Selective induction of expression of a ligand for the NKG2D receptor by proteasome inhibitors.

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. The expression of NKG2D ligands is induced by cellular "stress." However, the regulation of expression of these molecules is not well understood. Here, we show that cells treated with proteasome inhibitors can become more susceptible to cytotoxicity mediated by natural killer cells because of the induction of expression of ligands for NKG2D, specifically ULBP2, but not down-regulation of MHC class I. Treatment with proteasome inhibitors led to up-regulation of ULBP2 expression in multiple, but not all, cell lines tested. This increase in expression of ULBP2 at the cell surface correlated with induction of transcription of the ULBP2 gene and synthesis of ULBP2 protein. In contrast, treatment with inhibitors of histone deacetylases led to increased levels of mRNA and protein, for both ULBP2 and MHC class I-related chain A/B molecules. Thus, different types of stress can trigger up-regulated expression of different sets of NKG2D ligands. Proteasome inhibitors are proving to be of significant value in the treatment of hematologic malignancies and these observations may help to better understand the biology of therapy with these compounds.

Expression of ICP0 is sufficient to trigger natural killer cell recognition of herpes simplex virus-infected cells by natural cytotoxicity receptors.

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.

Transfer of NKG2D and MICB at the cytotoxic NK cell immune synapse correlates with a reduction in NK cell cytotoxic function.

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 receptor/ligand 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.

Recognition of vaccinia virus-infected cells by human natural killer cells depends on natural cytotoxicity receptors.

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.

CD161 (human NKR-P1A) signaling in NK cells involves the activation of acid sphingomyelinase.

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.