Ricciarda Galandrini

The adaptor protein shc is involved in the negative regulation of NK cell-mediated cytotoxicity

The activation of protein tyrosine kinase(s) (PTK) is a critical event required for the development of NK cell‐mediated cytotoxicity. Here we demonstrate that the adaptor protein shc undergoestyrosine phosphorylation during the generation of antibody‐dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we report that, upon direct or antibody‐dependent target cell interaction, shc coprecipitates with the Src homology 2 (SH2)‐containing inositol phosphatase, SHIP. To gain information on the functional role of shc in NK cytotoxicity, we overexpressed wild‐type or dominant negative shc constructs in the human NKL cell line. Our findings show a consistent shc‐mediated down‐regulation of ADCC and natural killing. Such functional effect correlates with a perturbation of the phoshoinositide (PI) metabolism by means of a shc‐mediated negative regulation of inositol 1, 4, 5 triphosphate (IP3) generation and intracellular calcium flux upon CD16 ligation. Furthermore, our data show that dominant‐negative shc‐mediated perturbation of shc/SHIP interaction leads to inhibition of ligand‐dependent SHIP recruitment to CD16 ζ chain. We suggest that shc plays a role of negative adaptor by modulating SHIP recruitment to activation receptors involved in the generation of NK cytotoxic function.

c-Cbl regulates MICA- but not ULBP2-induced NKG2D down-modulation in human NK cells.

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 down‐modulation 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 MICA‐induced 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.

Ubiquitin-dependent endocytosis of NKG2D-DAP10 receptor complexes activates signaling and functions in human NK cells

Signaling by an internalized natural killer cell receptor is required for the secretion of cytolytic granules and interferon-γ. Turning on natural killer cells Ligands present on the surface of tumor cells stimulate and activate natural killer (NK) cells of the innate immune system, which then trigger tumor cell death. Quatrini et al. found that the ligand-dependent internalization of a complex containing the stimulatory receptor NKG2D and the adaptor protein DAP10 depended on DAP10 ubiquitylation. Internalized receptor complexes in endosomes stimulated signaling by the kinase ERK, which was required for the secretion of cytolytic granules. Mutation of DAP10 to prevent its ubiquitylation resulted in decreased receptor internalization and defective cytotoxic responses, indicating that internalized receptors stimulate NK cell functions. 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.

Anti-CD20 Therapy Acts via FcγRIIIA to Diminish Responsiveness of Human Natural Killer Cells

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.

NKG2A inhibits NKG2C effector functions of γδ T cells: implications in health and disease.

The CD94/NKG2 complex is expressed on T and NK lymphocytes. CD94 molecules covalently associate to activating or inhibitory NKG2 molecules, and their expression finely tunes cell responses. Human γδ T cells express several NKRs. Expression of these receptors is confined to the cytolytic Vδ2 subset, which coexpresses the FcγRIII CD16 and CD45RA and has been defined as Vγ9Vδ2 TEMRA cells. We show that the CD94/NKG2C complex, associated with KARAP/DAP12, is fully functional in γδ T cells, as determined by measuring IFN‐γ production, T cell proliferation, and cytolytic activity by γδ lymphocytes. In contrast, NKG2A expression was found on all γδ T cell memory subsets, suggesting a crucial role of the inhibitory signal provided by this receptor on γδ T cell responses. Moreover, we found Vγ9Vδ2 TEMRA, NK, and CD8+ αβ T cells coexpressing NKG2A and NKG2C receptors. Functional experiments showed that the inhibitory signal mediated by the NKG2A receptor prevails when double‐positive cells are activated. Finally, NKG2A expression on γδ LDGL correlates with asymptomatic pathology, even in the presence of NKG2C coexpression, whereas in symptomatic patients affected by severe disease, the inhibitory NKG2A receptor is absent, and a variety of activatory NKRs was found. We propose that the silent behavior of γδ cells in LDGL patients is a result of effective inhibitory HLA class I receptors.

Regulation of NKG2D Expression and Signaling by Endocytosis

NKG2D is an activating receptor that can bind to a large number of stress-induced ligands that are expressed in the context of cancer or viral infection. This receptor is expressed on many cytotoxic lymphocytes, and plays a crucial role in antitumor and antiviral immune responses. However, exposure to NKG2D ligand-expressing target cells promotes receptor endocytosis, ultimately leading to lysosomal receptor degradation and impairment of NKG2D-mediated functions. Interestingly, before being degraded, internalized receptors can signal from the endosomal compartment, leading to the appropriate activation of cellular functional programs. This review summarizes recent findings on ligand-induced receptor internalization, with particular emphasis on the role of endocytosis in the control of both NKG2D-mediated intracellular signaling and receptor degradation.

PIP2-dependent regulation of Munc13-4 endocytic recycling: impact on the cytolytic secretory pathway

Cytotoxic lymphocytes clear infected and transformed cells by releasing the content of lytic granules at cytolytic synapses, and the ability of cytolytic effectors to kill in an iterative manner has been documented previously. Although bidirectional trafficking of cytolytic machinery components along the endosomal pathway has begun to be elucidated, the molecular mechanisms coordinating granule retrieval remain completely unexplored. In the present study, we focus on the lytic granule priming factor Munc13-4, the mutation of which in familial hemophagocytic lymphohistiocytosis type 3 results in a profound defect of cytotoxic function. We addressed the role of phosphatidylinositol (4,5)-bisphosphate (PIP2) in the regulation of Munc13-4 compartmentalization. We observed that in human natural killer cells, PIP2 is highly enriched in membrane rafts. Granule secretion triggering induces a transient Munc13-4 raft recruitment, followed by AP-2/clathrin-dependent internalization. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) γ gene silencing leads to the impairment of granule secretion associated with increased levels of raft-associated Munc13-4, which is attributable to a defect in AP-2 membrane recruitment. In such conditions, the ability to subsequently kill multiple targets was significantly impaired. These observations indicate that Munc13-4 reinternalization is required for the maintenance of an intracellular pool that is functional to guarantee the serial killing potential.

Attenuation of PI3K/Akt-Mediated Tumorigenic Signals through PTEN Activation by DNA Vaccine-Induced Anti-ErbB2 Antibodies

By studying BALB/c mice deficient in immune components, we show that the protective immunity to rat ErbB2+ tumors rests on the Ab response elicited by the electroporation of a DNA vaccine encoding the extracellular and transmembrane domains of rat ErbB2. In vivo, the adoptive transfer of vaccine-elicited anti-rat ErbB2 Abs protected against a challenge of rat ErbB2+ carcinoma cells (TUBO cells). In vitro, such Abs inhibited TUBO cell growth by impairing cell cycle progression and inducing apoptosis. To correlate intrinsic mechanisms of Ab action with their tumor-inhibitory potential, first we showed that TUBO cells constitutively express phosphorylated transgenic ErbB2/autochthonous ErbB3 heterodimers and exhibit a basal level of Akt phosphorylation, suggesting a constitutive activation of the PI3K/Akt pathway. Treatment with anti-ErbB2 Abs caused a drastic reduction in the basal level of Akt phosphorylation in the absence of an impairment of PI3K enzymatic activity. Notably, the same Ab treatment induced an increase in PTEN phosphatase activity that correlated with a reduced PTEN phosphorylation. In conclusion, vaccine-induced anti-ErbB2 Abs directly affected the transformed phenotype of rat ErbB2+ tumors by impairing ErbB2-mediated PI3K/Akt signaling.

Phosphatidylinositol 4-phosphate 5-kinase α activation critically contributes to CD28-dependent signaling responses.

CD28 is one of the most relevant costimulatory receptors that deliver both TCR-dependent and TCR-independent signals regulating a wide range of signaling pathways crucial for cytokine and chemokine gene expressions, T cell survival, and proliferation. Most of the CD28-dependent signaling functions are initiated by the recruitment and activation of class IA PI3Ks, which catalyze the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate, thus generating the docking sites for key signaling proteins. Hence, PIP2 is a crucial substrate in driving the PI3K downstream signaling pathways, and PIP2 turnover may be an essential regulatory step to ensure the activation of PI3K following CD28 engagement. Despite some data evidence that CD28 augments TCR-induced turnover of PIP2, its direct role in regulating PIP2 metabolism has never been assessed. In this study, we show that CD28 regulates PIP2 turnover by recruiting and activating phosphatidylinositol 4-phosphate 5-kinases α (PIP5Kα) in human primary CD4+ T lymphocytes. This event leads to the neosynthesis of PIP2 and to its consumption by CD28-activated PI3K. We also evidenced that PIP5Kα activation is required for both CD28 unique signals regulating IL-8 gene expression as well as for CD28/TCR-induced Ca2+ mobilization, NF-AT nuclear translocation, and IL-2 gene transcription. Our findings elucidate a novel mechanism that involves PIP5Kα as a key modulator of CD28 costimulatory signals.

Local Allergic Rhinitis in Children: Novel Diagnostic Features and Potential Biomarkers

Background Local allergic rhinitis (LAR) is a phenotype of rhinitis that has been poorly studied in children. It is characterized by the same symptoms of allergic rhinitis but with the absence of markers of systemic atopy. Objective To identify children affected by LAR and to analyze the pathogenesis of this disease. We chose to focus our attention on interleukin (IL) and thymic stromal lymphopoietin (TSLP). Methods We enrolled 20 children affected by nonallergic rhinitis (negative skin-prick test results and serum specific immunoglobulin E [sIgE] values). Each patient underwent a nasal allergen provocation test (NAPT) with dust mite and grass pollen. Before and after NAPT, nasal lavage was performed to detect sIgE, IL-5, and TSLP; anterior active rhinomanometry was used to evaluate changes in nasal obstruction. Results Two patients were positive to a nonspecific NAPT and, thus, were excluded from the study. Of the remaining 18 children, 12 (66.7%) had positive results to at least one NAPT. Among these 12 patients, nasal sIgE levels for Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Lolium perenne increased significantly after NAPT (D. pteronyssinus, p < 0.005; D. farinae, p < 0.05; L. perenne, p < 0.05). Nasal IL-5 levels showed a significant increase after NAPT (p ≤ 0.006), and this increase was significantly higher in children who had positive NAPT results than in those patients with negative NAPT results (p ≤ 0.03). Among the 12 children who had a positive NAPT result, nasal TSLP was detected in 4 patients (33.3%) and its levels showed a relevant increase after NAPT, even though the difference did not reach statistical significance (p ≤ 0.061). Conclusion Observed results raise the importance of better refining the diagnostic protocol for LAR in children. Nasal TSLP and IL-5 levels offer new insights concerning localized allergic inflammation, although the role of nasal sIgE has still to be clarified.