Endocytosis and Intracellular Trafficking of Human Natural Killer Cell Receptors

Natural killer (NK) cells play a vital role in the defense against viral infections and tumor development. NK cell function is primarily regulated by the sum of signals from a broad array of activation and inhibitory receptors. Key to generating the input level of either activating or inhibitory signals is the maintenance of receptor expression levels on the cell surface. Although the mechanisms of endocytosis and trafficking for some cell surface receptors, such as transferrin receptor and certain immune receptors, are very well known, that is not the situation for receptors expressed by NK cells. Recent studies have uncovered that endocytosis and trafficking routes characteristic for specific activation and inhibitory receptors can regulate the functional responses of NK cells. In this review, we summarize the current knowledge of receptor endocytosis and trafficking, and integrate this with our current understanding of NK cell receptor trafficking.     

New and potential strategies for the treatment of PMM2-CDG

BackgroundMutations in the PMM2 gene cause phosphomannomutase 2 deficiency (PMM2; MIM# 212065), which manifests as a congenital disorder of glycosylation (PMM2-CDG). Mutant PMM2 leads to the reduced conversion of Man-6-P to Man-1-P, which results in low concentrations of guanosine 5′-diphospho-D-mannose, a nucleotide-activated sugar essential for the construction of protein oligosaccharide chains. To date the only therapeutic options are preventive and symptomatic.Scope of reviewThis review covers the latest advances in the search for a treatment for PMM2-CDG.Major conclusionsTreatments based on increasing Man-1-P levels have been proposed, along with the administration of different mannose derivates, employing enzyme inhibitors or repurposed drugs to increase the synthesis of GDP-Man. A single repurposed drug that might alleviate a severe neurological symptom associated with the disorder is now in clinical use. Proof of concept also exists regarding the use of pharmacological chaperones and/or proteostatic regulators to increase the concentration of hypomorphic PMM2 mutant proteins.General significanceThe ongoing challenges facing the discovery of drugs to treat this orphan disease are discussed.     

PMM2-CDG: Phenotype and genotype in four affected family members

Congenital disorders of glycosylation (CDG) are genetic defects in protein and lipid glycosylation. PMM2-CDG is the most prevalent protein N-glycosylation disorder with more than 700 reported patients. Here we report on a large Italian family with four affected members and three mutations. Two young sisters are compound heterozygous for mutations p.Leu32Arg and p.Arg141His, while two paternal great-aunts are compound heterozygosity for p.Leu32Arg and p.Thr237Met. The latter association has not been reported before. The most severely affected member had in addition an ALG6 mutation known to exacerbate the phenotype of patients with PMM2-CDG.     

自然杀伤细胞受体及其配体表达的转录和表观遗传调控

自然杀伤(natural killer cell,NK)细胞受体及其配体在NK细胞发挥抗病毒、抗肿瘤和免疫调节作用中起重要作用.NK细胞功能的发挥取决于NK细胞受体及其配体的表达水平和其所传递信号的综合.病毒、肿瘤和热休克等刺激可以通过激活相应的转录调节因子,提高启动子活性而上调NKG2家族受体及其配体的表达,而启动子区DNA的甲基化状态、组蛋白的乙酰化和甲基化等表观遗传调控,在NK细胞受体及其配体的表达方面亦起重要作用,并决定NK细胞受体的克隆性分布.深入探讨NK细胞受体及其配体的表达调控机制,将为提高NK.细胞抗肿瘤和抗感染疗效提供新的策略.     

Natural Killer Cell Sensing of Infected Cells Compensates for MyD88 Deficiency but Not IFN-I Activity in Resistance to Mouse Cytomegalovirus

In mice, plasmacytoid dendritic cells (pDC) and natural killer (NK) cells both contribute to resistance to systemic infections with herpes viruses including mouse Cytomegalovirus (MCMV). pDCs are the major source of type I IFN (IFN-I) during MCMV infection. This response requires pDC-intrinsic MyD88-dependent signaling by Toll-Like Receptors 7 and 9. Provided that they express appropriate recognition receptors such as Ly49H, NK cells can directly sense and kill MCMV-infected cells. The loss of any one of these responses increases susceptibility to infection. However, the relative importance of these antiviral immune responses and how they are related remain unclear. In humans, while IFN-I responses are essential, MyD88 is dispensable for antiviral immunity. Hence, a higher redundancy has been proposed in the mechanisms promoting protective immune responses against systemic infections by herpes viruses during natural infections in humans. It has been assumed, but not proven, that mice fail to mount protective MyD88-independent IFN-I responses. In humans, the mechanism that compensates MyD88 deficiency has not been elucidated. To address these issues, we compared resistance to MCMV infection and immune responses between mouse strains deficient for MyD88, the IFN-I receptor and/or Ly49H. We show that selective depletion of pDC or genetic deficiencies for MyD88 or TLR9 drastically decreased production of IFN-I, but not the protective antiviral responses. Moreover, MyD88, but not IFN-I receptor, deficiency could largely be compensated by Ly49H-mediated antiviral NK cell responses. Thus, contrary to the current dogma but consistent with the situation in humans, we conclude that, in mice, in our experimental settings, MyD88 is redundant for IFN-I responses and overall defense against a systemic herpes virus infection. Moreover, we identified direct NK cell sensing of infected cells as one mechanism able to compensate for MyD88 deficiency in mice. Similar mechanisms likely contribute to protect MyD88- or IRAK4-deficient patients from viral infections.     

Fasting Enhances TRAIL-Mediated Liver Natural Killer Cell Activity via HSP70 Upregulation

Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)⁺ and CD69⁺ natural killer cells were significantly elevated (n = 7, p <0.01), as determined by flow cytometric analysis. Furthermore, we found that TRAIL⁻ natural killer cells that were adoptively transferred into Rag-2^−/− γ chain^−/− mice could convert into TRAIL⁺ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05) in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05). In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05). These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.     

Liver-Associated Natural Killer Activity in Cirrhotic Rats

An impaired host defense mechanism is well known in patients with liver cirrhosis (LC). Using a sinusoidal lavage method, lymphocytes were obtained from LC rats that were administered thioacetamide, and natural killer (NK) activity was measured by ^5lCr-release assay. The NK cell count was measured by flow cytometric analysis using monoclonal antibody (Mab) 3.2.3 and/or CD 3-8+ as markers for NK cells, and by immunohistochemical staining using Mab 3.2.3. Furthermore, interferon (IFN) α was administered to LC rats and the subsequent changes in hepatic NK activity and NK cell count were observed. In the large granular lymphocyte (LGL)-rich fraction (Fr.1, LGLs: 60-90%), the NK activity was significantly lower in the LC rats (40.0±3.8%) compared to that in the control rats (48.4±4.3%) (P<0.005). In addition, the number of NK cells in the liver tissues of the LC rats was significantly lower compared to that in the liver tissues of the control rats by morphometric analysis (P<0.05). For LC rats, NK activity of the Fr.1 24 hr after IFNα administration (5×10⁴ IU / 100 g body weight) increased significantly (P<0.005). Hepatic NK activity and NK cell count were reduced in the LC rats, and recovered following IFNα administration. The results obtained in this study may give clues to better understanding the impaired host defense mechanism in LC patients.     

Augmentation of Mouse Natural Killer Cell Activity by Lactobacillus casei and Its Surface Antigens

Lactobacillus casei YIT 9018 (LC 9018) augmented the natural killer (NK) cell activity of spleen cells from inbred BALB/c mice injected intravenously with LC 9018 or intraperitoneally with polyinosinate-polycytidylate. Augmentation of this activity by LC 9018 was also observed in male C3H/He, CBA/N, and C57BL/6 mice. The spleen cells exhibited no cytolytic activity against P815, a cell line insensitive to NK cells. The cytolytic activity of the spleen cells increased 2 days after the injection of 250 μg of LC 9018/mouse, peaked on day 3, and gradually declined thereafter. The increase caused by LC 9018 was also observed in normal and Meth A-bearing mice. In vitro treatment with anti-asialo GM1 antibody plus complement completely-abrogated the LC 9018-augmented murine NK cell activity. The NK activity on the 3rd day after LC 9018 injection was reduced by in vitro treatment with anti-Thy 1.2 monoclonal antibody plus complement to half of that observed when treatment was with complement alone. This suggests that there were two populations of NK cells in the spleen cell suspension derived from LC 9018-treated mice. One population was asialo GM1-positive and Thy 1-negative, the other was asialo GM1-positive and Thy 1-positive.     

CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation

Membrane nanotubes are thin membranous projections that physically connect two cells. While nanotubes have been studied in human natural killer (NK) cells and are implicated in aiding NK cell cytotoxic function, requirements for their formation to susceptible target cells remain incompletely understood. Here we demonstrate that the CD2-CD58/48 receptor-ligand interaction promotes and is required for nanotube formation in human NK cells. In the CD2⁻ NK cell line YTS, a stable CD2 expression variant enabled effective nanotube formation, and was associated with better cytotoxic function. Importantly, only interactions between an NK cell and a susceptible target cell were associated with multiple nanotubes and the number of nanotubes was inversely correlated with their length. Quantitative live cell fluorescence microscopy of CD2 nanotubes revealed time-dependent enrichment and localization of CD2 to the nanotube tip, and blocking CD2 receptor-ligand interactions prevented nanotube formation. Increased nanotube formation was not simply a feature of receptor-ligand pairing, as a KIR-MHC interaction in the same cell line system failed to promote nanotube formation. Additionally, blocking LFA-1-ICAM and 2B4-CD48 receptor-ligand interactions failed to inhibit nanotube formation. Thus only specific receptor-ligand pairs promote nanotubes. CD2 also promoted nanotube formation in ex vivo NK cells suggesting that CD2 plays a crucial role in the generation of nanotubes between an NK cell and its target.     

Occurrence of Nodular Lymphocyte-Predominant Hodgkin Lymphoma in Hermansky-Pudlak Type 2 Syndrome Is Associated to Natural Killer and Natural Killer T Cell Defects

Hermansky Pudlak type 2 syndrome (HPS2) is a rare autosomal recessive primary immune deficiency caused by mutations on β3A gene (AP3B1 gene). The defect results in the impairment of the adaptor protein 3 (AP-3) complex, responsible for protein sorting to secretory lysosomes leading to oculo-cutaneous albinism, bleeding disorders and immunodeficiency. We have studied peripheral blood and lymph node biopsies from two siblings affected by HPS2. Lymph node histology showed a nodular lymphocyte predominance type Hodgkin lymphoma (NLPHL) in both HPS2 siblings. By immunohistochemistry, CD8 T-cells from HPS2 NLPHL contained an increased amount of perforin (Prf) + suggesting a defect in the release of this granules-associated protein. By analyzing peripheral blood immune cells we found a significant reduction of circulating NKT cells and of CD56^brightCD16⁻ Natural Killer (NK) cells subset. Functionally, NK cells were defective in their cytotoxic activity against tumor cell lines including Hodgkin Lymphoma as well as in IFN-γ production. This defect was associated with increased baseline level of CD107a and CD63 at the surface level of unstimulated and IL-2-activated NK cells. In summary, these results suggest that a combined and profound defect of innate and adaptive effector cells might explain the susceptibility to infections and lymphoma in these HPS2 patients.     

CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56^bright, NK cells. Most NK cells in healthy human donors are CD45RA⁺CD45RO^-. The CD45RA^-RO⁺ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RA^dimRO^- (CD45RA^dim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RA^dim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RA^dim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.     

LFA-1 and CD2 Synergize for the Erk1/2 Activation in the Natural Killer (NK) Cell Immunological Synapse

Natural killer (NK) cell recognition and formation of a conjugate with target cells, followed by intracellular signal pathway activation and degradation of cytolytic granules, are essential for NK cell cytotoxicity. In this study, NK92 cells were used to investigate synapse formation and subsequent signaling after binding to the target cell. The binding rate of the NK92-target cell was associated with NK92 cell cytotoxicity. Confocal results showed that adhesion molecules, LFA-1 (CD11a) and CD2, accumulated at the interface of the NK92-K562 contact. Ligation with K562 cells activated the Erk1/2 signal pathway of NK92 cells. The blocking of the NK-target conjugate by EDTA or anti-CD11a or/and anti-CD2 antibody decreased the phosphorylation of Erk1/2 and NK cell cytotoxicity. Inhibition of Erk1/2 phosphorylation by the chemical inhibitor U0126 suppressed the cytolytic activity of NK92 cells, but had no effect on NK-target conjugate formation. Thus, conjugate formation of the NK92-target cell was prerequisite to NK cell activation, and subsequent signal transduction was also required for NK cell cytotoxicity.Natural killer (NK)³ cells are a population of granular lymphocytes that play an essential role in cellular immune defense against a variety of tumor cells, virus-infected cells, or allogeneic cells (1–3). NK cells are critical for host immunity for their ability for a quick cytotoxic response and to produce a wide variety of cytokines and chemokines to modulate other cellular components of the immune system (4, 5). NK cells express two functional types of receptors: activating and inhibitory receptors (6–8). The effector function of NK cells is regulated by a balance between opposite signals delivered by the MHC class I-specific inhibitory receptors and the activating receptors responsible for NK cell triggering to permit elimination of pathogens (6).NK cell recognition and binding to target cells, as well as formation of conjugates, are essential for NK cell cytotoxicity (9). Conjugate formation by the NK cell with a target cell is a process mediated by integrins and immunoglobulin superfamily molecules including CD2, CD11a (LFA-1), CD11b, CD11c, and CD28, which also participate in the promotion of NK cell function (10–12). They participate in adhesion between the NK cell and the target cell, and blocking antibodies suppress the adhesion. In addition to possessing an adhesive role, ligation of CD2 induces kinase function and lipid raft polarization (11), whereas ligation of CD11a, CD11b, and CD11c induces phosphorylation-dependent NK cell activation (13, 14). The interaction of specific cell surface receptors with their ligands on a target cell at their interface forms specific activating NK cell immunological synapses and leads to the activation of a cascade of intracellular signals, resulting in Ca²⁺ flux, polarization of granules, and subsequent release of lytic molecules (13, 15, 16). The Erk1/2 (p44/42 mitogen-activated protein kinase) pathway plays an important role in NK cell cytotoxicity (17–21). Inhibition of Erk1/2 might block NK cell cytolytic activity by compromising the release of perforin (22). In this study, the roles of adhesion molecules in NK92-target cell conjugate formation of immunological synapse, and subsequent Erk1/2 activation in NK92 cells was investigated.     

艾氏腹水癌细胞核糖核酸体外对C_(57)BL/6脾自然杀伤活性的影响

本文采用~(51)Cr释放法,研究了艾氏腹水癌细胞核糖核酸(EAC-RNA)体外对C_(57)BL/6小鼠脾自然杀伤细胞(NK)对YAC-1靶细胞杀伤活性的影响。结果表明,EAC-RNA能显著抑制NK活性。经RNase处理后,其抑制活性消失,DNase或Pronase的处理不改变其抑制活性。     

Human and Mouse Type I Natural Killer T Cell Antigen Receptors Exhibit Different Fine Specificities for CD1d-Antigen Complex

Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4′,4″-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4′-OH position in comparison with the 3′-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4′-OH > 3′-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.     

The Traffic of the NKG2D/Dap10 Receptor Complex during Natural Killer (NK) Cell Activation

NKG2D is an important activating receptor for triggering the NK cell cytotoxic activity, although chronic engagement of specific ligands by NKG2D is also known to provoke decreased cell surface expression of the receptor and compromised NK cell function. We have studied the dynamics of surface NKG2D expression and how exposure to the specific ligand major histocompatibility complex class I chain-related molecule B (MICB) affects receptor traffic and fate. While in the NKL cell line and “resting” NK cells NKG2D was found principally at the cell surface, in activated primary NK cells an intracellular pool of receptor could also be found recycling to the plasma membrane. Exposure of NK cells to targets expressing MICB resulted in degradation of ∼50% of total NKG2D protein and lysosomal degradation of the DAP10 adaptor molecule. Consistent with these observations, confocal microscopy experiments demonstrated that DAP10 trafficked to secretory lysosomes in both transfected NKL cells and in activated primary NK cells upon interaction with MICB-expressing target cells. Interestingly, polarization to the synapse of secretory lysosomes containing DAP10 was also observed. The implications of the intracellular traffic of the NKG2D/DAP10 receptor complex for NK cell activation are discussed. We propose that the rapid degradation of NKG2D/DAP10 observed coincident with recruitment of the receptor to the cytotoxic immune synapse may explain the loss of NKG2D receptor expression after chronic exposure to NKG2D ligands.The killer cell lectin-like receptor NKG2D is one of the best characterized NK³ cell-activating receptors. Signaling via NKG2D depends on its association with DAP10, a transmembrane adaptor molecule containing the sequence YINM, which signals via recruitment of phosphatidylinositol 3-kinase and Grb2 (growth factor receptor-bound protein 2) (1, 2). Effector cell activation mediated by NKG2D has been described as immune recognition of the “induced self,” because the cellular ligands for NKG2D (NKG2D-L): the polymorphic MHC class I chain-related molecules (MIC) A and MICB and the UL16-binding proteins are not normally expressed but instead are up-regulated on target cells after pathogen infection or tumor transformation to render these cells susceptible to NK cell lysis (3). Strikingly, however, although induced expression of NKG2D-L acts as a danger signal to provoke an immune response, a number of studies performed in mouse models have shown that chronic exposure to NKG2D-L can also lead to down-modulation of the surface expression of NKG2D and impaired NK cell cytotoxic function (4–6).In humans, a common feature of patients with multiple different tumors is the presence in the serum of high levels of soluble MICA and -B or UL16-binding proteins, released by tumor cells, that are associated with an impairment of CTL and NK cell cytotoxic function (7–10). These observations have been interpreted as suggesting that the release of soluble NKG2D-L is a strategy of tumor immune evasion (11). However, recent data show that receptor interaction with cell membrane-anchored MICB can also lead to impaired NKG2D function. We have shown that brief cytotoxic interactions between NK cells and MICB-expressing target cells trigger a synaptic interchange of NKG2D and MICB as well as a rapid down-modulation of surface NKG2D and compromised NK cell cytotoxicity suggesting that NKG2D traffic is rapidly altered upon recognition of MICB expressed on target cell (12).The surface level of a receptor is dictated by the relative rates of synthesis and transport to the plasma membrane and endocytosis, recycling, and degradation. The loss of cell surface NKG2D observed after NKG2D-L binding (7–10, 12) raises the question of what is the intracellular fate of the receptor on interaction with NKG2D-L. However, the traffic of this receptor has not been previously studied. Here we describe the dynamics of surface NKG2D expression and examine how cytotoxic interactions between NK cells and the MHC class I- 721.221 (here called 221) cells that express MICB (here called 221B) affect the traffic and fate of the NKG2D/DAP10 receptor complex. In NKL cells and resting primary NK cells NKG2D is mainly expressed at the cell surface; however, in activated primary NK cells an intracellular pool of receptor recycling to the cell surface is detected. During cytotoxic interactions the recognition of MICB expressed on target cells results in a rapid degradation of NKG2D/DAP10 that is associated with the traffic of DAP10 to secretory lysosomes (SLs) (13, 14). Our data provide new insights into the dynamics of NKG2D receptor expression in NK cells and suggest a plausible model to explain how chronic exposure to NKG2D-L could lead to NKG2D down-modulation and compromised NK cell function.