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...

Engagement of the high affinity receptor for IgE (Fc⑀RI) on mast cells and basophils results in Fc⑀RI ␤ and ␥ subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon Fc⑀RI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of Fc⑀RI ␤ and ␥ chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of Fc⑀RI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated Fc⑀RI and activated Syk to the proteasome for degradation.The activation of protein-tyrosine kinases (PTKs) 1 is an essential event in the transduction of intracellular signals from immune receptors (IR), including the T and B cell antigen receptors (TCR and BCR, respectively), the high affinity receptor for IgE (Fc⑀RI), and the widely distributed receptors for IgG.These IRs contain multiple subunits: some, distinct for each receptor, are used for ligand binding, whereas others share conserved cytoplasmic motifs that are critical for the process of cell activation (immune receptor tyrosine-based activation motif, ITAM) (1-6). The IRs lack intrinsic kinase activity; however, within seconds of their engagement, PTKs are activated leading to phosphorylation of various substrates, including IR subunits (7-12). ITAM phosphorylation by the Src family PTKs provides docking sites for the tandem pair of Src homology 2 (SH2) domains of a second class of PTKs belonging to the Syk family (3-6). This family includes only two members: Syk, which is present in most hematopoietic cells and ZAP-70, which is exclusively expressed in T and NK cells. As documented by several studies, the expression of Syk and ZAP-70 is essential for lymphocyte development and signal transduction via IRs (13-16). The association of phosphorylated ITAMs with SH2 domains of Syk family PTKs leads to the activation of Syk and ZAP-70 mainly by autophosphorylation (17), thus allowing the propagation of IR signaling.We and others have demonstrated that Syk and ZAP-70 as well as IR subunits are subjected to an additional covalent modification following IR engagement in that they become modified by ubiquitin (Ub) (18 -22). Moreover, we have also provided evidence suggesting a direct correlation between IRinduced Syk and ZAP-70 ubiquitination and degradation (21).Ubiquitination, which consists in the covalen...

IntroductionNatural killer (NK) cells are a small lymphocyte subpopulation resident in peripheral blood and in some lymphoid and nonlymphoid organs, capable of rapidly migrating to peripheral sites in response to infections or to neoplastic transformation. 1 They represent an important component of innate immunity by exerting both a constitutive cytotoxic activity, directed against infected or transformed cells and immature hematopoietic precursors, and the antibody-dependent cytolytic activity (ADCC), thanks to the presence of CD16, the low-affinity Fc receptor for immunoglobulin G (IgG) (Fc␥RIII), on the vast majority of them. 2,3 Besides their cytolytic function, NK cells also rapidly secrete a variety of cytokines and chemokines in response to stimulation, by means of which they amplify the recruitment and activation of other effector cell populations. 1,4,5 NK cell activation is regulated by the fine balance of positive and negative signaling pathways initiated by multiple receptors displaying either activating, costimulatory, or inhibitory activity, whose expression and/or functional capability can be modulated during NK cell activation/differentiation. 6-8 NK cell functions are rapidly augmented by a vast array of both innate and adaptive cytokines and a number of other biologic response modifiers. 1,5 NK cells play a crucial role in the natural resistance against viral infections, both by exerting an effector function in the early containment of the infection and by participating in the instructive phase of the adaptive immune response. 9-11 NK cells rely on a variety of cues to precociously sense and respond to the presence of viral infections, such as the up-regulation of viral or virus-induced peptidic ligands for different activating receptors, the virusinduced down-modulation of major histocompatibility complex (MHC) class I ligands for inhibitory receptors, and the presence of proinflammatory cytokines, such as type I interferon (IFN), which promptly up-regulate NK cytolytic activity and cytokine production. 5,[9][10][11][12][13] In particular, the synthetic copolymer polyinosinicpolycytidylic acid (poly I:C), which mimics double-stranded (ds) RNA viral products, has been previously shown to rapidly promote NK cell responses in vivo through its capacity to induce type I IFN production. 1,[9][10][11]14 Innate immunity effector cells recognize the presence of different pathogens mainly through a recently identified family of 10 genetically invariant receptors, the Toll-like receptors (TLR1-10), capable of recognizing distinct molecular components of microbes. 15,16 In particular, TLR3 is the specific receptor for dsRNA, a common intermediate in the reproductive cycle of many viruses; indeed, responsiveness to viral dsRNA or poly I:C is severely compromised in TLR3 knock-out mice. 17 TLR3 expression in the human hematopoietic compartment has been initially reported to be restricted mainly to myeloid dendritic cells, where it induces cell maturation, and the production of inflammatory and antimicrobial...

The MAP kinase (MAPK) p38 plays a key role in regulating inflammatory responses. Here, we demonstrate that beta1 integrin ligation on human NK cells results in the activation of the p38 MAPK signaling pathway, which is required for integrin-triggered IL-8 production. In addition, we identified some of the upstream events accompanying the beta1 integrin-mediated p38 MAPK activation, namely, the activation of the Rac guanine nucleotide exchange factor (GEF) p95 Vav, the small G protein Rac1, and the cytoplasmic kinases Pak1 and MKK3. Finally, we provide direct evidence that p95 Vav and Rac control the activation of p38 MAPK triggered by beta1 integrins.

Herein, we provide the first evidence on the capsaicin (CPS) receptor vanilloid receptor type-1 (VR1) by rat thymocytes, and its involvement in CPS-induced apoptosis. VR1 mRNA was identified by quantitative RT-PCR in CD5 þ thymocytes. By immunofluorescence and flow cytometry, we found that a substantial portion of CD5 þ thymocytes, namely CD4 þ and double negative (DN) cell subsets, express VR1 that was present on plasma membrane on discrete spots. By Western blot, VR1 protein was identified as a single band of 95 kDa. We also described that CPS could trigger two distinct pathways of thymocyte death, namely apoptosis and necrosis depending on the dose of CPS exposure. CPS-induced apoptosis involved intracellular free calcium (Ca 2 þ ) influx, phosphatidylserine exposure, mitochondrial permeability transmembrane pore (PTP) opening and mitochondrial transmembrane potential (DW m ) dissipation leading to cytochrome c release, activation of caspase-9 and -3 and oligonucleosomal DNA fragmentation. VR1 was functionally implicated in these events as they were completely abrogated by the VR1 antagonist, capsazepine (CPZ). Finally, we demonstrated that VR1 expression on distinct thymocytes was associated with the selective ability of CPS to trigger DNA fragmentation in VR1 þ CD4 þ and DN thymocytes. Overall, our results suggest that the expression of VR1 on thymocytes may function as a sensor of harmful stimuli present in the thymic environment.

SummaryRecent evidence indicates that integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that ␤ 1 integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogenactivated protein kinase pathways. Formation of Shc-growth factor receptor-bound protein 2 (Grb2) and Shc-proline-rich tyrosine kinase 2-Grb2 complexes are the receptor-proximal events accompanying the ␤ 1 integrin-mediated Ras activation. In addition, we demonstrate that ligation of ␤ 1 integrins results in the stimulation of interferon ␥ (IFN-␥ ) production, which is under the control of extracellular signal-regulated kinase 2 activation. Overall, our data indicate that ␤ 1 integrins, by delivering signals capable of triggering IFN-␥ production, may function as NK-activating receptors.

Fas (APO-1/CD95) and its ligand (FasL/CD95L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T lymphocytes, the Fas/FasL system regulates activation-induced cell death, a fundamental mechanism for negative selection of immature T cells in the thymus and for maintenance of peripheral tolerance. Aberrant expression of Fas and FasL has also been implicated in diseases in which the lymphocyte homeostasis is compromised, and several studies have described the pathogenic functions of Fas and FasL in vivo, particularly in the induction/regulation of organ-specific autoimmune diseases. The 1,25(OH)2D3 is a secosteroid hormone that activates the nuclear receptor vitamin D3 receptor (VDR), whose immunosuppressive activities have been well studied in different models of autoimmune disease and in experimental organ transplantation. We and others have recently described the molecular mechanisms responsible for the negative regulation of the IFN-γ and IL-12 genes by 1,25(OH)2D3 in activated T lymphocytes and macrophages/dendritic cells. In this study, we describe the effect of 1,25(OH)2D3 on the activation of the fasL gene in T lymphocytes. We show that 1,25(OH)2D3 inhibits activation-induced cell death, fasL mRNA expression, and that 1,25(OH)2D3-activated VDR represses fasL promoter activity by a mechanism dependent on the presence of a functional VDR DNA-binding domain and ligand-dependent transcriptional activation domain (AF-2). Moreover, we identified a minimal region of the promoter containing the transcription start site and a noncanonical c-Myc-binding element, which mediates this repression. These results place FasL as a novel target for the immunoregulatory activities of 1,25(OH)2D3, and confirm the interest for a possible pharmacological use of this molecule and its derivatives.

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcεRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcεRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcεRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.