CD38, an ectoenzyme and a signaling receptor, is a novel marker of human mature monocyte-derived dendritic cells (MDDCs). The working hypothesis is that CD38 is not only a marker but also contributes to functions specifically gained by MDDCs with maturation. This was tested by assessing the role(s) of CD38 after signaling with agonistic anti-CD38 monoclonal antibodies or by blocking the interactions taking place between CD38 and CD31, its counterreceptor. IntroductionCD38 is a pleiotropic cell surface molecule acting as an ectoenzyme and a receptor. The enzymatic activity ends in the synthesis of Ca 2ϩ -mobilizing metabolites (ie, ADP-ribose [ADPR], nicotinic acid adenine dinucleotide phosphate [NAADP], and cyclic ADP-ribose [cADPR]) involved in the regulation of calcium-dependent calcium release. 1,2 CD38 has a unique pattern of surface expression among cells of the human immune system, being present on lymphoid and myeloid progenitors, lost during differentiation, and re-expressed at high density in activated T, B, and natural killer (NK) cells. 3,4 The molecule shows a broad distribution in different tissues. 5 CD38 ligation in immune cells delivers activation signals and induces cytokine production and secretion by T, 6 B, 7 and NK cells 8 and monocytes. 9 CD38 also regulates cell viability by preventing human germinal center B cells from undergoing apoptosis 10 and contributing to increased survival of B-cell chronic lymphocytic leukemia (B-CLL) cells. 11 This panoply of different functional roles may be explained considering some nonconventional features of CD38 as a receptor. The intrinsic ineptitude of CD38 to transduce signals is overcome by working in physical and functional associations with specialized signaling molecules, such as T-cell receptor on T cells, 12-14 B-cell receptor on B cells, 15,16 and CD16 on NK cells. 17 CD38 can sustain adhesion and rolling of CD38 ϩ lymphocytes on endothelial cells through interaction with CD31 (identified as a specific counterreceptor 18 ), suggesting its possible role in lymphocyte trafficking. 19 All the signals mediated by monoclonal antibody (mAb) ligation of CD38 can be reproduced by its interaction with CD31.In murine models, CD38 is involved in chemotaxis and transendothelial migration of both polymorphonuclear leukocytes (PMNs) and dendritic cells (DCs) and this function requires its enzymatic activities. 20,21 Consequently, CD38 knockout mice have impaired capacity to respond to infections. 20 The evaluation of the expression of the molecule also has applications in clinical diagnosis, such as in AIDS (where CD38 is one of the earliest indicators of infection 22 ) and B-CLL (where the expression is generally associated with poor prognosis 15 ). Autoantibodies specific for CD38 are found in diabetes and thyroid disorders. [23][24][25][26] The agonistic properties of these autoantibodies envisage pathogenic role(s) in these diseases.We recently reported a pulsatile pattern of surface CD38 expression in human monocyte-derived dendritic cells (MDDCs). 27 ...

Natural killer (NK) immune cells mediate antibody-dependent cellular cytotoxicity (ADCC) by aggregating FcγRIIIA/CD16, contributing significantly to the therapeutic effect of CD20 monoclonal antibodies (mAb). In this study, we show that CD16 ligation on primary human NK cells by the anti-CD20 mAb rituximab or ofatumumab stably impairs the spontaneous cytotoxic response attributable to cross-tolerance of several unrelated NK-activating receptors (including NKG2D, DNAM-1, NKp46, and 2B4). Similar effects were obtained from NK cells isolated from patients with chronic lymphocytic leukemia in an autologous setting. NK cells rendered hyporesponsive in this manner were deficient in the ability of these cross-tolerized receptors to phosphorylate effector signaling molecules critical for NK cytotoxicity, including SLP-76, PLCγ2, and Vav1. These effects were associated with long-lasting recruitment of the tyrosine phosphatase SHP-1 to the CD16 receptor complex. Notably, pharmacologic inhibition of SHP-1 with sodium stibogluconate counteracted CD20 mAb-induced NK hyporesponsiveness, unveiling an unrecognized role for CD16 as a bifunctional receptor capable of engendering long-lasting NK cell inhibitory signals. Our work defines a novel mechanism of immune exhaustion induced by CD20 mAb in human NK cells, with potentially negative implications in CD20 mAb-treated patients where NK cells are partly responsible for clinical efficacy.

Cytotoxic lymphocytes share the presence of the activating receptor NK receptor group 2, member D (NKG2D) and the signaling-competent adaptor DNAX-activating protein 10 (DAP10), which together play an important role in antitumor immune surveillance. Ligand stimulation induces the internalization of NKG2D-DAP10 complexes and their delivery to lysosomes for degradation. In experiments with human NK cells and cell lines, we found that the ligand-induced endocytosis of NKG2D-DAP10 depended on the ubiquitylation of DAP10, which was also required for degradation of the internalized complexes. Moreover, through combined biochemical and microscopic analyses, we showed that ubiquitin-dependent receptor endocytosis was required for the activation of extracellular signal-regulated kinase (ERK) and NK cell functions, such as the secretion of cytotoxic granules and the inflammatory cytokine interferon-γ. These results suggest that NKG2D-DAP10 endocytosis represents a means to decrease cell surface receptor abundance, as well as to control signaling outcome in cytotoxic lymphocytes.

The NKG2D activating receptor on human NK cells mediates "altered self" recognition, as its ligands (NKG2DLs) are upregulated on target cells in a variety of stress conditions. Evidence collected in the past years shows that, even though expression of NKG2DLs acts as a danger signal that renders tumor cells susceptible to cytotoxicity, chronic exposure to soluble or membrane-bound NKG2DLs can lead to down-modulation of receptor expression and impairment of NKG2D-mediated cell functions. Here, we evaluated whether different cell-bound NKG2DLs, namely MICA and ULBP2, are equivalently able to induce NKG2D down-modulation on human NK cells. We found that although both ligands reduce NKG2D surface expression, MICA promotes a stronger receptor downmodulation than ULBP2, leading to a severe impairment of NKG2D-dependent NK-cell cytotoxicity. We also provide evidence that the ubiquitin pathway and c-Cbl direct MICAinduced but not ULBP2-induced NKG2D internalization and degradation, thus identifying a molecular mechanism to explain the differential effects of MICA and ULBP2 on NKG2D expression. A better understanding of the molecular mechanisms employed by the different NKG2DLs to control NKG2D surface expression could be useful for the development of anti-tumor strategies to restore a normal level of NKG2D receptors on human NK cells. Keywords:Endocytosis r NK cells r NKG2D ligands r NKG2D receptor Additional supporting information may be found in the online version of this article at the publisher's web-site Introduction NK cells are an important arm of the innate immune system directly involved in the recognition and lysis of virus-infected and tumor cells. NK-cell functional activation mainly depends on the interplay between inhibitory receptors for MHC class I molecules Correspondence: Prof. Rossella Paolini e-mail: rossella.paolini@uniroma1.it and a wide array of activating receptors that act in concert to induce efficient target cell elimination [1][2][3]. One of the best characterized activating receptors is NKG2D (NK receptor group 2, member D), whose expression is not restricted to NK cells, as it is also found on many T-cell subsets [4,5]. NKG2D mediates "altered self" recognition, in that it recognizes ligands absent or poorly expressed on most tissues and upregulated by conditions of stress or infection, in autoimmune diseases and malignant transformation, thus rendering damaged cells susceptible to NKG2D-mediated cellular cytotoxicity [6,7]. Eur. J. Immunol. 2014Immunol. . 44: 2761Immunol. -2770 The early hypothesis claiming a prominent role of NKG2D in tumor immune surveillance [8] has been supported by studies using various animal tumor models and NKG2D-deficient mice [9][10][11][12]. In humans, a remarkable characteristic of NKG2D resides in the interaction of a single invariant receptor with two structurally distinct families of MHC class I-related cell surface glycoproteins that differ in modes of membrane anchor and affinities for their common receptor: the polymorphic MHC class I-related Chain (MIC...