IntroductionNatural killer (NK) cells are present in the bloodstream, spleen, bone marrow, and in nonlymphoid organs and represent one of the main effectors of the immunosurveillance against tumors, by exerting 2 major effector functions, cytolysis of target cells and production of cytokines and chemokines. 1,2 The activity of NK cells depends on the interplay between inhibitory receptors for major histocompatibility complex (MHC) class I molecules and activating receptors, which operate in concert to induce the elimination of tumor cells. 3,4 Among the activating receptors particularly relevant for tumor cell recognition and killing is NKG2D, the receptor for the MHC I-related molecules MICA/B, and ULBPs (UL16-binding proteins), belonging to the C-type lectin-like receptor family. 3,[5][6][7] The NKG2D activating receptor is expressed not only on NK cells, but also on ␥␦ T cells, CD8 ϩ T cells, and a subset of CD4 ϩ T cells. The expression of NKG2D ligands is largely confined to virus-infected, tumor, and stressed cells. 7 To promote escape of tumors from NKG2D-mediated immunosurveillance, NKG2D ligands undergo proteolytic shedding. Soluble NKG2D ligands (NKG2DLs) have been shown to down-regulate the cell surface NKG2D expression on NK cells, resulting in impaired killing of tumor cells. 8,9 Another activating receptor involved in NK-cell-mediated tumor cell killing is DNAX accessory molecule-1 (DNAM-1), a transmembrane glycoprotein constitutively expressed on the majority of T cells, NK cells, and macrophages; its ligands are Nectin-2 (Nec-2, CD112) and the poliovirus receptor (PVR, CD155), which belong to the nectin/nectin-like family. 10-12 DNAM-1 ligands have been initially described as adhesion molecules mainly regulating trans-endothelial migration 13 and only recently they have been found on a variety of tumor cells. 12,14 Both DNAM-1 and NKG2D cooperate in the induction of NK-cell killing against tumor cells of different histotypes, including those of hematopoietic origin. 14,15 Similarly to the NKG2D ligands, soluble isoforms of PVR have also been found in human serum and in the culture supernatant of tumor cell lines, and their role in tumor immunoevasion has been considered. 12 It has recently been demonstrated that agents that produce a genotoxic stress or DNA-replication inhibitors up-regulate NKG2D ligand expression through the activation of ATM (ataxia telangiectasia mutated) and ATR (ATM-and Rad3-related) protein kinases on human fibroblasts and on mouse tumor cell lines, and enhance their destruction by NK cells. 16,17 Increased ligand expression is regulated by the activation of the DNA damage response (DDR). This is a cellular program devoted to the maintenance of genome integrity through the inhibition of cell cycle and activation of the DNA repair systems, or by the induction of apoptosis or a protracted cell-cycle arrest known as cellular senescence. 17,18 No information is so far available on the regulation of DNAM-1 ligand expression through the DDR pathway.Submitted August 11, 2008; acc...

Recent evidence indicates that natural killer (NK) cells can negatively regulateT-cell responses, but the mechanisms behind this phenomenon as a consequence of NK-T-cell interactions are poorly understood. We studied the interaction between the NKG2D receptor and its ligands (NKG2DLs), and asked whether T cells expressed NKG2DLs in response to superantigen, alloantigen, or a specific antigenic peptide, and if this rendered them susceptible to NK lysis. As evaluated by FACS, the major histocompatibility complex (MHC) class I chain-related protein A (MICA) was the ligand expressed earlier on both CD4 ؉ and CD8 ؉ T cells in 90% of the donors tested, while UL16-binding protein-1 (ULBP)1, ULBP2, and ULBP3 were induced at later times in 55%-75% of the donors. By carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, we observed that NKG2DLs were expressed mainly on T cells that had gone through at least one division. Real-time reverse-transcription polymerase chain reaction confirmed the expression of all NKG2DLs, except ULBP4. In addition, T-cell activation stimulated phosphorylation of ataxia-telangiectasia mutated (ATM), a kinase required for IntroductionThe negative regulation of adaptive immunity is relevant to maintain lymphocyte homeostasis and to prevent inappropriate T-cell activation, which can ultimately result in autoimmune or lymphoproliferative diseases. Although it is well-documented that natural killer (NK) cells are important effectors of innate immunity, and their role against virally infected and tumor cells has been studied over the years, 1,2 much attention has also been focused on their ability to promote adaptive immunity by secretion of immunomodulatory cytokines and chemokines. [3][4][5] More recently, however, a previously unappreciated negative immunoregulatory role has emerged. In fact, NK cells can downregulate T-cell-mediated immune responses by their killing capacity and by secreting inhibitory cytokines such as interleukin (IL)-10 and transforming growth factor beta (TGF)-␤. [6][7][8] In vitro experiments have shown that activated human NK cells can kill dendritic cells (DCs), 9,10 probably contributing to inhibition of T-cell activation in inflamed tissues. During murine cytomegalovirus (MCMV) infection, the presence of NK cells limits CD4 ϩ and CD8 ϩ IFN-␥ production and proliferation. 11 In addition, in a major histocompatibility complex (MHC) class I-positive host grafted with MHC class I-negative bone marrow, development of MHC class I-deficient thymocytes is delayed as a result of NK-cell cytotoxicity. 12 Furthermore, studies in humans and animal models have demonstrated that NK cells can prevent the initiation and progression of autoimmune diseases: 13 lack of NK cells correlates with severe forms of experimental autoimmune encephalomyelitis and CD4 ϩ T-cell-mediated colitis, suggesting that NK cells can actively inhibit proliferation and cytokine production by autoreactive T cells. 14,15 In accordance, reduced NK-cell numbers and compromised NK-cell functions are foun...

It is important to understand which molecules are relevant for linking innate and adaptive immune cells. In this study, we show that OX40 ligand is selectively induced on IL-2, IL-12, or IL-15-activated human NK cells following stimulation through NKG2D, the low affinity receptor for IgG (CD16) or killer cell Ig-like receptor 2DS2. CD16-activated NK cells costimulate TCR-induced proliferation, and IFN-γ produced by autologous CD4+ T cells and this process is dependent upon expression of OX40 ligand and B7 by the activated NK cells. These findings suggest a novel and unexpected link between the natural and specific immune responses, providing direct evidence for cross-talk between human CD4+ T cells and NK receptor-activated NK cells.

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (−/−) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8−/− mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.

Natural killer (NK) cells are a major component of the host innate immune defence against various pathogens. Several viruses, including Human immunodeficiency virus 1 (HIV-1), have developed strategies to evade the NK-cell response. This study was designed to evaluate whether HIV-1 could interfere with the expression of NK cell-activating ligands, specifically the human leukocyte antigen (HLA)-I-like MICA and ULBP molecules that bind NKG2D, an activating receptor expressed by all NK cells. Results show that the HIV-1 Nef protein downmodulates cell-surface expression of MICA, ULBP1 and ULBP2, with a stronger effect on the latter molecule. The activity on MICA and ULBP2 is well conserved in Nef protein variants derived from HIV-1-infected patients. In HIV-1-infected cells, cell-surface expression of NKG2D ligands increased to a higher extent with a Nef-deficient virus compared with wild-type virus. Mutational analysis of Nef showed that NKG2D ligand downmodulation has structural requirements that differ from those of other reported Nef activities, including HLA-I downmodulation. Finally, data demonstrate that Nef expression has functional consequences on NK-cell recognition, causing a decreased susceptibility to NK cell-mediated lysis. These findings provide a novel insight into the mechanisms evolved by HIV-1 to escape from the NK-cell response.

In vivo liposomes, like other types of nanoparticles, acquire a totally new ‘biological identity’ due to the formation of a biomolecular coating known as the protein corona that depends on and modifies the liposomes’ synthetic identity. The liposome–protein corona is a dynamic interface that regulates the interaction of liposomes with the physiological environment. Here we show that the biological identity of liposomes is clearly linked to their sequestration from peripheral blood mononuclear cells (PBMCs) of healthy donors that ultimately leads to removal from the bloodstream. Pre-coating liposomes with an artificial corona made of human plasma proteins drastically reduces capture by circulating leukocytes in whole blood and may be an effective strategy to enable prolonged circulation in vivo. We conclude with a critical assessment of the key concepts of liposome technology that need to be reviewed for its definitive clinical translation.

The activating receptor NKG2D is peculiar in its capability to bind to numerous and highly diversified MHC class I-like self-molecules. These ligands are poorly expressed on normal cells but can be induced on damaged, transformed or infected cells, with the final NKG2D ligand expression resulting from multiple levels of regulation. Although redundant molecular mechanisms can converge in the regulation of all NKG2D ligands, different stimuli can induce specific cellular responses, leading to the expression of one or few ligands. A large body of evidence demonstrates that NK cell activation can be triggered by different NKG2D ligands, often expressed on the same cell, suggesting a functional redundancy of these molecules. However, since a number of evasion mechanisms can reduce membrane expression of these molecules both on virus-infected and tumor cells, the co-expression of different ligands and/or the presence of allelic forms of the same ligand guarantee NKG2D activation in various stressful conditions and cell contexts. Noteworthy, NKG2D ligands can differ in their ability to down-modulate NKG2D membrane expression in human NK cells supporting the idea that NKG2D transduces different signals upon binding various ligands. Moreover, whether proteolytically shed and exosome-associated soluble NKG2D ligands share with their membrane-bound counterparts the same ability to induce NKG2D-mediated signaling is still a matter of debate. Here, we will review recent studies on the NKG2D/NKG2D ligand biology to summarize and discuss the redundancy and/or diversity in ligand expression, regulation, and receptor specificity.

IntroductionIn addition to their role in providing antitumor and antiviral immunity, 1 natural killer (NK) cells are also able to regulate the T-cell arm of the adaptive immune response by secreting different cytokines and chemokines. 2 Moreover, several studies have provided evidence of cognate cell-cell interactions between NK cells and various leukocyte types, including dendritic cells (DCs) and B and T lymphocytes. [3][4][5] Although NK cells have been thought to mainly promote adaptive immune responses, recent in vivo studies suggest that they can also restrain T cell-mediated immune responses. Therefore, the depletion of NK cells results in enhanced T-cell proliferation and effector functions during murine cytomegalovirus infection 6 and in an antitumor response against lymphoma cells. 7 Conversely, several studies have indicated that the depletion of NK cells is associated with increased severity of autoimmune diseases. In fact, NK cell-depleted mice develop a more severe form of experimental autoimmune encephalomyelitis, 8 and NK-cell-mediated downregulation of autoreactive cytotoxic T lymphocytes has been shown to have a protective role in type 1 diabetes. 9 These findings suggest that NK cells may be crucial for terminating T cell-mediated responses and for preventing inappropriate T-cell activation and effector functions leading to the development of autoimmune diseases.NK cell-mediated attenuation of T-cell responses can involve several mechanisms, including the production of inhibitory cytokines (eg, TGF-␤ and IL-10) 10,11 and killing of DCs and/or activated T cells. 4,12,13 In regard to the NK cell-mediated killing of T cells, IL-2-activated mouse and human NK cells recognize and lyse T-cell blasts in a perforin-dependent manner through the activating receptor NKG2D. 4,12 Interestingly, the results of our previous study indicated that Ag stimulation of human T cells was sufficient to induce the surface expression of the NKG2D ligands (NKG2DLs) MHC class I-related chain A (MICA), MICB, and UL16-binding proteins 1-3 (ULBP1-3). 4,14 To date, little is known about the existence of additional receptor-ligand interactions that might contribute to the NK cell-mediated recognition of T lymphocytes.DNAX accessory molecule-1 (DNAM-1) is an activating receptor belonging to the Ig superfamily that is constitutively expressed by most NK cells, T cells, macrophages, and DCs. 15,16 DNAM-1 interacts with lymphocyte function-associated antigen 1 (LFA-1), and this association is required for its functional activity on both NK and cytotoxic T cells. 17 Ligands for DNAM-1 (DNAM1Ls) include Nectin-2 and poliovirus receptor (PVR), which belong to the Nectin/Nectin-like family of adhesion molecules. 18 DNAM1Ls are often expressed by tumor cells and can activate or enhance tumor cell lysis in vitro. 15,18 Recent studies have reported that they can also be expressed by monocytes, DCs, and phytohemagglutinin (PHA)-stimulated CD4 ϩ T lymphocytes. 19,20 Little information is available about the molecular mechanisms regulating...