A phenotypic and functional characterization of NK cells in adenoids

Adenoids are part of the MALT. In the present study, we analyzed cell surface markers and cytolytic activity of adenoidal NK (A-NK) cells and compared them with NK cells derived from blood of the same donors (B-NK). NK cells comprised 0.67% (0.4-1.2%) of the total lymphoid population isolated from adenoids. The majority (median=92%) of the A-NK cells was CD56(bright)CD16(-). A-NK cells were characterized by the increased expression of activation-induced receptors. NKp44 was detected on >60%, CD25 on >40%, and HLA-DR on >50% of freshly isolated A-NK cells. Functional assays indicated that the cytotoxic machinery of A-NK is intact, and sensitive target cells are killed via natural cytotoxicity receptors, such as NKG2D. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66) expression was up-regulated in 23% (median) of the A-NK cells by IL-2 activation but unchanged in B-NK cells. CEACAM1 inhibited the A-NK killing of target cells. CXCR4 was expressed on more than 40% A-NK cells prior to activation. Its ligand, CXCL12, was found in endothelial cells of the capillaries within the adenoid and in cells of the epithelial lining. In addition, A-NK cells migrated in vitro toward a gradient of CXCL12 in a dose-responsive manner, suggesting a role for this chemokine in A-NK cell recruitment and trafficking. We conclude that the A-NK cells are unique in that they display an activated-like phenotype and are different from their CD16(-) B-NK cell counterparts. This phenotype presumably reflects the chronic interaction of A-NK cells with antigens penetrating the body through the nasal route.     

NK cells developing in vitro from fetal mouse progenitors express at least one member of the Ly49 family that is acquired in a time-dependent and stochastic manner independently of CD94 and NKG2

NK cells developing in vitro from fetal progenitors in the presence of IL-2 are phenotypically and functionally indistinguishable from mature adult NK cells, with the exception that they generally lack surface expression of any of the Ly49 molecules that have previously been examined. Using two recently developed anti-Ly49 mAb, we show here that most of these NK cells in fact express high levels of at least one previously uncharacterized member of the Ly49 family, most likely Ly49E. Detailed kinetic and clonal analysis revealed that these Ly49 molecules were acquired in a progressive and stochastic manner independently of CD94 and NKG2. CD94 and NKG2 were both expressed early in NK cell development, sometimes in the absence of NK1.1, with CD94 invariably being expressed at two different levels. IL-4 differentially inhibited the expression of CD94 and Ly49 receptors, but had little or no effect on the expression of NKRP1 molecules.     

Palmitoylation of MICA, a ligand for NKG2D, mediates its recruitment to membrane microdomains and promotes its shedding

MICA and MICB (MHC-class-I-related chain A/B) are transmembrane proteins expressed in pathological conditions that are ligands for NKG2D, an activating receptor found on cytotoxic lymphocytes. The recognition on target cells of NKG2D ligands leads to the activation of lysis and cytokine secretion by NK cells and T cells. Besides being expressed at the cell surface, MICA/B can be released as soluble proteins. Soluble NKG2D ligands downmodulate expression of the NKG2D receptor on lymphocytes, leading to a diminished cytotoxic response. Prior studies suggested that recruitment of MICA/B molecules to cholesterol-enriched microdomains was an important factor regulating the proteolytic release of these molecules. We now show that recruitment of MICA to these microdomains depends on palmitoylation of two cysteine residues that allow MICA molecules to reside in the membrane in the same domains as caveolin-1. Compared with WT molecules, nonpalmitoylated mutant MICA molecules were shed to the supernatant with low efficiency; however, both WT and mutant MICA were able to trigger NK cell cytotoxicity. These data suggest that the presence of NKG2D ligands at the plasma membrane is sufficient to activate cytotoxicity and reflect the need of different ligands to exploit different cellular pathways to reach the cell surface upon different stress situations.     

Evidence of NK cell dysfunction in SIV-infected rhesus monkeys: impairment of cytokine secretion and NKG2C/C2 expression

Defects in the adaptive immune response have been extensively characterized in human immunodeficiency virus type-1 (HIV-1)-infected individuals; however, much less is known about the function of natural killer (NK) cells during the course of HIV-1 infection. In the present study, we demonstrate that the NK cells from simian immunodeficiency virus (SIV)-infected rhesus monkeys are significantly impaired in their ability to secrete IFN-gamma, TNF-alpha, and IL-2, while NK cell function in SIV-infected long-term non-progressor monkeys is similar to that of normal monkeys. These findings suggest that abnormal NK cell activity may contribute to the global immune dysfunction observed in HIV-1-infected individuals. NK cell function is modulated by several families of cell surface receptors, including the CD94/NKG2 family. We evaluated the messenger RNA levels of these inhibitory and activating NKG2 molecules in SIV-infected rhesus monkeys. These experiments demonstrate that the activating molecules NKG2C and NKG2C2 are significantly down-regulated in peripheral blood mononuclear cells of SIV-infected rhesus monkeys, suggesting that the dysregulation of these molecules may contribute to the abnormal NK cell function observed in the setting of infection.     

IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.     

Clonal analysis of NK cell development from bone marrow progenitors in vitro: orderly acquisition of receptor gene expression

In the mouse, two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2, have been identified on NK cells. Individual NK cells can express several Ly49 molecules as well as members of the CD94/NKG2 family. The expression of multiple receptors with different specificities for MHC class I is thus thought to generate NK cells with diverse recognition patterns. To delineate the mechanism by which NK cells begin to express different patterns of Ly49 and CD94/NKG2 molecules, we developed a clonal assay in which NK1.1(-), IL-2/ IL-15 receptor beta+ NK precursors generated by culture of multipotential Lin(-), c-kit+ progenitors in IL-7, stem cell factor and flt3 ligand are induced to differentiate into NK1.1+ , Ly49+ NK cells. Examination of the clonal populations thus generated revealed heterogeneity in the pattern of Ly49 and CD94/NKG2 gene expression. In addition, a distinct kinetic pattern of expression was observed. CD94, NKG2A, NKG2C and Ly49B were expressed first followed by Ly49G, then Ly49C and I and finally, Ly49A, D, E and F. The data suggest a stochastic but ordered acquisition of class I receptors on NK cells in which developing NK cells become capable of expressing distinct receptors at different times but show no absolute prerequisite to express the receptors that are acquired early in NK development for the expression of those that are acquired later.     

MHC class I chain-related molecules induced on monocytes by IFN-gamma promote NK cell activation

NKG2D receptor-ligand interaction triggers NK cell-mediated cytolysis and IFN-gamma secretion. IFN-gamma produced by NK cells has been found to promote the interaction between NK cells and monocytes; however, the underlying mechanism remains elusive. We demonstrate here that IFN-gamma exclusively induced or upregulated the expression of MHC class I chain-related (MIC) molecules, which are ligands of the NKG2D receptor, on the surface of human monocytes of the PBMC population. The IFN-gamma-induced MIC molecules on monocytes played an essential role in triggering the activation of NK cells because mAb-mediated masking of the MIC molecules and the inhibition of cell-to-cell contact using transwell inserts significantly abolished NK cell activation. Meanwhile, membrane-bound IL-15 (mIL-15) was concomitantly induced with MIC molecules on IFN-gamma-treated monocytes and played an essential role in protecting NK cells cocultured with monocytes from MIC-induced NKG2D down-modulation. Therefore, we conclude that the IFN-gamma-induced MIC molecules participated in monocyte/NK cell interaction and that this interaction also involved mIL-15.     

NKG2D ligands: key targets of the immune response

NKG2D is an activating receptor expressed by NK and T cells. NKG2D ligands show a restricted expression in normal tissues, but they are frequently overexpressed in cancer and infected cells. The binding of NKG2D to its ligands activates NK and T cells and promotes cytotoxic lysis of the cells expressing these molecules. The mechanisms involved in the expression of the ligands of NKG2D play a key role in the recognition of stressed cells by the immune system and represent a promising therapeutic target for improving the immune response against cancer or autoimmune disease. In this review, we analyse the recent advances in understanding the regulation of NKG2D ligand expression and their therapeutic implications.     

High expression of NKG2A/CD94 and low expression of granzyme B are associated with reduced cord blood NK cell activity

Human umbilical cord blood （CB） has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease （GVHD） and graft-versus-leukemia （GVL）. It was reported that the activity of CB NK cells was lower than that of adult peripheral blood （PB） NK cells. In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-γ, TNF-α and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.     

Two genes in the rat homologous to human NKG2

Two different lectin-like receptors for MHC class I molecules have so far been identified on natural killer (NK) cells, the Ly-49 homodimeric receptors in mice and the NKG2/CD94 heterodimeric receptors in humans. The recent identification of a rat CD94 orthologue implied that NK cell receptors equivalent to NKG2/CD94 also exist in rodents. Here we describe the cDNA cloning of two rat genes homologous to members of the human NKG2 multigene family. The deduced rat NKG2A protein contains a cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM), whereas the cytoplasmic tail of rat NKG2C lacks ITIM. The genes map to the rat NK gene complex and are selectively expressed by NK cells. The expression is strain dependent, with high expression in DA and low in PVG NK cells, correlating with the expression of rat CD94. Ly-49 genes have previously been identified in the rat, and the existence of rat NKG2 genes in addition to a CD94 orthologue suggests that NK cell populations utilize different C-type lectin receptors for MHC class I molecules in parallel.     

Tumor-derived soluble MICs impair CD3(+)CD56(+) NKT-like cell cytotoxicity in cancer patients

Upon ligation with its ligands, the activating receptor NKG2D stimulates or costimulates CD8(+) T cells or NK cells. The inducible gene MHC class I chain-related molecules (MICs), which belong to the NKG2D ligand family and usually initiate the process of lymphocyte activation, have been found to be broadly expressed on epithelial tumor cells. Sustained localized expression or release of soluble forms of MICs (sMICs) by tumor cells play key roles in tumor evasion via the impairment of T cell and NK cell functions. NKG2D is also expressed on the surface of CD3(+)CD56(+) NKT-like cells, which participate in tumor rejection via direct cytolysis. We speculated whether sMICs have the same impact on NKT-like cells. In this study, we demonstrated that in vitro killing by freshly isolated NKT-like cells was principally mediated by NKG2D, and the cytotoxic function of NKT-like cells isolated from cancer patients was obviously compromised. We found a significant correlation between elevated tumor-derived sMICs and down-modulation of NKG2D expression on NKT-like cell surfaces in human ovarian cancer and prostate cancer patients. We determined that elevated serum sMIC impairs the lytic activity via downregulation of the NKG2D receptor because incubation of NKT-like cells with sera obtained from cancer patients down-modulated surface NKG2D expression, whereas the addition of neutralizing anti-MIC mAbs restored surface NKG2D expression. We suggest that tumors shedding MICs may promote immune evasion by impairing NKT-like cell cytotoxicity.     

CMV drives clonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronic hepatitis patients

Natural killer (NK) cells are affected by infection with human cytomegalovirus (HCMV) manifested by increased expression of the HLA-E binding activating receptor NKG2C. We here show that HCMV seropositivity was associated with a profound expansion of NKG2C(+) CD56(dim) NK cells in patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Multi-color flow cytometry revealed that the expanded NKG2C(+) CD56(dim) NK cells displayed a highly differentiated phenotype, expressed high amounts of granzyme B and exhibited polyfunctional responses (CD107a, IFN-γ, and TNF-α) to stimulation with antibody-coated as well as HLA-E expressing target cells but not when stimulated with IL-12/IL-18. More importantly, NKG2C(+) CD56(dim) NK cells had a clonal expression pattern of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self-HLA class I molecules, with predominant usage of KIR2DL2/3. KIR engagement dampened NKG2C-mediated activation suggesting that such biased expression of self-specific KIRs may preserve self-tolerance and limit immune-pathology during viral infection. Together, these findings shed new light on how the human NK-cell compartment adjusts to HCMV infection resulting in clonal expansion and differentiation of educated and polyfunctional NK cells.     

Reciprocal age related change in natural killer cell receptors for MHC class I

Natural killer (NK) cells are essential for healthy aging. NK cell activation is controlled by MHC class I-specific CD94/NKG2 receptors and killer immunoglobulin-like receptors (KIR). To assess NK cytotoxic function in isolation from MHC receptor engagement, we measured the ability of purified NK cells to kill mouse P815 target cells in the presence of anti-CD16 mAb. CD16-mediated cytotoxicity did not change with age, indicating that NK activation and cytotoxic granule release remained functional. We then investigated MHC class I receptor expression on NK cells. There was an age related decrease in CD94 and NKG2A expression and a reciprocal age related increase in KIR expression. NKG2A expression also declined with age on CD56(+) T cells. CD94/NKG2A receptor function was proportional to expression, indicating that NK cell inhibitory signaling pathways were intact. NKG2A and KIR expression were complementary, suggesting that CD94/NKG2A function could substitute for inhibitory KIR function during polyclonal NK cell development in both young and elderly adults. The distinct roles of CD94/NKG2A and KIR receptors suggest that shifting MHC class I receptor expression patterns reflect age related changes in NK cell and CD56(+) T cell turnover and function in vivo.     

Human Natural Killer cell expression of ULBP2 is associated with a mature functional phenotype

NKG2D is an important activating receptor expressed on NK cells. Ligands (termed NKG2DL) for this receptor include ULBP1-6, MICA and MICB in humans; they are upregulated in stressed, cancerous or infected cells where they engage NKG2D to induce NK cell cytotoxicity and cytokine production.Expression of NKG2DL on effector cells has been described in mice and more recently in human cells. We confirm that NK cell lines and IL-2 stimulated primary human NK cells also express the NKG2DL, ULBP2. However, expression of ULBP2 was not a result of transfer from a non-NK cell to an NK cell and in contrast to recent reports we saw no evidence that ULBP2 expression targeted these NK cells for fratricide or for cytotoxicity by NKG2D-expressing, non-NK effector cells.ULBP2 expression was however linked to expression of mature CD57⁺ NK cells. In particular, expression of ULBP2 was strongest on those NK cells that had evidence of recent activation and proliferation. We suggest that ULBP2 could be used to identify recently activated “mature” NK cells. Defining this phenotype would be useful for understanding the ontogeny on human NK cells.     

The balancing act: inhibitory Ly49 regulate NKG2D-mediated NK cell functions

NK cells use NKG2D receptor to recognize 'induced-self'. In apparent violation of the 'missing-self' hypothesis, NK cells stimulated through NKG2D can lyse target cells despite normal expression levels of MHC class I molecules. Although, 'overriding' of the inhibitory by the activating signals had been postulated the precise role of inhibitory Ly49 receptors on NKG2D-mediated activation has only started emerging. We propose that NKG2D-mediated activation is a function of 'altering the balance' in the signaling strength between the activating NKG2D and inhibiting Ly49 receptors. Balance in the signaling strength depends on the expression levels of activating ligands on the target cells. Qualitative and quantitative variations of MHC class I molecules expressed on the target cells also plays a major role in determining this 'altered-balance'. Consequently, the nature of Ly49 receptors expressed on specific NK subsets determines the level of NKG2D-mediated NK cell activation. These observations provide a firm basis of 'altered-balance' in NK signaling and describe an active interplay between inhibitory Ly49 and activating NKG2D receptors.