The role of natural killer cells in hematopoietic stem cell transplantation

Natural killer (NK) cells are important elements of innate immunity, and a large body of evidence supports the significant role of NK in immune surveillance against infections and tumors. Regulation of cytotoxic activity is mediated through activating and inhibitory receptors expressed on the cell surface. NK cells are key players of allogeneic hematopoietic stem cell transplantation (allo-SCT), and previous studies showed the beneficial effect of NK alloreactivity in prevention of relapse, especially in the setting of haploidentical SCT. Biology of human NK cells is an area of active research. Exploitation of the molecular mechanisms regulating NK maturation, tolerance to self, and NK-mediated cytotoxicity will help in the development of innovative NK cell immunotherapy methods.     

Possible role for C-reactive protein in the human natural killer cell response

Functional NK activity can be removed from human PBL and from phagocyte- and T cell-depleted LGL preparations by treatment with antisera specific for C-reactive protein (CRP) in the presence of complement (C). Pretreatment of NK effector cells with high concentrations of anti- CRP in the absence of C also depletes functional activity. These results indicate that CRP or an antigenically similar molecule is present on a population of NK effector cells. Fluorescent antibody studies in which biotin-avidin amplification was used confirm the presence of surface CRP (S-CRP) on a small percentage of nonphagocytic peripheral blood mononuclear cells. S-CRP readily caps off, which suggest that removal by capping obviates killing by this cell population. This indicates that S-CRP or a molecule that co-caps with S- CRP may be required for successful effector-target cell interaction. The addition of exogenous CRP or CRP-CPS complexes, however, does not alter NK responses. A subpopulation of lymphoid cells responsible for functional NK activity therefore appears to bear surface CRP.     

Evidence for discrete stages of human natural killer cell differentiation in vivo

Human natural killer (NK) cells originate from CD34(+) hematopoietic progenitor cells, but the discrete stages of NK cell differentiation in vivo have not been elucidated. We identify and functionally characterize, from human lymph nodes and tonsils, four NK cell developmental intermediates spanning the continuum of differentiation from a CD34(+) NK cell progenitor to a functionally mature NK cell. Analyses of each intermediate stage for CD34, CD117, and CD94 cell surface expression, lineage differentiation potentials, capacity for cytokine production and natural cytotoxicity, and ETS-1, GATA-3, and T-BET expression provide evidence for a new model of human NK cell differentiation in secondary lymphoid tissues.     

Natural killer T cell activation inhibits hepatitis B virus replication in vivo

We have previously reported that hepatitis B virus (HBV)-specific CD8(+) cytotoxic T lymphocytes and CD4(+) helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting interferon (IFN)-gamma when they recognize viral antigen. To determine whether an activated innate immune system can also inhibit HBV replication, in this study we activated natural killer T (NKT) cells in the liver of HBV transgenic mice by a single injection of alpha-galactosylceramide (alpha-GalCer), a glycolipid antigen presented to Valpha14(+)NK1.1(+) T cells by the nonclassical major histocompatibility complex class I-like molecule CD1d. Within 24 h of alpha-GalCer injection, IFN-gamma and IFN-alpha/beta were detected in the liver of HBV transgenic mice and HBV replication was abolished. Both of these events were temporally associated with the rapid disappearance of NKT cells from the liver, presumably reflecting activation-induced cell death, and by the recruitment of activated NK cells into the organ. In addition, prior antibody-mediated depletion of CD4(+) and CD8(+) T cells from the mice did not diminish the ability of alpha-GalCer to trigger the disappearance of HBV from the liver, indicating that conventional T cells were not downstream mediators of this effect. Finally, the antiviral effect of alpha-GalCer was inhibited in mice that are genetically deficient for either IFN-gamma or the IFN-alpha/beta receptor, indicating that most of the antiviral activity of alpha-GalCer is mediated by these cytokines. Based on these results, we conclude that alpha-GalCer inhibits HBV replication by directly activating NKT cells and by secondarily activating NK cells to secrete antiviral cytokines in the liver. In view of these findings, we suggest that, if activated, the innate immune response, like the adaptive immune response, has the potential to control viral replication during natural HBV infection. In addition, the data suggest that therapeutic activation of NKT cells may represent a new strategy for the treatment of chronic HBV infection.     

异源反应性NK细胞对输血相关免疫抑制作用的初步研究

目的探讨供血者KIR与受血者HLA-Ⅰ类分子不同程度匹配情况下的异源反应性NK细胞对输血相关免疫抑制的作用。方法模拟临床输血状态,采用miniMACS免疫磁珠分选方法,从外周血单个核细胞(PBMC)中得到纯化的NK细胞,体外培养并采用PCR-SSP法进行5种KIR抑制基因的分型。将已经KIR分型的NK细胞(4ml共1×106个细胞)与已知HLA分型的血液病患者骨髓移植供者的全血(20 ml)按比例(相当于60 kg体重个体输入800 ml全血)混合,不同时间点取样检测免疫指标的变化。根据KIR和HLA的不相合程度分为20组,比较不同相合程度对血液免疫功能的影响。结果从0—24 h,IL-2、IL-4、PGE2、TNF-α、TNF-β、IFN-γ、GM-CSF的浓度分别由(1.97±0.12)、(0.22±0.03)、(0.22±0.06)、(0.19±0.02)、(0.13±0.02)、(0.12±0.02)、(0.19±0.02)μg/L增加到(2.94±0.06)、(0.55±0.09)、(0.53±0.05)、(3.54±0.07)、(3.08±0.14)、(2.65±0.11)、(2.37±0.16)μg/L(P<0.05),IgA、IgM、IgG的浓度都随时间的增加而分别由(1.20±0.20)、(1.28±0.07)、(9.89±0.37)g/L减少到(0.60±0.07)、(0.72±0.06)、(6.66±0.91)g/L(P<0.05);KIR与HLA相合程度的不同与IL-2、IL-4、PGE2、IgA、IgM、IgG浓度无关(P>0.05);随KIR与HLA不相合程度的加大,TNF-α、TNF-β、IFN-γ、GM-CSF的浓度逐渐增加,分别从(0.42±0.04)、(0.37±0.04)、(0.46±0.08)、(0.43±0.02)μg/L增加到(3.54±0.07)、(3.08±0.14)、(2.65±0.11)和(2.37±0.16)μg/L(P<0.05)。结论 KIR与HLA不相合程度越高越能激发异源反应性NK细胞的免疫功能,可更好地逆转输血相关免疫抑制作用。     

Potential mechanisms of human natural killer cell expansion in vivo during low-dose IL-2 therapy

The continuous, in vivo infusion of low-dose IL-2 selectively expands the absolute number of human natural killer (NK) cells after 4-6 weeks of therapy. The mechanism responsible for this expansion is unknown and was examined in this study. NK cells cultured at low concentrations of IL-2, comparable to those found during in vivo therapy, proliferate for 6 days and then exit the cell cycle. However, NK cells in vivo did not traverse the S/G(2)/M phase of the cell cycle during low-dose IL-2 therapy. Low concentrations of IL-2 delay programmed cell death of NK cells but have the same effect on resting T cells that do not expand in vivo. When CD34(+) bone marrow hematopoietic progenitor cells are cultured for 21 days with low concentrations of IL-2, they differentiate into CD56(+)CD3(-) NK cells, not T cells. Thus, the selective expansion of human NK cells during continuous in vivo infusion of low-dose IL-2 likely results from enhanced NK-cell differentiation from bone marrow progenitors, combined with an IL-2-dependent delay in NK-cell death, rather than proliferation of mature NK cells in the periphery.     

A potential role for interleukin-15 in the regulation of human natural killer cell survival.

Resting lymphocyte survival is dependent upon the expression of Bcl-2, yet the factors responsible for maintaining lymphocyte Bcl-2 protein expression in vivo are largely unknown. Natural killer (NK) cells are bone marrow-derived lymphocytes that constitutively express the beta and common gamma(c) subunits of the IL-2 receptor (R) as a heterodimer with intermediate affinity for IL-2. IL-15 also binds to IL-2Rbeta gamma(c) and is much more abundant in normal tissues than IL-2. Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2R gamma(c) do not have NK cells. Further, treatment of mice with an antibody directed against IL-2Rbeta results in a loss of the NK cell compartment. These data suggest that a cytokine other than IL-2, which binds to IL-2Rbeta gamma(c), is important for NK cell development and survival in vivo. In the current report, we show that the recently described IL-15R(alpha) subunit cooperates with IL-2Rbeta gamma(c) to transduce an intracellular signal at picomolar concentrations of IL-15. We demonstrate that resting human NK cells express IL-15R(alpha) mRNA and further, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 d in the absence of serum. NK cell survival was not sustained by other monocyte-derived factors (i.e., TNF-alpha, IL-1beta, IL-10, IL-12) nor by cytokines known to use gamma(c) for signaling (i.e., IL-4, IL-7, IL-9, IL- 13). One mechanism by which IL-15 promotes NK cell survival may involve the maintenance of Bcl-2 protein expression. Considering these functional properties of IL-15 and the fact that it is produced by bone marrow stromal cells and activated monocytes, we propose that IL-15 may function as an NK cell survival factor in vivo.     

Potential therapeutic role of natural killer cells in cancer

Cancer immunotherapy is a growing field that aims at restoring and enhancing immune function to combat oncogenic conditions. One target of this field is natural killer (NK) cells. Part of innate immunity, NK cells are able to kill tumor cells without previous priming. Results from stem cell transplants containing alloreactive donor NK cells and in vitro work have evidenced a great antitumor potential. In addition, NK cells are likely to interact with dendritic cells, potent antigen-presenting cells, thus forming a bridge between innate and adaptive immunity. This review aims to provide an overview of NK cells with particular emphasis on properties that can and are being targeted in order to potentiate the antitumor activity of these cells.     

The host defense system in the human newborn: the role of interferon and the natural killer cell

The human newborn is particularly susceptible to infections in the neonatal period, due in part to the immaturity and naive state of the immune system. The role of interferon and natural killer cells in host defense in this age group is poorly defined. We have studied natural killer cells and the activation of the interferon system in cord blood. Most newborns (75%) had low natural killer cell activity, and this was unrelated to levels of the intracellular interferon-associated enzyme, 2'-5' oligoadenylate synthetase (2-5A synthetase). Newborn cells responded to interferon in vitro with an increase in natural killer activity, suggesting that this low natural killer cell activity is the result of fewer numbers of effector cells rather than a functional immaturity. Circulating serum interferon was detected in greater than 50% of maternal samples tested; however, this did not correlate with 2-5A synthetase levels in maternal or the corresponding cord blood mononuclear cells. Natural killer cell activity was low in the newborn in spite of activation of the interferon system in 39% of these individuals. This may be an important factor in susceptibility to infections in this period.     

Elimination in vivo of developing T cells by natural killer cells

Natural killer cells gauge the absence of self class I MHC on susceptible target cells by means of inhibitory receptors such as members of the Ly49 family. To initiate killing by natural killer cells, a lack of inhibitory signals must be accompanied by the presence of activating ligands on the target cell. Although natural killer cell-mediated rejection of class I MHC-deficient bone marrow (BM) grafts is a matter of record, little is known about the targeting in vivo of specific cellular subsets by natural killer cells. We show here that development of class I MHC-negative thymocytes is delayed as a result of natural killer cell toxicity after grafting of a class I MHC-positive host with class I MHC-negative BM. Double positive thymocytes that persist in the presence of natural killer cells display an unusual T cell receptor-deficient phenotype, yet nevertheless give rise to single positive thymocytes and yield mature class I MHC-deficient lymphocytes that accumulate in the class I MHC-positive host. The resulting class I MHC-deficient CD8 T cells are functional and upon activation remain susceptible to natural killer cell toxicity in vivo. Reconstitution of class I MHC-deficient BM precursors with H2-K(b) by retroviral transduction fully restores normal thymic development.     

The changing role of natural killer cells in cancer metastasis

Natural killer (NK) cells are innate immune cells that are critical to the body’s antitumor and antimetastatic defense. As such, novel therapies are being developed to utilize NK cells as part of a next generation of immunotherapies to treat patients with metastatic disease. Therefore, it is essential for us to examine how metastatic cancer cells and NK cells interact with each other throughout the metastatic cascade. In this Review, we highlight the recent body of work that has begun to answer these questions. We explore how the unique biology of cancer cells at each stage of metastasis alters fundamental NK cell biology, including how cancer cells can evade immunosurveillance and co-opt NK cells into cells that promote metastasis. We also discuss the translational potential of this knowledge.     

丙型病毒性肝炎病毒重叠结核杆菌感染患者的自然杀伤性细胞的分析

目的 研究丙型病毒性肝炎(丙肝)重叠结核杆菌感染对NK细胞的作用,探讨NK细胞在丙肝重叠结核杆菌感染患者体内的免疫学功能。 方法 选择单纯丙肝感染患者47例,单纯结核杆菌感染患者66例,丙肝重叠结核杆菌感染患者27例,应用流式细胞仪检测患者外周血中NK细胞的数量及分泌的细胞因子的含量。 结果 丙肝重叠结核杆菌感染患者的NK细胞数量明显低于单纯感染组,二者比较差异有统计学意义(PP结论 重叠感染组的NK细胞数量及活性下降,细胞免疫机制参与了丙肝重叠结核杆菌感染的发病及发展。     

Adoptive transfer studies demonstrating the antiviral effect of natural killer cells in vivo

We carried out adoptive transfer studies to determine the role of natural killer (NK) cells in resistance to murine cytomegalovirus (MCMV) and lymphocytic choriomeningitis virus (LCMV). We transferred leukocytes from adult mice into suckling mice 1 d before injecting them with virus. Resistance was measured by enhancement of survival and reduction of virus multiplication in the spleens of recipient mice. The phenotype of the cell population capable of mediating resistance to MCMV was that of a nylon wool-nonadherent, asialo GM1+, NK 1.2+, Ly-5+, Thy-1-, Ia-, low density lymphocyte; this is the phenotype of an NK cell. Cloned NK cells, but not cloned T cells, provided resistance to MCMV in suckling mice. Cloned NK cells also provided resistance to MCMV in irradiated adult mice, and antibody to asialo GM1, which depletes NK cell activity in vivo, enhanced the synthesis of MCMV in athymic nude mice. Neither adult leukocytes nor cloned NK cells influenced LCMV synthesis in suckling mice. We conclude that a general property of NK cells may be to provide natural resistance to virus infections, and that NK cells can protect mice from MCMV but not from LCMV.     

In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes.

Natural killer (NK) receptor signaling can lead to reduced cytotoxicity by NK cells and cytolytic T lymphocytes (CTLs) in vitro. Whether T cells are inhibited in vivo remains unknown, since peptide antigen-specific CD8(+) T cells have so far not been found to express NK receptors in vivo. Here we demonstrate that melanoma patients may bear tumor-specific CTLs expressing NK receptors. The lysis of melanoma cells by patient-derived CTLs was inhibited by the NK receptor CD94/NKG2A. Thus, tumor-specific CTL activity may be decreased through NK receptor triggering in vivo.     

Polymorphic Hh genes in the HLA-B(C) region control natural killer cell frequency and activity

We demonstrated earlier that individuals homozygous for conserved major histocompatibility complex (MHC)-extended haplotypes have low natural killer (NK) activity as measured by cytolysis of the K562 tumor cell lines. In the present study, we investigated the segregation and MHC linkage of NK activity in families in which MHC haplotypes of human histocompatibility leukocyte antigens (HLA)-A, -C, and -B, complotype, and DR specificities are known. In two informative families, low activity was inherited as a recessive trait linked to the MHC. By using individuals homozygous for specific fragments of extended haplotypes or for HLA-B alleles, we found that the HLA-C and -B and not the complotype or HLA-DR region contains genes controlling NK activity. The majority of the unrelated individuals with low NK activity were homozygous or doubly heterozygous for HLA-B7 (Cw7), B8 (Cw7), B44 (Cw5), B18, or B57 (Cw6). Thus, these alleles form one complementation group designated NKB1. Another less frequent group, NKB2, was also identified, and consisted of individuals homozygous for B35 (Cw4). NK activity was correlated with the number of circulating NK (CD16+ CD56+) cells. Individuals homozygous for the NKB complementation groups have fewer circulating NK cells than individuals heterozygous for these alleles and alleles of other complementation groups, possibly explaining the low activity of cells in these subjects. These findings suggest that during the maturation of NK cells there is NK cellular deletion in donors homozygous for NKB genes resulting in low NK cell numbers and activity.     

Preservation of natural killer and interleukin-2 activated killer cell activity in ataxia-telangiectasia with T cell deficiency

Peripheral blood mononuclear cells (PBMs) from 6 patients with ataxia-telangiectasia (AT) were studied by 5 kinds of cell-mediated cytotoxicity systems. Decrease in cell mediated lympholysis (CML) activity to allogeneic lymphocytes was observed in all 6 AT patients who had low numbers of OKT-3+ cells. These patients also showed decreased proliferative responses to phytohemagglutinin stimulation and allogeneic lymphocytes. In contrast, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and natural killer (NK) activity were comparable with those in normal controls. In addition, PBMs from these AT patients activated by in vitro stimulation with allogeneic PBMs or interleukin-2 were able to acquire lytic activity against NK-insensitive target cells. The phenotypes of these effectors determined by complement-mediated lysis were OKT-3- and Leu-11+, suggesting that they were derived from NK cell lineage. Thus, AT patients with severe T cell defects were found to maintain a normal range of NK, ADCC, MLC-activated and lymphokine-activated killer activity.     

The role of invariant natural killer T cells in microbial immunity

Invariant natural killer T cells (iNKT cells) are unique lymphocytes with characteristic features, such as expression of an invariant T-cell antigen receptor (TCR) α-chain, recognition of glycolipid antigens presented by CD1d molecules, and ability to rapidly produce large amounts of cytokines, including interferon-γ (IFN-γ) and interleukin 4 (IL-4) upon TCR stimulation. Many studies have demonstrated that iNKT cells participate in immune response against diverse microbes, including bacteria, fungi, protozoan parasites, and viruses. Generally, these cells play protective roles in host defense against infections. However, in some contexts they play pathogenic roles, by inducing or augmenting inflammation. Recent reports show that iNKT cells recognize glycolipid antigens from pathogenic bacteria including Streptococcus pneumoniae, and they contribute to host defense against infection. iNKT cell responses to these microbial glycolipid antigens are highly conserved between rodents and humans, suggesting that iNKT cells are evolutionally conserved because their invariant TCR is useful in detecting certain pathogens. Furthermore, glycolipid-mediated iNKT cell activation during immunization has adjuvant activity, enhancing humoral and cell-mediated responses. Therefore, iNKT cell activation is an attractive target for developing new vaccines for infectious diseases.     

Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions

Infection with hepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide. Little is known about how this virus is able to persist or whether this persistence might be because of its ability to alter the early innate immune response. The major HCV envelope protein E2 has been shown to bind to CD81. Thus, HCV binding to natural killer (NK) cells could result in the cross-linking of CD81. To explore this possibility, we investigated whether cross-linking CD81 on NK cells could alter NK cell function. CD81 cross-linking by monoclonal antibody (mAb) specific for CD81 or by immobilized E2 have been shown to result in costimulatory signals for human T cells. In this study, we show that CD81 cross-linking via immobilized E2 or mAbs specific for CD81 inhibits not only non major histocompatibility complex-restricted cytotoxicity mediated by NK cells but also interferon (IFN)-gamma production by NK cells after exposure to interleukin (IL)-2, IL-12, IL-15, or CD16 cross-linking. These results show that CD81 cross-linking mediates completely different signals in NK cells versus T cells. Importantly, these results suggest that one mechanism whereby HCV can alter host defenses and innate immunity is via the early inhibition of IFN-gamma production by NK cells.     

Endogenous opioids and the exercise-induced augmentation of natural killer cell activity

Based on prior observations that both beta-endorphin and exercise stimulate natural killer (NK) cell activity, we have examined the hypothesis that the release of endogenous opioids during the stress of acute exercise may mediate this NK cell augmentation. Eight healthy young women underwent a maximal bicycle ergometer exercise test with prior in vivo administration of a placebo and an opioid antagonist, naloxone (100 micrograms/kg), in a randomized, blind protocol. Exercise after the placebo injection was accompanied by a dramatic rise in NK activity, as well as an increase in the percentage of lymphocytes bearing the NK cell surface markers Leu 11a and Leu 19. Significant stimulation of NK activity was observed with beta-endorphin in vitro before exercise, but after exercise, beta-endorphin had a nonsignificant inhibitory effect. When these experiments were carried out in the presence of naloxone in vivo, the rise in NK activity after exercise was no longer significant. Naloxone did not significantly alter the rise in Leu 11a+ or Leu 19+ cell after exercise, as compared with the placebo. Finally, when naloxone was given to the subjects beforehand, exercise no longer completely blocked the in vitro beta-endorphin stimulation of NK cells. In conclusion, our observations that the exercise-induced augmentation of NK activity and the lack of effect of beta-endorphin in vitro on NK activity after exercise are both significantly attenuated by prior administration of naloxone suggest that the opioid system may play a major role in the modulation of NK cells during physiologic stress.