The recruitment and activation of phosphatidylinositol 4-phosphate 5-kinases α critically regulate CD28-dependent signaling responses

CD28 costimulatory receptor is a crucial determinant of the outcome of T lymphocyte activation. The engagement of CD28 by its natural ligands, B7.1/CD80 or B7.2/CD86, expressed on the surface of professional APC, lowers T cell receptor (TCR) activation threshold, thus leading to the enhancement of early signalling events necessary for efficient cytokine production, cell cycle progression, survival and regulation of T cells effector responses. CD28 is also able to act as a unique signalling receptor and to deliver TCR-independent autonomous signals, which account for its critical role in the regulation of pro-inflammatory cytokine/chemokine production and T cell survival. Most of the CD28-dependent signalling functions are initiated by the recruitment and activation of class IA phosphatidylinositol 3-kinase (PI3K), The intracytoplasmic domain of CD28 contains a N-terminal YMNM motif that following phosphorylation binds the p85 subunit of phosphatidylinositol 3-kinase (PI3K). Once activated, PI3K catalyzes the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) and generates the docking sites for key signalling proteins. PIP2 plays a critical role in the regulation of both cytoskeleton dynamics and second messenger generation. Indeed, PIP2 is the common source for two major distinct signalling cascades involving PI3K and PLCγ1 that often colocalize in the same signalling complexes competing for the common pool of substrate. Consequently, PIP2 levels decrease following receptor activation, thus suggesting that stimulation of PIP2 synthesis may be an essential regulatory step to sustain the activation of both PI3K and PLCγ1 following CD28 engagement. The main biosynthetic pathway of PIP2 involves phosphorylation of phosphatidylinositol 4-monophosphate (PI4P) at the D5 position of the inositol ring by PIP5K. Three PIP5K isoforms (α, β and γ) have been identified. Several data obtained in different cell systems evidenced differential subcellular localizations of each isoform. PIP5Kα, for instance, is localized at the plasma membrane, where it guarantees the local availability of PIP2. Here we show that CD28 stimulation by both B7.1/CD80 or agonistic Abs induces the recruitment and activation of PIP5Kα in human primary CD4+ T lymphocytes. This event leads to the neo-synthesis of PIP2 that is consumed by CD28-activated PI3K. By either small interference RNA (siRNA)-driven cell silencing or overexpressing a kinase dead mutant, we evidenced that PIP5Kα activation is required for both CD28 autonomous 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 identify PIP5Kα as a critical mediator of CD28-dependent responses

Memory NK cell features exploitable in anticancer immunotherapy

Besides their innate ability to rapidly produce effector cytokines and kill virus-infected or transformed cells, natural killer (NK) cells display a strong capability to adapt to environmental modifications and to differentiate into long-lived, hyperfunctional populations, dubbed memory or memory-like NK cells. Despite significant progress in the field of NK cell-based immunotherapies, some factors including their short life span and the occurrence of a tumor-dependent functional exhaustion have limited their clinical efficacy so that strategies aimed at overcoming these limitations represent one of the main current challenges in the field. In this scenario, the exploitation of NK cell memory may have a considerable potential. This article summarizes recent evidence in the literature on the peculiar features that render memory NK cells an attractive tool for antitumor immunotherapy, including their long-term survival and in vivo persistence, the resistance to tumor-dependent immunosuppressive microenvironment, the amplified functional responses to IgG-opsonized tumor cells, and in vitro expansion capability. Along with highlighting these issues, we speculate that memory NK cell-based adoptive immunotherapy settings would greatly take advantage from the combination with tumor-targeting therapeutic antibodies (mAbs), as a strategy to fully unleash their clinical efficacy

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. Cancer Res; 75(19); 1-12. ©2015 AACR

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

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 progra

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

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

Vaccine hesitancy among religious groups: reasons underlying this phenomenon and communication strategies to rebuild trust

The research leading to these results has been performed in the framework of the European training programme in Pharmacovigilance and Pharmacoepidemiology, Eu2

Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming

Natural Killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human Cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C+Fc∝RI≥- long-lived “memory” NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterised yet. Here we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells, and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansion. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting, and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy

Preventable Cases of Oral Anticoagulant-Induced Bleeding: Data From the Spontaneous Reporting System

Background: Despite the risk of bleeding is a well-known adverse effect of oral anticoagulants, there is scarce evidence on the preventability of oral anticoagulant-induced bleedings. Therefore, we investigated the potential risk factors related to preventable cases of oral anticoagulant-induced bleedings. Methods: We performed a study using Individual Case Safety Reports (ICSRs) with an oral anticoagulant as suspected drug among those reported through the spontaneous reporting system of Campania Region from 1 July 2012 to 31 December 2017. The P-method was used for the preventability assessment of all cases of bleeding. Results: In total, 58 cases out of 253 (22.9%) were preventable, and the most reported suspected drug was an indirect oral anticoagulant (warfarin). Sixty-eight critical criteria for preventability were identified, all related to healthcare professionals' practices. The most detected risk factor related to healthcare professionals' practices was the labeled drug-drug interaction for both direct and indirect oral anticoagulants. Conclusion: Our findings describe the most reported risk factors for preventability of oral anticoagulant-induced bleedings. These factors may be useful for targeting interventions to improve pharmacovigilance activities in our regional territory and to reduce the burden of medication errors and inappropriate prescription

Gender Differences in Outpatient Pediatric Drug Utilization: A Cohort Study From Southern Italy

Objective: The aim of this retrospective population-based cohort study is to in-depth investigate gender-specific drug utilization pattern in pediatric outpatient population.Methods: By using a large administrative database of the Local Health Unit of Caserta (Southern Italy), a pediatric cohort from the birth to 18 years was observed over 6 years (from 1st January 2010 to 31st December 2015). Yearly prevalence of drug use per 100 inhabitants as well as the median number of prescriptions was stratifying by gender. Prevalence of acute and recurrent use of the most frequently used active substances was calculated for the year 2015.Results: A decreasing trend in prevalence of drug use (−3.2%, with a reduction of median number of drugs dispensed) was observed in children for both sexes, from 2010 to 2015. In 2015, the drug classes most commonly used among children of any age were modestly but consistently prescribed more to males than to females: systemic anti-infective drugs (M = 43.5%; F = 42.3%), respiratory tract drugs (M = 29.0%; F = 26.1%), and hormones (M = 13.1%; F = 11.3%). Irrespective of gender, beclomethasone was the most utilized active substance in the first 2 years of life, while thereafter amoxicillin/clavulanate in combination.Conclusions: In a large population of pediatric outpatients no major difference was seen between genders, although commonly used drug classes; in particular, antibiotics, respiratory tract drugs and Hormones with corticosteroids for systemic use prescribed modestly but consistently to larger extent in males than females