Dok1 and Dok2 proteins regulate natural killer cell development and function

Natural killer (NK) cells are involved in immune responses against tumors and microbes. NK‐cell activation is regulated by intrinsic and extrinsic mechanisms that ensure NK tolerance and efficacy. Here, we show that the cytoplasmic signaling molecules Dok1 and Dok2 are tyrosine phosphorylated upon NK‐cell activation. Overexpression of Dok proteins in human NK cells reduces cell activation induced by NK‐cell‐activating receptors. Dok1 and Dok2 gene ablation in mice induces an NK‐cell maturation defect and leads to increased IFN‐γ production induced by activating receptors. Taken together, these results reveal that Dok1 and Dok2 proteins are involved in an intrinsic negative feedback loop downstream of NK‐cell‐activating receptors in mouse and human.     

Evaluation of natural killer cell activity

Natural killer (NK) cells are being appreciated not only for their ability to recognize and lyse tumor cells and virus-infected cells but also for their immunoregulatory properties. NK cells provide a first line of defense against invading pathogens with a two pronged attack, lysis of infected cells and secretion of cytokines and chemokines with potent antipathogen effects. This article describes the standard chromium release assay, which measures the ability of NK cells derived from the peripheral blood to lyse appropriate target cells.     

Chemokine networks modulating natural killer cell trafficking to solid tumors

Natural killer (NK) cell-based cell therapy has been emerging as a powerful weapon in the treatment of multiple malignancies. However, the inadequate infiltration of the therapeutic NK cells into solid tumors remains a big challenge to their clinical utility. Chemokine networks, which play essential roles in the migration of lymphocytes, have been recognized as critical in driving the intratumoral infiltration of NK cells via interactions between soluble chemokines and their receptors. Often, such interactions are complex and disease-specific. In the context of NK cells, chemokine receptors of note have included CCR2, CCR5, CCR7, CXCR3, and CX3CR1. The immunobiology of chemokine-receptor interactions has fueled the development of approaches that hope to improve the infiltration of NK cells into the microenvironment of solid tumors. Stimulation of NK cells ex vivo in the presence of various cytokines (such as IL-2, IL-15, and IL-21) and genetic engineering of NK cells have been utilized to alter the chemokine receptor profile and generate NK cells with higher infiltrating capacity. Additionally, the immune-suppressive tumor microenvironment has also been targeted, by introducing, either directly or indirectly, chemokine ligands which NK cells are able to respond to, ultimately creating a more hospitable niche for NK cell trafficking. Such strategies have promoted the infiltration and activity of infused NK cells into multiple solid tumors. In this review, we discuss how chemokine receptors and their ligands coordinate and how they can be manipulated to regulate the trafficking, distribution, and residence of NK cells in solid tumors.     

自然杀伤细胞受体的研究进展

自然杀伤细胞（ＮＫ细胞）可表达两类功能相悖的识别受体,即活化受体（ＫＡＲ）和抑制受体（ＫＩＲ）。ＫＩＲ能识别自身细胞上的ＭＨＣⅠ类分子与自身或外来肽形成２的复合物,所产生的抑制信号可阴断ＫＡＲ的活化,以此抑制ＮＫ细胞的细胞毒作用。如果靶细胞失去ＫＩＲ所识别的配体,ＮＫ细胞即可通过ＫＡＲ对靶细胞进行攻击。本文将介绍此类受体的结构及基识别与信号转导机制的研究进展。     

Soluble ULBP suppresses natural killer cell activity via down-regulating NKG2D expression

Our previous studies show that growth hormone overexpression (GHo) upregulates the expression of the IGF-1R and IGF-2R resulting in the protection of the EL4 lymphoma cell line from apoptosis. In this study, we report that GHo also increases TGF-beta1 protein expression measured by luciferase promoter assay, Western analysis, and ELISA. Further, the data show that antibody to TGF-betaR2 decreases TGF-beta1 promoter activity to the level of vector alone control cells. GHo cells treated with (125)I-rh-latent TGF-beta1 showed increased activation of latent TGF-beta1 as measured by an increase in the active 24kDa, TGF-beta1 compared to vector alone control cells. The ability of endogenous GH to increase TGF-beta1 expression is blocked in EL4 cells by antisense but not sense oligodeoxynucleotides or in cells cultured with antibody to growth hormone (GH). The data suggest that endogenous GH may protect from apoptosis through the IGF-1R receptor while limiting cellular growth through increased expression and activation of TGF-beta1.     

Tolerant and diverse natural killer cell repertoires in the absence of selection

Natural killer (NK) cells are innate lymphocytes that participate in the early control of viruses and tumors. The function of NK cells is under tight regulation by two complementary inhibitory receptor families that bind to classical and non-classical HLA class I molecules: the CD94/NKG2A receptors and the killer cell immunoglobulin-like receptors (KIRs). In this mini-review, recent data on the structure of human NK cell receptor repertoires and its relation to functional responses and tolerance to self are discussed. We propose that no active selection is required to generate diverse NK cell repertoires characterized by a dominant expression of receptors with specificity for self-HLA class I. Instead, the primary consequence of interactions with HLA class I molecules is a functional tuning of randomly generated NK cell repertoires.     

Expression of chemokine receptors on natural killer cells in HIV-infected individuals

Chemokine receptors CCR5 and CXCR4 are of major importance in the pathogenesis of HIV-1 infection because they are co-receptors for human immunodeficiency virus (HIV) entry. We examined the frequency of CD3⁻CD56⁺CCR5⁺ and CD3⁻CD56⁺CXCR4⁺ in HIV-infected long-term slow progressors (SPs), HIV typical progressors (TPs) with or without highly active antiretroviral therapy (HAART), and HIV-seronegative controls. The results showed that the frequency of CD3⁻CD56⁺CCR5⁺ was up-regulated, and frequency of CD3⁻CD56⁺CXCR4⁺ was down-regulated in HAART-naïve HIV TPs group compared with HIV SPs group and HIV-seronegative controls (P < 0.05). The frequency of CD3⁻CD56⁺CCR5⁺ was down-regulated by HAART therapy (P < 0.05). The frequency of CD3⁻CD56⁺CCR5⁺ was lower in HIV SPs compared with controls (P < 0.05). Lower frequency of CD3⁻CD56⁺CXCR4⁺ and higher frequency of CD3⁻CD56⁺CCR5⁺ positively correlated with the level of HIV viral loads and negatively correlated with CD4 T cell counts (P < 0.05). These results indicated that the expression of chemokine receptors on NK cells correlated with HIV disease progression.     

Unravelling natural killer cell function: triggering and inhibitory human NK receptors

Natural killer (NK) cells represent a highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, NKp30, NKp44 and NKG2D. The NK-mediated lysis of tumor cells involves several such receptors, while killing of dendritic cells involves only NKp30. The target-cell ligands recognized by some receptors have been identified, but those to which major receptors bind are not yet known. Nevertheless, functional data suggest that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the ability of NK cells to lyse target cells requires both the lack of surface MHC class I molecules and the expression of appropriate ligands that trigger NK receptors.     

NK细胞和NKT细胞发育的转录调控

自然杀伤（natural killer,NK）细胞和自然杀伤T（natural killer T,NKT）细胞是参与机体抗病毒免疫和肿瘤免疫的两群淋巴细胞亚群,是介导先天性免疫（innate immunity）应答和调节适应性免疫（adaptive immunity）应答的重要效应细胞。近年来,随着对NK细胞和NKT细胞及其转录调控因子研究的不断深入,NK细胞和NKT细胞的发育机制逐步被阐明,这将为提高NK细胞和NKT细胞的抗病毒和肿瘤免疫疗效提供新的策略。     

Natural killer,natural killer T,helper and cytotoxic T cells in the decidua from recurrent spontaneous abortion with normal and abnormal chromosome karyotypes

Problem

Recurrent spontaneous abortion (RSA) is associated with immune imbalance at the maternal–fetal interface. Decidual immune cells and cytokines expressed at this interface regulate the response of the maternal immune system to the fetus. However, the populations and cytokine expression levels of these lymphocytes in miscarriage with normal and abnormal chromosome karyotypes remain unclear.

Effect of phenols on natural killer (NK) cell-mediated death in the K562 human leukemic cell line

Cancer disease is a major cause of death in Western societies. Epidemiologically, antioxidant phenols have been associated with diminished incidence of cancer, while experimentally, they have cytotoxic effects on cancer cells. The aim of this study was to clarify whether natural antioxidant phenols render K562 human leukemic cells more susceptible to natural killer (NK) cell apoptosis and/or necrosis. K562 cells were pre-incubated with 7 different phenols (p-hydroxy benzoic acid, syringic acid, ferulic acid, p-coumaric acid, o-coumaric acid, gallic acid, and rutin) individually and afterwards targeted with NK cells at a ratio 1/5. Percentages of apoptotic and necrotic cells were assayed via flow cytometric analysis of annexin V and PI-stained cells. For the morphological assessment, cells were stained with acridine orange and ethidium bromide and were examined under a fluorescence microscope. Pre-treatment with gallic acid significantly rendered K562 cells more susceptible to NK cell-mediated necrosis, while pre-treatment with rutin significantly rendered K562 cells more susceptible to apoptosis. Gallic acid and rutin exert anticarcinogenic activity via the enhancement of K562 cell susceptibility to NK cell-mediated necrosis and apoptosis, respectively.     

A single major chromosomal region controls natural killer cell-like activity in rainbow trout

We report the identification of a single major chromosomal region controlling natural killer (NK) cell-like activity in rainbow trout (Oncorhynchus mykiss). A genetic map based on 484 AFLP and 39 microsatellite genotypes from 106 doubled haploid fish was constructed. These fish were produced by androgenesis from a hybrid of two clonal lines divergent in NK-like activity. NK-like activities for 75 of the doubled haploids were quantified by an in vitro chromium release assay utilizing ⁵¹Cr-labeled YAC-1 target cells. Composite interval mapping revealed a single major quantitative trait locus (QTL) associated with NK-like activity in this rainbow trout model. Genetic mapping revealed this QTL to also be unlinked to: fragmented MHC class I and MHC class II regions, the leukocyte receptor cluster, the natural killer cell enhancement factor (NKEF) gene, the RAG-1 gene, and two QTL associated with resistance to infectious pancreatic necrosis virus in rainbow trout. Collectively, these results extend the utility of rainbow trout as an immunological model and are consistent with the idea that a single chromosomal region homologous to the natural killer cell complex (NKC) located on syntenic portions of mouse chromosome (Chr) 6, human Chr 12, and rat Chr 4 may exist in a lower vertebrate model.     

Lysophosphatidylglycerol stimulates chemotactic migration in human natural killer cells

We observed that lysophosphatidylglycerol (LPG) stimulates chemotactic migration in human natural killer (NK) cells. The LPG-induced chemotactic migration of NK cells was completely inhibited by pertussis toxin (PTX). LPG also stimulated the extracellular signal-regulated kinase (ERK) and Akt activities in NK cells. LPG-stimulated ERK activity was inhibited by PTX, indicating the involvement of PTX-sensitive G-proteins. The preincubation of NK cells with an ERK inhibitor (PD98059) or phosphoinositide-3-kinase (PI3K) inhibitors (wortmannin and LY294002) completely inhibited LPG-induced chemotactic migration, suggesting the essential role of ERK and PI3K in the process. Moreover, LPG-induced chemotactic migration in NK cell was inhibited by Ki16425, an LPA_1/3 receptor-selective antagonist, suggesting the involvement of the Ki16425-sensitive G-protein coupled receptor (GPCR) in the process. Taken together, the results indicate that LPG stimulates chemotactic migration in NK cells through GPCR, suggesting a new function of LPG as a modulator of NK cell functioning.     

Heatstroke induces a reduction of natural killer cell activity in rats

Experiments were carried out to determine the changes of natural killer (NK) cell activity that occurred during heatstroke in rats pretreated with or without interleukin-1 (IL-1) receptor antagonist (IL-1ra). After the onset of heatstroke, all the splenic NK cell activity, the effector-target cell conjugation, and the NK cell numbers were decreased in rats. Additionally, an increase in the plasma IL-1 level was associated with arterial hypotension, cerebral ischemia and hyperthermia during rat heatstroke. Pretreatment with an IL-1ra reversed in part the heatstroke-induced inhibition of NK cell activity. Thus it appears that the inhibition of NK cell activity produced by activation of IL-1 receptor mechanism is associated with the increased susceptibility to infection that is well described in heatstroke.     

Ccr5 deficiency regulates the proliferation and trafficking of natural killer cells under physiological conditions

Chemokines were shown to govern the trafficking of immune cells and may also play important roles in the survival and activation of these cells. We report here that under physiological conditions, the bone marrow (BM), spleen, blood and liver of Ccr5, but not of Ccr1-deficient mice, contain reduced numbers of NK cells. NK cells in the BM of Ccr5-deficient mice proliferate to a lesser extent compared to WT mice. Furthermore, spleen NK cells derived from Ccr5-deficient mice that were transplanted into irradiated recipients failed to proliferate in the host. Ccr5, but not Ccr1-deficient NK cells, failed to migrate in vitro in response to RANTES and MIP-1β but not MIP-1β or SDF-1 and had reduced activation, lower expression levels of NK cell markers and a slightly reduced capacity to adhere to target cells and stimulate their killing. Using the polyI:C mouse model for NK trafficking, we found that in the absence of Ccr5, but not Ccr1, NK cells failed to accumulate in the liver. In contrast, using the influenza viral infection as a model to evaluate NK cell proliferation, we found that Ccr5-deficient NK cells in the BM had a higher proliferation rate than WT NK cells. These results suggest a role for Ccr5 in NK cell proliferation and circulation under physiological conditions and a complex role for Ccr5 in determining the fate of NK cells under pathological conditions.     

Renal carcinoma cell lines inhibit natural killer activity via the CD94 receptor molecule

MHC class I molecules protect normal and transformed cells from lysis by natural killer (NK) cells through recognition of receptors expressed on leucocytes. Defects in NK cell activity and lymphokine activated killer (LAK) cell generation have been previously demonstrated in patients with renal cell carcinoma (RCC). However, to date, the importance of NK receptor/MHC class I interactions for immune evasion by RCC cells has not been described. In this study, human RCC cell lines (HTB46, HTB47, ACHN, CRL 1933 and HTB44) were found to be susceptible to lysis by both NK cells and interleukin-15 (IL-15)-derived LAK cells from normal donors in vitro. However, when NK cells were co-cultured with RCC cells their expression of the CD94 NK receptor molecule was significantly increased and their cytolytic activity against RCC targets was reduced. The cytolytic activity of NK cells was restored by the addition of IL-15, which further augmented the expression of CD94 on CD56+ NK cells. Disruption of NK receptor-MHC class I interactions by the addition of blocking antibodies to CD94 had no effect on the lysis of K562 or HTB47 targets by NK cells. However, the sensitivity of HTB46 cells to NK-mediated lysis was increased by blocking the CD94 receptor molecule, but only when the NK cells had not been previously co-cultured with RCC cells. This was independent of the presence of IL-15. These results show that RCC cells can inhibit NK activity via CD94 and suggest that disruption of interactions between receptor and ligand on RCC cells in vivo may augment the immune response against tumours by innate effector cells.     

Sooty mangabeys and rhesus macaques exhibit significant divergent natural killer cell responses during both acute and chronic phases of SIV infection

A correlation between NK cells and rate of disease progression in HIV-1-infected individuals has been documented. The role NK cells play in disease outcome can optimally be studied in SIV-infected disease susceptible rhesus macaques (RM) and SIV-infected disease resistant sooty mangabeys (SM). In this study, three main subsets of CD16⁺CD56⁻, CD16^−/dimCD56⁺ and CD16⁻CD56⁻ NK cells have been identified with the predominant CD16⁺CD56⁻ subset being primarily responsible for cytolytic activity in both species. Cross-sectional studies revealed a significant decline in the frequency and function of this cytolytic subset in SIV-infected RM while an increase occurred in SIV-infected SM. Longitudinal studies revealed that an earlier NK response during acute infection occurred in all SIV-infected SM and in select SIV-infected RM that eventually controlled viral load set point during chronic infection, suggesting that early NK activity and continued maintenance of this cell lineage may indirectly contribute to disease resistance.     

Phenotypically and functionally distinct subsets of natural killer cells in human PBMCs

Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hi CD16(-), CD56lo CD16(-), CD56+CD16+ and CD56(-)CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)-2 stimulation, interferon-gamma was preferentially produced by CD56+CD16(-) NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16(-) and CD56+/-CD16+ subsets could be augmented in response to IL-2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.