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.

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...

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches.