Role of natural killer cells in HIV pathogenesis

Although the majority of research on immune cell recognition of HIV-infected cells has focused on CD8⁺ T cells with an eye towards vaccine development, innate immune recognition by natural killer (NK) cells has become a focus in recent years. Genetic and mechanistic data indicate that NK cells play a role during pathogenesis, and research on NK biology in the context of the broader immune response shows that NK cells are required to mount an effective antiviral response. HIV is able to escape cytotoxic T lymphocyte recognition by downmodulation of major histocompatibility complex class I receptors, which should enhance NK cytotoxicity against infected targets. However, the virus has evolved elaborate mechanisms to evade NK cell responses. Moreover, NK cell function as a whole is compromised through poorly understood mechanisms as a result of viremia. Further work on the role of NK cells during all stages of disease will further our understanding of the immune response against HIV.     

Involvement of natural killer cells in endogenous biological retranslation

Analysis of functional properties of natural killer cells displayed in reactions of natural cytotoxicity and noncytotoxic regulatory intercellular interactions suggests that this population of lymphocytes is involved in endogenous biological retranslation. In the immune system, retranslation is the production of regulatory immunoactive cytokines by a cell, cellular complex, or functional complex. The substances produced are identical to those affecting these structures. Various forms of endogenous biological retranslation in humans and higher animals, as well as its phylogenetic and ontogenetic manifestations (on the basis of noncytotoxic regulatory interactions of natural killer cells with cells of lymphoid or nonlymphoid nature) during evolution of the complex of immunobiological surveillance are considered. The axiomatic basis of retranslation realized through the system of natural cytotoxicity was established. Prospects for application of the methodology of endogenous biological retranslation to experimental and clinical studies of functioning of natural killer cells are considered. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 8, pp. 124–135, August, 1998     

Compartmentalization of human natural killer cells

Human natural killer (NK) cells are bone marrow-derived cells that are found in the bloodstream, but can extravasate into various tissue sites upon inflammation. NK cells that migrate toward inflamed sites must be activated prior to their extravasation. However, the factors responsible for NK cell compartmentalization are not clearly defined. Resting human NK cells (CD16(-) and CD16(+)) express constitutive chemokine receptors, as well as receptors that have both constitutive and inflammatory functions. Upon activation, NK cells up-regulate the expression of the inflammatory chemokine receptors which facilitate their distribution into inflammatory sites. However, chemokines are not expected to play any role in maintaining resting NK cells in the blood circulation. In contrast, members of the lysolipids which are abundant in the bloodstream may be the major factors responsible for maintaining resting NK cells in the bloodstream, and also for facilitating their extravasation into inflamed tissues. Both resting and activated NK cells express receptors for various lysolipids. Hence, chemoattractants which include chemokines and lysolipids have important roles in determining the compartmentalization of NK cells where resting NK cells are found in the blood circulation, and activated NK cells extravasate into inflamed sites.     

Dendritic cells and natural killer cells in the pathogenesis of HIV infection

Dendritic cells (DC) and natural killer (NK) cells, the main cellular components of the innate immune system, participate in the most ancient first line of defense against infections. Both types of cells patrol peripheral tissues, whereas their rapid recruitment and activation at mucosal surfaces [the major entry point for the human immunodeficiency virus (HIV)] is a hallmark of acute inflammatory response. The ability of HIV to survive and replicate in the human host relies upon several molecular mechanisms eluding the immune surveillance of both adaptive immunity and of DC and NK cells beginning with the acute phase of primary HIV infection. DC and NK cells, unlike CD4⁺ T cells, are impaired more functionally rather than being depleted by HIV infection. In this article we will review some of the aspects of DC/NK cells interaction with HIV infection both in vitro and in vivo, and we will also speculate on the potential consequence for HIV pathogenesis and for the capacity of the virus to escape the surveillance of the innate immune system.     

Pro- and antiinflammatory cytokine signaling: reciprocal antagonism regulates interferon-gamma production by human natural killer cells

Activated monocytes produce proinflammatory cytokines (monokines) such as interleukin (IL)-12, IL-15, and IL-18 for induction of interferon-gamma (IFN-gamma) by natural killer (NK) cells. NK cells provide the antiinflammatory cytokine transforming growth factor (TGF)-beta, an autocrine/negative regulator of IFN-gamma. The ability of one signaling pathway to prevail over the other is likely important in controlling IFN-gamma for the purposes of infection and autoimmunity, but the molecular mechanism(s) of how this counterregulation occurs is unknown. Here we show that in isolated human NK cells, proinflammatory monokines antagonize antiinflammatory TGF-beta signaling by downregulating the expression of the TGF-beta type II receptor, and its signaling intermediates SMAD2 and SMAD3. In contrast, TGF-beta utilizes SMAD2, SMAD3, and SMAD4 to suppress IFN-gamma and T-BET, a positive regulator of IFN-gamma. Indeed, activated NK cells from Smad3(-/-) mice produce more IFN-gamma in vivo than NK cells from wild-type mice. Collectively, our data suggest that pro- and antiinflammatory cytokine signaling reciprocally antagonize each other in an effort to prevail in the regulation of NK cell IFN-gamma production.     

Glycogen synthetase kinase 3 inhibition drives MIC-A/B to promote cytokine production by human natural killer cells in Dengue virus type 2 infection

Dengue virus (DENV) is the most widespread arbovirus worldwide and is responsible for major outbreaks. The host's immune response plays a crucial role in controlling this infection but might also contribute to the promotion of viral spread and immunopathology. In response to DENV infection, NK cells preferentially produce cytokines and are cytotoxic in the presence of specific antibodies. Here, we identified that DENV-2 inhibits glycogen synthase kinase 3 (GSK-3) activity to subsequently induce MHC class-1-related chain (MIC) A and MIC-B expression and IL-12 production in monocyte-derived DCs, independently of the STAT-3 pathway. The inhibition of GSK-3 by DENV-2 or small molecules induced MIC-A/B expression on monocyte-derived DCs, resulting in autologous NK cells of a specific increase in IFN-γ and TNF-α production, in the absence of direct cytotoxicity. Together, these findings identified GSK-3 as a regulator of MIC-A/B expression and suggested its role in DENV-2 infection to specifically induce cytokine production by NK cells.     

2B4-mediated activation of human natural killer cells

2B4 is a member of the CD2 subset of the immunoglobulin superfamily of cell surface receptors. Other members of this family include CD2, CD48, CD58, CD84, signaling lymphocytic activation molecule and Ly-9. Some of these molecules are activating structures expressed by natural killer cells and T cells. We have recently cloned and characterised the human homologue of 2B4 and found that the cytoplasmic domain of 2B4 can interact with SAP, a signaling adaptor protein that is mutated in the immunodeficiency X-linked lymphoproliferative disease (XLP). Additionally, the natural ligand of 2B4 has been identified as CD48. These findings have facilitated the investigation of the functional role of this receptor-ligand pair, and associated signal transduction pathways, on immune cells. In this study, it was found that the interaction between 2B4 on effector cells and CD48 on target cells induced NK-cell activation, as evidenced by increased cytotoxicity and secretion of IFN-gamma. The responses induced by ligation of 2B4 could be reduced by the co-ligation of inhibitory receptors expressed by NK cells, demonstrating that activation signals delivered via 2B4 can be regulated by the action of certain inhibitory receptors. Because the signalling pathway of 2B4 involves SAP, it is possible that 2B4-mediated NK-cell activation may be compromised in patients with XLP due to mutations in SAP. This may contribute to the phenotype and progression of this disease.     

Involvement of transferrin and transferrin receptors in human natural killer effector:target interaction

In this current study one of the determinants of natural killer cell specificity in immunosurveillance against cancer, may be the recognition of transferrin receptors on neoplastic cells by the NK effectors. Human transferrin, when saturated with iron (FeTf), was found to inhibit human natural killer (NK) activity against K562 tumor cells, if included in assay mixtures at physiologically relevant levels. Whereas both FeTf and iron-free transferrin (ApoTf) inhibited initial conjugate formation at the level of the target cell, only FeTf inhibited NK cytolytic activity, as judged by release of chromium from the targets. This suggested a functional role for FeTf on either NK or tumor cells. When either targets or effector lymphocytes were pre-incubated with FeTf, inhibition of killing was strongest when the targets were first exposed to FeTf. The evidence suggested that NK-associated transferrin mediated the interaction with target cells through free target-associated transferrin receptors. The finding that rabbit anti-human transferrin antibody (RaHTf) inhibited killing, when reacted with effector lymphocytes but not with target cells, supported this hypothesis.     

Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3-) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3- LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Involvement of fibronectin and interacting serum components in the regulation of activity of human natural killer cells

The cytotoxic activity of natural killer cells and the intensity of conjugate formation are studiedin vitro in the natural cytotoxicity reaction against³H-uridine-labeled human erythromyeloleukotic cells K-562 in the presence of fibronectin, γ-globulin, and fibronectin/γ-globulin combination. It is demonstrated that fibronectin does not change natural cytotoxicity, γ-globulin increases the activity of human natural killer cells, and the fibronectin — γ-globulin combination increases both the intensity of conjugate formation and the cytotoxic activity of natural killer cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 7, pp. 54–59, July, 1994     

Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3⁻) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3⁻ LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Co-stimulation of human decidual natural killer cells by interleukin-2 and stromal cells

At the late secretory phase of the menstrual cycle and in early pregnancy, the uterine mucosa is infiltrated by large numbers of natural killer (NK) cells with a distinctive phenotype (CD56bright CD16- CD3-) and large granular lymphocyte (LGL) morphology. Circulating CD56bright NK cells generally proliferate in the presence of interleukin-2 (IL-2), but it is clear that cofactors besides IL-2 are required for optimal response. In the bone marrow, this co-stimulating signal is provided by stromal cells. In the present study we observe that uterine CD56+ cells from early pregnancy decidua similarly proliferate vigorously when cultured with decidual stromal cells and a suboptimal dose of IL-2. This response is dependent on cell-cell contact, as no proliferation of decidual NK cells was observed when they were separated from stromal cells by a permeable cyclopore membrane. In addition, we have studied the expression of Bcl-2 by decidual CD56+ cells. Our results show that the microenvironment of the uterus is likely to have a significant influence on the proliferation and survival of uterine CD56+ cells.     

Selective cross-talk among natural cytotoxicity receptors in human natural killer cells

The cytolytic activity of human natural killer cells is induced by several triggering cell surface receptors upon interaction with specific cellular ligands. These receptors include NKp46, NKp30 and NKp44, collectively termed natural cytotoxicity receptors (NCR). Co-operation among NCR has been shown to occur for optimal recognition and killing of most tumor target cells. In this study, we show that the mAb-mediated engagement and clustering of one or another NCR results in the activation of an identical set of tyrosine kinases. These kinases are included in the signaling cascade leading to tyrosine phosphorylation of different receptor-associated signal transducing molecules i.e. CD3 zeta (associated with NKp46 and NKp30) and KARAP/DAP12 (associated with NKp44). In line with the notion that the engagement of inhibitory receptors prevents NCR-mediated responses, we show that the engagement of CD94/NKG2A virtually abrogates the tyrosine phosphorylation of the NCR-associated signaling molecules, i.e. it acts at the very early steps of the signaling cascade. Importantly, the engagement of a single NCR resulted in the activation of the signaling cascades associated with the other NCR. This "cross-talk" is confined to NKp46, NKp30 and NKp44 since neither CD16-nor KIR2DS4-associated signaling polypeptides were phosphorylated following the NCR engagement. These results suggest that a functional cross-talk specifically occurs among different NCR, possibly resulting in the amplification of the activating signals.     

Activation of natural killer cells and cytokine production in man by bacterial extracts

Broncho-Vaxon (OM-85 BV) is a bacterial extract of eight bacterias usually involved in the respiratory tract infections. Since Broncho-Vaxom is clinically active in decreasing the incidence of such infections, its immunological effect was investigated, in vitro, using peripheral blood mononuclear cells (PBMC). The experimental data indicate that Broncho-Vaxom can modulate various immune functions. It was shown, using a radioimmunoassay for these cytokines, that Broncho-Vaxom will spontaneously enhance TNF alpha and IL-2 production whereas it has no action on IF gamma production. However, when the PBMC are stimulated with PHA, an increased production for IF gamma, TNF alpha and IL-2 was observed suggesting that, under appropriate conditions, Broncho-Vaxom enhances the production of these cytokines. In other experiments, Broncho-Vaxom was shown to markedly increase the natural killer activity of PBMC. All these results demonstrate that Broncho-Vaxom is an immunomodulator affecting multiple immunological mechanisms including the activation of natural killer cells, of monocytes and of T cells through direct mechanisms or through the cytokine cascade.     

人NK细胞体外高效扩增的实验研究

目的:建立人NK细胞体外大量扩增的方法。方法:采用基因工程方法,在K562细胞上同时表达IL-15、IL-18、4-1BBL3种基因,构建特定的K562工程细胞作为刺激细胞。IL-15、IL-18基因分别与一段特殊的跨膜蛋白基因融合,4-1BBL直接跨膜表达,使这3种蛋白在K562细胞中表达后锚定于细胞膜表面。其次,以照射致死的该K562工程细胞作为刺激细胞,以人外周血单个核细胞(PBMC)为扩增培养对象,通过与IL-2的共刺激作用,使NK细胞在体外培养条件下得到大量的扩增。结果:经过21d的刺激培养后,CD56 CD3-细胞数量扩增了(520±75)倍。CD56 CD3-细胞的纯度从培养前占PBMC的7%±4%到扩增后占总细胞比例的93%±3%。PBMC中的T细胞基本上没有得到扩增,扩增后的细胞中CD3 细胞只占2%±1.2%。扩增的细胞具备了NK细胞的基本特征和生物学特性,除了CD56 CD3-外,还对扩增的NK细胞上NKG2D、NKp46、NKp44、NKp30、CD94、CD158b、CD158a、NKB1、NKAT2等标记进行了验证。细胞毒实验表明,在效应细胞∶靶细胞为5∶1时,扩增的NK细胞的杀伤率达到了95%±4%。结论:建立的NK细胞体外扩增方法,达到了较高的扩增水平,且扩增的细胞活性较好。本方法以PBMC为原始材料,能够实现NK细胞体外的大规模制备,这将为抗病毒与抗肿瘤的NK细胞免疫治疗奠定基础。     

Virus-infected colostral cell cytokine stimulation of human leukocyte natural killer cytotoxicity.

Natural killer cytotoxicity is an important antiviral defense mechanism. Human peripheral blood mononuclear cells cultured with herpes simplex virus (HSV)-infected cells produced a cytokine. This substance stimulated adult natural killer cytotoxicity from 53.0 +/- 10.5% to 79.8% (P less than 0.01) against HSV-infected target cells. These data resulted in a calculated cytokine-dependent cellular cytotoxicity (CDCC) value of 65.8%. Cytokine production was not stimulated by uninfected cells and was independent of the presence or absence of antibodies to HSV in sera of donors and mononuclear cells. Cells from human colostrum also produced an HSV-stimulated cytokine which mediated CDCC by using both adult (19.8 +/- 3.9%) and neonatal (18.6 +/- 3.4%) mononuclear effectors cells. Colostral cell cytokine production was also independent of donor HSV serology. Not all colostral cultures produced the cytokine, and in general colostrum-stimulated CDCC was lower than peripheral blood leukocyte-stimulated CDCC. Colostral cell cytokine stimulation of neonatal natural killer cytotoxicity may account in part for the increased nonspecific resistance of breast-fed infants to viral infection.     

Lectin-binding characteristics of human natural killer cells.

Human natural killer (NK) cells separated initially by density centrifugation of lymphocytes (E+) forming rosettes with sheep red blood cells (SRBC), were further fractionated on gradients of bovine serum albumin (BSA). Low density fractions contained effector cells which displayed high cytotoxicity against the NK-sensitive erythroleukaemic cell line, K562. These low density cells, which expressed receptors for Fc and the monoclonal antibody OKMI, showed enhanced cytotoxicity when treated with lymphoblastoid interferon (IFN-alpha). They also showed an increased response to phytomitogen in comparison with unseparated cells or those recovered from high density fractions. Two lymphocyte subsets one of high and one of low lectin binding capacity were identified in the E+ populations by their reactivity with Lens culinaris agglutinin (LCA). High LCA binding was observed only in low density fractions and was associated with a marked enrichment of NK activity. This property was used to separate the NK active population in E+ cells by fluorescence-activated cell sorting (FACS). These data add a new dimension to the cell surface properties of human NK cells and suggest the presence of LCA-reactive glycoproteins which are either enriched in, or uniquely associated with, cells of the NK subset. The experiments indicate that lectins can serve as useful probes of lymphocyte function and provide the basis for effective cell sorting.