No abstract

Although transfer of membrane proteins has been shown to occur during immune cell interactions, the functional significance of this process is not well understood. Here we describe the intercellular transfer of NKG2D and MHC class I chain-related molecule (MIC) B proteins at the cytotoxic natural killer cell immune synapse (cNK-IS). MICB expressed on the 721.221 cell line induced clustering of NKG2D at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing F-actin. Moreover, natural killer (NK) cell membrane-connective structures formed during cytotoxic interactions contained F-actin, perforin, and NKG2D. NKG2D transfer depended on binding to MICB and was specific because transfer of other molecules not involved in NK-IS formation was not observed. Transfer of MICB to NK cells also was noted, suggesting a bidirectional exchange of receptor͞ligand pairs at cNK-IS. Experiments designed to test the functional significance of these observations revealed that brief interactions between NK cells and MICB expressing target cells led to a reduction in NKG2D-dependent NK cytotoxicity. These data demonstrate interchange of an activating receptor and its ligand at the cNK-IS and document a correlation between synapse organization, intercellular protein transfer, and compromised NK cell function after interaction with a susceptible target cell.cytotoxicity ͉ protein transfer ͉ innate immunity ͉ NKG2D N KG2D, a member of the killer cell lectin-like receptor family, is one of the best characterized natural killer (NK) cellactivating receptors, but it also is expressed on TCR␥␦ ϩ and CD8 ϩ TCR␣␤ ϩ T cells, where it can act as a costimulatory molecule for T cell activation. NKG2D associates with DAP10, a transmembrane adaptor molecule containing a YINM motif that binds and activates via phosphatidylinositol 3-kinase and Grb2 (1, 2). In humans, NKG2D-immune activation can be triggered by interaction with its ligands: the polymorphic MHC class I chain-related molecule (MIC) A and MICB and the UL16-binding proteins (3). NKG2D ligands are absent, or expressed only at low levels, on normal cells, but their expression is often enhanced͞induced on tumors and cells infected by various pathogens. Dysregulation of NKG2D ligand expression with inappropriate activation of NKG2D ϩ T cells also has been reported recently to be a feature of various autoimmune diseases (4).Receptor-ligand interactions between cells do not occur randomly, rather the groups of interacting molecules are found in ordered structures, named immune synapses (IS), providing a platform for intercellular communication among cells of the immune system (5). Upon NK cell-target cell interaction, highly organized supramolecular clusters are formed at the contact area of both cells, termed the NK cell IS (NK-IS) (6, 7). When NK cells interact with susceptible target cells, the redistribution of signaling molecules at the central (c) SMAC (supramolecular activation cluster, SMAC), as well as LFA-1 and talin at th...

A glycoprotein H (gH)-deleted herpes simplex virus type 2 (HSV-2) was evaluated as a vaccine for the prevention of HSV-induced disease. This virus, which we term a DISC (disabled infectious single cycle) virus, can only complete one replication cycle in normal cells and should thus be safe yet still able to stimulate broad humoral and cell-mediated antiviral immune responses. A gH-deleted HSV-2 virus that has been tested as a vaccine in the guinea pig model of recurrent HSV-2 infection was constructed. Animals vaccinated with DISC HSV-2 showed complete protection against primary HSV-2-induced disease, even when challenged 6 months after vaccination. In addition, the animals were almost completely protected against recurrent disease. Even at low vaccination doses, there was a high degree of protection against primary disease. A reduction in recurrent disease symptoms was also observed following therapeutic vaccination of animals already infected with wild type HSV-2.

NK and NKT cells play a major role in both innate immunity and in influencing the development of adaptive immune responses. CD161 (human NKR-P1A), a protein encoded in the NK gene complex, is a major phenotypic marker of both these cell types and is thought to be involved in the regulation of NK and NKT cell function. However, the mechanisms of action and signaling pathways of CD161 are poorly understood. To identify molecules able to interact with the cytoplasmic tail of human CD161 (NKR-P1A), we have conducted a yeast two-hybrid screen and identified acid sphingomyelinase as a novel intracellular signaling pathway linked to CD161. mAb-mediated cross-linking of CD161, in both transfectants and primary human NK cells, triggers the activation of acid, but not neutral sphingomyelinase. The sphingomyelinases represent the catabolic pathway for N-acyl-sphingosine (ceramide) generation, an emerging second messenger with key roles in the induction of apoptosis, proliferation, and differentiation. These data therefore define a novel signal transduction pathway for the CD161 (NKR-P1A) receptor and provide fresh insights into NK and NKT cell biology.

Natural Killer (NK) cells are important in the immune response to a number of viruses; however, the mechanisms used by NK cells to discriminate between healthy and virus-infected cells are only beginning to be understood. Infection with vaccinia virus provokes a marked increase in the susceptibility of target cells to lysis by NK cells, and we show that recognition of the changes in the target cell induced by vaccinia virus infection depends on the natural cytotoxicity receptors NKp30, NKp44, and NKp46. Vaccinia virus infection does not induce expression of ligands for the activating NKG2D receptor, nor does downregulation of major histocompatibility complex class I molecules appear to be of critical importance for altered target cell susceptibility to NK cell lysis. The increased susceptibility to lysis by NK cells triggered upon poxvirus infection depends on a viral gene, or genes, transcribed early in the viral life cycle and present in multiple distinct orthopoxviruses. The more general implications of these data for the processes of innate immune recognition are discussed.Natural killer cells are important components of the immune responses to many viruses, yet with the exception of NK recognition of cytomegalovirus-infected cells (17,25), the mechanisms used by NK cells to distinguish between healthy and virus-infected cells are not well understood. The behavior of an NK cell confronted by a target cell is thought to depend on a delicate balance of signals transduced by activating and inhibitory receptors (36). Major histocompatibility complex (MHC) class I molecules are key ligands transmitting inhibitory signals to NK cells, while NKG2D and the natural cytotoxcity receptors (NCR), NKp46, NKp44, and NKp30, seem to be the major receptors driving activation of NK cells. The importance of the different activating receptors for NK cell recognition of different types of target cells varies, and it is clear that the basis of this phenomenon is differential ligand expression by target cells (43,58). These observations are obviously important but derive mainly from studies using tumor cell targets (36). It is thus important to explore the mechanisms controlling NK cell recognition of virus-infected cells, since it seems likely that the mechanisms used by NK cells for target cell recognition have evolved in response to the pressure of pathogens, such as viruses, rather than in response to cancer. The few NK cell-deficient humans identified have suffered from recurrent virus infections (8), while in various murine systems, loci associated with resistance to infection with cytomegalovirus, herpes simplex virus, and ectromelia have been mapped to the NK gene complex on chromosome 6 (12, 31, 44). Thus, we decided to explore the mechanisms used by NK cells to discriminate between uninfected targets and cells infected with poxviruses.Vaccinia virus infection elicits NK activation, proliferation, and accumulation at the site of infection (14,18,21,40), and NK cells have long been proposed to mediate an important element ...

The interaction of the activating receptor NKG2D with its ligands plays an important role in immunosurveillance of tumors and infectious pathogens, but dysregulation of this system may lead to autoimmunity.

Natural killer (NK) cells are an important component of the immune response to a number of viruses; however, the molecular basis of how NK cells discriminate between healthy and virus-infected cells is largely unknown. Here, we show that expression of the immediate-early gene product ICP0 is sufficient to produce an increased susceptibility to NK lysis of herpes simplex virus (HSV)-infected cells. This effect does not depend on down-regulation of major histocompatibility complex class I molecules or on the induction of expression of ligands for the activating NKG2D receptor. Detection by NK cells of the changes in the target cell induced by HSV ICP0 gene expression depends on the natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46. To our knowledge, this is the first identification of a viral gene that triggers the up-regulation of cellular ligands for the NCR; moreover, these observations highlight the importance of the NCR for immunosurveillance of viral infection by NK cells.

The neutralizing and enhancing activities of respiratory syncytial virus (RSV)-specific antibodies were examined. These two biological activities were measured for a panel of six monoclonal antibodies (MAbs) specific to the RSV surface F and G glycoproteins. Four MAbs specific for the F protein possessed both neutralizing and enhancing activities. One MAb (11-2-D2), specific to the G protein, enhanced RSV infection of U937 cells, a human macrophage cell line, but did not neutralize virus infectivity. One MAb (11-3-A3), specific to the F protein, efficiently neutralized virus infectivity but did not enhance RSV infection of U937 cells. MAb 11-3-A3 neutralized representative strains of the two antigenic subtypes of RSV. Assays performed with mixtures of MAbs showed that high concentrations of MAb 11-3-A3 masked the enhancing activity of MAb 11-2-D2. The assay of mixtures of two MAbs possessing only enhancing activities demonstrated that this response was synergistic. The role of neutralizing and enhancing antibodies in determining the outcome of RSV infection was examined for infants from whom cord blood serum samples were collected at birth. There was no significant difference in the magnitude of the serum-enhancing activities between infants who were hospitalized with RSV infections and a group of age-and sex-matched control infants with no reported respiratory illness requiring hospitalization. However, the results indicated a possible correlation between RSV infection of the infants and the occurrence of in vitro antibody-dependent enhancement of the cord blood sera at a serum dilution of 10 ؊2. A significant inverse correlation was found between the plaque-neutralizing and enhancing activities of the cord blood sera from infants, irrespective of subsequent RSV infection. These data are discussed in relation to the possible contribution of antibody-dependent enhancement to the normal course of RSV pathology in vivo.