Natural killer cells: integrating diversity with function

The key role of natural killer cells in many aspects of the immune response is now being recognized. The last decade has seen an exponential increase in our understanding of the workings of these cells. Receptor diversity is crucial in allowing natural killer cells to respond effectively to a variety of different pathogens. This article reviews aspects of natural killer cell diversity that combine to generate populations of functional natural killer cells that exist within both the individual and throughout the population at large.     

Innate and adaptive immune response to apoptotic cells

The immune system is constantly exposed to dying cells, most of which arise during central tolerance and from effete circulating immune cells. Under homeostatic conditions, phagocytes (predominantly macrophages and dendritic cells) belonging to the innate immune system, rapidly ingest cells and their debris. Apoptotic cell removal requires recognition of altered self on the apoptotic membrane, a process which is facilitated by natural antibodies and serum opsonins. Recognition, may be site and context specific. Uptake and ingestion of apoptotic cells promotes an immunosuppressive environment that avoids inflammatory responses to self-antigens. However, it does not preclude a T cell response and it is likely that constant exposure to self-antigen, particularly by immature dendritic cells, leads to T cell tolerance. Tolerance occurs by several different mechanisms including anergy and deletion (for CD8+T cells) and induction of T regulatory cells (for CD4+T cells). Failed apoptotic cell clearance promotes immune responses to self-antigens, especially when the cellular contents are leaked from the cell (necrosis). Inflammatory responses may be induced by nucleic acid stimulation of Toll like receptors and other immune sensors, specific intracellular proteins and non-protein (uric acid) stimulation of inflammasomes.     

Natural killer cells as effectors of selection and differentiation of stem cells: Role in resolution of inflammation

Abstract

Evidence has previously been demonstrated for the role of NK cells in specific elimination of healthy stem cells (e.g. hMSC, hDPSC, hESC, hiPSC) as well as cancer stem cells, but not their differentiated counterparts. There is also a stage-wise susceptibility to NK cell-mediated cyto-toxicity in tumors, in which case the poorly-differentiated tumors are lysed much more than moderately-differentiated tumors. Well-differentiated tumors were lysed the least compared to either moderately- or poorly-differentiated tumors. It has also been reported that inhibition of differentiation or reversion of cells to a less-differentiated stage by blocking NF-κB or by gene deletion of COX2 significantly augmented NK cell cytotoxicity against both transformed and healthy cells. Additionally, the cytotoxic function of NK cells was severely inhibited against stem cells when they were cultured in the presence of monocytes. Therefore, it is proposed that CD16⁺CD56^dimCD69^? NK cells were important for the selection of stem cells, whereas the CD16^dim/?CD56^dim/+CD69⁺ anergized NK cells were important for differentiation and eventual regeneration of the tissues and the resolution of inflammation, thus potentially serving as regulatory NK (NK_reg) cells. The concept of ‘split anergy’ in NK cells and the generation of NK_reg cells with regard to contributions to cell differentiation, tissue repair and regeneration and in tumor resistance are discussed in this review.     

Natural killer cell-endothelial cell interactions in xenotransplantation

Interest in xenotransplantation derives from the documented need for more organs and tissues than can be expected from living or cadaveric donors. Although the barriers to xenotransplantation are formidable, the scientific rewards in addressing these problems have been significant. The first and most potent barrier to xenotransplantation is hyperacute rejection mediated by xenoreactive natural antibodies and serum complement. The majority of the xenoreactive antibodies appear to be directed at terminal galactose epitopes, especially gal α 1–3 gal. Significant progress has been made in surmounting hyperacute rejection, and this has led to an examination of underlying mechanisms of delayed xenograft rejection. One of these delayed mechanisms concerns the potential role of graft recipient, natural killer (NK) cells. NK cells can cause variable, low-level cytotoxicity of xenogeneic endothelial cells in vitro that may be enhanced in the presence of xenoreactive IgG. The specificity of NK cell-mediated cytotoxicity appears to overlap with a major subset of xenore active natural antibodies. These cytotoxic interactions can be regulated by “humanizing” the endothelial cells through expression of the appropriate human MHC class I genes. More important, NK cells induce endothelial cell activation, which results in changing the nature of the endothelial cell surface from an anticoagulant surface to a procoagulant surface. These findings parallel those observed in allogeneic NK cell-endothelial cell interactions and suggest these important observations may be extended to NK cell-endothelial cell interactions in general.     

Uptake of apoptotic DC converts immature DC into tolerogenic DC that induce differentiation of Foxp3+ Treg

DC apoptosis has been observed in patients with cancer and sepsis, and defects in DC apoptosis have been implicated in the development of autoimmune diseases. However, the mechanisms of how DC apoptosis affects immune responses, are unclear. In this study, we showed that immature viable DC have the ability to uptake apoptotic DC as well as necrotic DC without it being recognized as an inflammatory event by immature viable DC. However, the specific uptake of apoptotic DC converted immature viable DC into tolerogenic DC, which were resistant to LPS‐induced maturation. These tolerogenic DC secreted increased levels of TGF‐β1, which induced differentiation of naïve T cells into Foxp3⁺ Treg. Furthermore, induction of Treg differentiation only occurred upon uptake of apoptotic DC and not apoptotic splenocytes by viable DC, indicating that it is specifically the uptake of apoptotic DC that gives viable immature DC the potential to induce Foxp3⁺ Treg. Taken together, these findings identify uptake of apoptotic DC by viable immature DC as an immunologically tolerogenic event.     

Soluble HLA-G molecules induce apoptosis in natural killer cells

Membrane-bound human leukocyte antigen-G (HLA-G) molecules are primarily expressed by cytotrophoblasts of the fetus. They are thought to protect the fetus from immunologic attack by the maternal immune system and have recently been associated with transplantation graft acceptance. In addition, soluble HLA-G molecules (sHLA-G) have been shown to play a role in the success of pregnancies, but are upregulated in certain cancers. However, the exact mechanism for this regulation has remained elusive. The aim of this study was to examine the mechanism by which sHLA-G interact with natural killer (NK) cells in vitro. sHLA-G effectively blocked NK lysis of target cells via fracticide killing of NK cells by apoptosis. These studies support the protective role of sHLA-G in immunologic reactions by interacting with NK cells, thus providing a regulatory function.     

早期凋亡的T淋巴细胞诱导生成耐受性树突状细胞的实验研究

目的： 探讨早期凋亡T淋巴细胞的抑制性免疫调节性能。方法： 采用定时紫外线照射诱导T淋巴细胞早期凋亡，深低温反复冻融获得坏死T淋巴细胞。体外诱导、纯化并培养骨髓源性不成熟树突状细胞（imDCs）,imDCs分别和早期凋亡或坏死T淋巴细胞共培养。用流式细胞仪、双夹心ELISA、［³H］掺入混合淋巴细胞反应等方法分析imDCs吞噬早期凋亡或坏死T淋巴细胞后，在不同处理条件下，MHC-Ⅱ、CD40、CD80、CD86的表达水平、分泌IL-12 p70以及刺激T淋巴细胞增殖能力的差异。结果： imDCs和坏死细胞碎片共培养后明显趋于成熟，其MHCⅡ和CD40、CD80、CD86的表达水平显著上调；分泌较高水平的IL-12 p70；和同种异体处女T淋巴细胞混合培养后显著刺激处女T淋巴细胞增殖。 imDCs和早期凋亡的T淋巴细胞共培养后,其MHC-Ⅱ、CD40、CD80和CD86的表达维持较低水平；仅分泌较低水平IL-12 p70；和同种异体处女T淋巴细胞混合培养后不能刺激淋巴细胞增殖。此外，早期凋亡的T淋巴细胞孵育上清显著抑制了吞噬坏死细胞碎片后的imDCs表达共刺激分子CD40、CD80、CD86。当TGFβ₁中和抗体和早期凋亡T淋巴细胞同加入imDCs，在表达MHC-Ⅱ、CD40、CD80、CD86，分泌IL-12 p70，刺激处女T淋巴细胞增殖等方面和吞噬坏死T淋巴细的DCs相比无显著差异。结论： 早期凋亡T淋巴细胞通过释放免疫抑制性细胞因子TGFβ₁，诱导imDCs呈现出耐受性DCs（TolDCs）的免疫表型及生物学特征，从而发挥抑制性免疫调节作用。     

Modification of anti-tumor immunity by tolerogenic dendritic cells

Immunosuppressive functions of glucocorticoids (GC) can be mediated via various mechanisms, including the modulation of dendritic cells (DC). Our study investigates the effects of tolerogenic GC-treated DCs on NK and T cell anti-tumor responses in OT-1/Rag^?/? mice, expressing a transgenic TCR in CD8⁺ T cells. The effects caused by GC-treated DCs were compared to the responses to immunogenic, CpG-activated DCs. The effects of DCs on anti-tumor immune responses were analyzed using the EG7 tumor model, where the tumor cells express the peptide epitope recognized by OT-1 T cells. We observed that immunization with CpG and peptide-treated DCs protected against tumor growth by activation of NK cell response. Also, immunogenic DCs induced the expansion of cytotoxic CD8⁺OT-1 cells, expressing activation markers CD44 and CD69 and producing IFNγ. In contrast, the peptide and GC-treated DCs in OT-1 mice increased the numbers of immature Mac-1⁺CD27^? NK cells as well as Foxp3⁺ and IL-10 secreting CD8⁺OT-1 cells with suppressive properties. We conclude that the generation of tolerogenic DCs is one of many immunosuppressive mechanisms that can be induced by GC. Our study demonstrated that tolerogenic DCs modify anti-tumor immune response by suppressing NK cell activity and stimulating the formation of IL-10-secreting CD8⁺ Tregs.     

Cytotoxic granule disruption is a late event in chemotherapy-induced apoptosis in natural killer YT cells

Cytotoxic lymphocytes such as Natural Killer (NK) cells can result in leukemias and lymphomas with aggressive clinical course. The cytoplasmic granules of NK cells contain molecules that cause apoptosis of their target cells. In this study, we examined changes of the cytotoxic granules of the Natural Killer cell line YT during Etoposide-induced apoptosis. Etoposide treatment resulted in an early upregulation of Fas and cytoplasmic release of mitochondrial cytochrome c. The cytotoxic granules remain intact in the early stages of apoptosis induction with no significant cytoplasmic release of granzyme B, which is one of the components of the cytotoxic granules. These results suggest that disruption of the cytotoxic granules is not involved in the induction stage of Etoposide-induced apoptosis in the NK cell line YT.     

Natural killer cells: versatile roles in autoimmune and infectious diseases

Natural killer (NK) cells are essential members of innate immunity and they rapidly respond to a variety of insults via cytokine secretion and cytolytic activity. Effector functions of NK cells form an important first line of innate immunity against viral, bacterial and parasitic infections, as well as an important bridge for the activation of adaptive immune responses. The control of NK-cell activation and killing is now understood to be a highly complex system of diverse inhibitory and activatory receptor–ligand interactions, sensing changes in MHC expression. NK cells have a functional role in innate immunity as the primary source of NK-cell-derived immunoregulatory cytokines, which have been identified in target organs of patients suffering from autoimmune diseases, and play a critical role in early defense against infectious agents. This review focuses on recent research of NK cells, summarizing their potential immunoregulatory role in modulating autoimmunity and infectious diseases.     

Interaction of natural killer cells with Trypanosoma cruzi-infected fibroblasts

The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and invades a variety of cells to multiply intracellularly as amastigotes. The acute phase triggers an immune response that restricts the proliferation of the parasite. However, parasites are able to persist in different tissues causing the pathology of Chagas' disease. Natural killer (NK) cells play an important role in innate resistance to a variety of pathogens. In the present study we demonstrate that NK cells trigger trypanocidal mechanisms in infected L929 cells that are critically dependent on inducible nitric oxide (NO) synthase (iNOS) induction which is, to a major degree, triggered by interferon (IFN)-gamma provided by NK cells. This work provides a more detailed analysis of how NK cells as a part of the innate immune system participate in the control of parasites that reside intracellularly in fibroblast-like L929 cells.     

健康人外周血单个核细胞诱导的NK细胞对脑胶质瘤杀伤作用的探讨

目的 对体外培养的NK细胞杀伤脑胶质瘤细胞进行探讨,为脑胶质瘤的免疫细胞疗法提供理论基础.方法 从4个健康人外周血提取单个核细胞,在KRATM培养环境下,诱导产生NK细胞和LAK细胞.分别用细胞计数法和流式细胞仪检测NK细胞增殖和细胞表面特异性标志CD3/CDl6/CD56,并用MTr法检测NK细胞及LAK细胞对脑胶质瘤细胞的杀伤.结果 经过体外培养,平均可得到7.93×109个以上细胞.和LAK细胞相比,NK细胞对脑胶质瘤细胞LNI8、LN229、T98G和U87MG的敏感性更高.结论 NK细胞能够对脑胶质瘤细胞产生有效免疫应答,为过继性免疫细胞治疗脑胶质瘤提供可能.     

Natural killer cells are required for accelerated type 1 diabetes driven by interferon-beta

The destruction of beta cells by the islet infiltrating lymphocytes causes type 1 diabetes. Transgenic mice models expressing interferon (IFN)-beta in beta cells, in the non-obese diabetic (NOD) strain and in a diabetes-free, major histocompatibility complex-matched, homologous strain, the non-obese resistant (NOR) mice, developed accelerated type 1 diabetes after 3 weeks of age. Our aim was to determine if natural killer (NK) cells could affect the acceleration of the disease. We determined the amount of NK cells in the pancreas, spleen and lymph nodes from NOD rat insulin promoter (RIP)-IFN-beta mice. Pancreatic cytokines were assessed by quantitative real-time polymerase chain reaction and protein arrays. To confirm the relevance of NK cells in the acceleration of autoimmune diabetes this subset was depleted with anti-asialo GM1 antibodies. An increase of intrapancreatic NK cells characterized the accelerated onset of diabetes both in NOD and NOR RIP-IFN-beta transgenic models. Cytokines involved in NK function and migration were found to be hyperexpressed in the pancreas from accelerated diabetic mice. Interestingly, the depletion of NK cells in vivo abolished completely the acceleration of diabetes. NK cells connect innate to adaptive immunity and might play a role in autoimmunity. We report here that NK cells are required critically in the pancreas for accelerated diabetes. This model links inflammation to acceleration of beta cell-specific autoimmunity mediated by NK cells.     

Pertussis toxin-sensitive GTP-binding proteins regulate activation-induced apoptotic cell death of human natural killer cells

Apoptosis of natural killer (NK) cells can be induced by non-specific physical damage (UV irradiation, heat shock) or by simultaneous ligation of the CD 16 and the interleukin-2 receptor (IL-2R) molecules, but not with either anti-CD 16 or IL-2 alone. Whereas blockade of GTP-binding protein (G protein)-mediated signal transduction using ADP-ribosylating bacterial toxins or the GTPase-resistant GTP analog guanosine 5′-0-(3-thiotriphosphate (GTPγS) does not affect non-specific induction of NK cell apoptosis, such interventions do inhibit induction of apoptosis by anti-CD16/IL-2. The G proteins involved in the regulation of activation-induced NK apoptosis are sensitive to pertussis toxin (PTX) and to the non-specific GTP analog GTPγS but not to cholera toxin, Pseudomonas exotoxin A or diphtheria toxin. A pertussis toxin mutant that lacks ADP-ribosylating activity, but conserves the membrane translocating and T cell-mitogenic effects of the native molecule, fails to inhibit NK apoptosis. To exert their apoptosis-inhibitory effect, PTX and GTPγS must be employed before cells are activated. Later addition has no effect, suggesting the implication of G proteins in the transmission of apoptosis-inducing signals, but not in the effector stage of apoptosis. Pre-incubation with PTX or GTPγS does not affect the activation of NK cells by CD 16 cross-linking, IL-2 stimulation - or both, as assessed by the induction of CD69 expression, protein tyrosine phosphorylation and calcium mobilization. Moreover, neither PTX nor GTPγS compromise the effector function of NK cells or the susceptibility of target cells to NK-mediated lysis. These data suggest apoptosis as a novel mechanism by which NK responses may be controlled in vivo, as well as an experimental and therapeutical strategy to counteract endogenous down-regulation of NK responses.     

Natural killer (NK) cell activity during HIV infection: a decrease in NK activity is observed at the clonal level and is not restored after in vitro long-term culture of NK cells.

NK cell activity is impaired in HIV-infected patients. The mechanisms behind the altered NK functions are not clear, and conflicting data concerning NK and antibody-dependent cellular cytotoxicity (ADCC) activity have been reported. In order to investigate whether this impairment is also observed at the clonal level and whether it is related to a defect at the target cell binding and/or the post-binding level, we evaluated highly purified NK cell lines and cloned NK cells obtained from 22 HIV-infected patients at different stages of disease and compared them with normal controls for their ability to: (i) kill K-562 and U-937 cell lines using a 51Cr release assay; (ii) bind and kill K-562 and U-937 cells at the single cell binding level; (iii) release NK cytotoxic factor (NKCF), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma); (iv) kill anti-IgM preincubated Daudi cell line (ADCC activity). This study with cloned NK cells or NK cell lines from HIV-infected individuals showed: (i) a decrease in their lytic capability against target cell lines; (ii) a low ability to form conjugates with K-562 and U-937 cell lines with respect to controls; (iii) a decreased ability to kill bound target cells; (iv) low levels of released NKCF, TNF-alpha and IFN-gamma after incubation with U-937 cells. Taken together, these findings suggest that the impaired NK cell function during HIV infection is also observed at the clonal level and is related to defects both at the target and post-binding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long-term culture in the presence of high levels of recombinant IL-2 is in agreement with the hypothesis of a 'general anergic process' during HIV infection.     

Expression and regulation of complement receptors by human natural killer cells

Integration of cellular and humoral arms of the innate immune response is fundamental to the development of powerful effector functions in host defence as well as aberrant immune responses. Here, we provide evidence in support of the relationship between complement activation and NK cell functional modulation. We demonstrate that human NK cells and both CD56^brightCD16⁻ and CD56^dimCD16⁺ populations express receptors known to detect the biologically active peptides C3a and C5a (i.e. C3aR, C5aR, C5L2) and the covalently-bound fragments C3b and metabolites iC3b and C3d which serve in immune adhesion (e.g. CR3, CR4). We also show that several pathogen- or tumour/inflammation-related stimuli differentially regulated those complement receptor expression. Furthermore, our results suggest that C3 fragments (C3a, iC3b) have a negative regulatory effect on IFN-γ production in NK cells. This work provides extensive information of human complement receptors relevant to the integrated actions of complement and NK cells which has been suggested by animal studies. The observations may act as a resource that allows further understanding and exploitation of role of complement in human health and immune mediated diseases.     

Natural killer (NK) cells from killers to regulators: distinct features between peripheral blood and decidual NK cells

Natural killer (NK) cells are a key component of innate immunity, particularly crucial during the early phase of immune responses against certain viruses, parasites, and microbial pathogens. The role of NK cell during pregnancy has been vividly discussed over the past years and it is now becoming increasingly clear that NK cells control pregnancy maintenance at several levels. In normal pregnancy, it appears that they provide benefit by properly secreting cytokines, chemokines and angiogenic factors rather than functioning as cytotoxic effector cells. However, as they are endowed with all the cytolytic weapons, they promptly become capable of attacking fetal and maternal tissues during infection and inflammation.