nodes, a phenotype that is thought to be the result of defective migration. Here, we revisit the role of Tbet in NK cell bone marrow egress. We definitively show that the accumulation of NK cells in the bone marrow of Tbet-deficient Tbx21^−/− animals occurs because of a migration defect and identify a module of genes, co-ordinated by Tbet, which affects the localization of NK cells in the bone marrow. Cxcr6 is approximately 125-fold underexpressed in Tbx21^−/−, compared with wild-type, immature NK cells. Immature NK cells accumulate in the bone marrow of CXCR6-deficient mice, and CXCR6-deficient progenitors are less able to reconstitute the peripheral NK cell compartment than their wild-type counterparts. However, the CXCR6 phenotype is largely confined to immature NK cells, whereas the Tbet phenotype is present in both immature and mature NK cells, suggesting that genes identified as being more differentially expressed in mature NK cells, such as S1pr5, Cx3cr1, Sell and Cd69, may be the major drivers of the phenotype.     

卵巢肿瘤患者自然杀伤细胞活化性受体NKG2D与其配体MICA表达的初步研究

目的:研究卵巢癌、良性卵巢肿瘤患者外周血NK细胞表面活化性受体NKG2D的表达及局部组织中相应配体MICA的表达情况,并结合临床病理因素分析探讨宿主NK细胞受体NKG2D在抗卵巢癌中的作用及其与肿瘤免疫逃逸的关系。方法:对4 2例卵巢癌、2 3例良性卵巢肿瘤及2 0例正常妇女,采用流式细胞术检测外周血NK细胞NKG2D的表达状况,RT PCR技术检测在上述部分相应组织标本中MICA的表达。结果:恶性、良性卵巢肿瘤患者及正常人外周血NK细胞NKG2D的表达水平分别为( 94 2 3±6 0 2 ) %、( 98 70±0 98) %、( 98 6 1±1 5 9) % ,恶性组与另两组之间比较,差异有统计学意义(P <0 0 5 ) ;相应配体MICA在卵巢癌组织中的表达率较良性卵巢肿瘤中明显增高,差异有统计学意义(P <0 0 1) ;在卵巢癌病人是否绝经、不同组织类型、分化程度、手术分期及是否淋巴转移等各组临床病理情况下,其表达率未见明显差异(P >0 0 5 )。结论:卵巢恶性肿瘤患者外周血NK细胞活性降低,其活化性受体NKG2D表达的下降是NK细胞活性下降的原因之一。NKG2D的配体MICA的基因表达可能与卵巢癌的恶性转化有一定的相关性,卵巢癌的免疫逃逸可能与NKG2D表达下调及其配体MI CA的表达升高有关