Cristina Capuano

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.

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.

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.

Cancer-Associated CD43 Glycoforms as Target of Immunotherapy

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer–mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy. Mol Cancer Ther; 13(3); 752–62. ©2013 AACR.

Phosphatidylinositol 4–Phosphate 5–Kinase α and Vav1 Mutual Cooperation in CD28-Mediated Actin Remodeling and Signaling Functions

Phosphatidylinositol 4,5–biphosphate (PIP2) is a cell membrane phosphoinositide crucial for cell signaling and activation. Indeed, PIP2 is a pivotal source for second messenger generation and controlling the activity of several proteins regulating cytoskeleton reorganization. Despite its critical role in T cell activation, the molecular mechanisms regulating PIP2 turnover remain largely unknown. In human primary CD4+ T lymphocytes, we have recently demonstrated that CD28 costimulatory receptor is crucial for regulating PIP2 turnover by allowing the recruitment and activation of the lipid kinase phosphatidylinositol 4–phosphate 5–kinase (PIP5Kα). We also identified PIP5Kα as a key modulator of CD28 costimulatory signals leading to the efficient T cell activation. In this study, we extend these data by demonstrating that PIP5Kα recruitment and activation is essential for CD28-mediated cytoskeleton rearrangement necessary for organizing a complete signaling compartment leading to downstream signaling functions. We also identified Vav1 as the linker molecule that couples the C-terminal proline-rich motif of CD28 to the recruitment and activation of PIP5Kα, which in turn cooperates with Vav1 in regulating actin polymerization and CD28 signaling functions.

Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production

ABSTRACT Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses. Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcϵRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcϵRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli. These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional program.

Activation of Lymphocyte Cytolytic Machinery: Where are We?

Target cell recognition by cytotoxic lymphocytes implies the simultaneous engagement and clustering of adhesion and activating receptors followed by the activation of an array of signal transduction pathways. The cytotoxic immune synapse represents the highly specialized dynamic interface formed between the cytolytic effector and its target that allows temporal and spatial integration of signals responsible for a defined sequence of processes culminating with the polarized secretion of lytic granules. Over the last decades, much attention has been given to the molecular signals coupling receptor ligation to the activation of cytolytic machinery. Moreover, in the last 10 years the discovery of genetic defects affecting cytotoxic responses greatly boosted our knowledge on the molecular effectors involved in the regulation of discrete phases of cytotoxic process at post-receptor levels. More recently, the use of super resolution and total internal reflection fluorescence imaging technologies added new insights on the dynamic reorganization of receptor and signaling molecules at lytic synapse as well as on the relationship between granule dynamics and cytoskeleton remodeling. To date we have a solid knowledge of the molecular mechanisms governing granule movement and secretion, being not yet fully unraveled the machinery that couples early receptor signaling to the late stage of synapse remodeling and granule dynamics. Here we highlight recent advances in our understanding of the molecular mechanisms acting in the activation of cytolytic machinery, also discussing similarities and differences between Natural killer cells and cytotoxic CD8+ T cells.