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Background Natural killer (NK) cell therapy is considered an attractive and safe strategy for anticancer therapy. Nevertheless, when autologous or allogenic NK cells are used alone, the clinical benefit has been disappointing. This is partially due to the lack of target specificity. Recently, CD19-specific chimeric antigen receptor (CAR)-NK cells have proven to be safe and potent in patients with B-cell tumors. However, the generation of CAR-NK cells is a complicated manufacturing process. We aim at developing a targeted NK cell therapy without the need for cellular genetic modifications. We took advantage of the natural expression of the IgG Fc receptor CD16a (FcγRIIIa) to induce strong antigen-specific effector functions through antibody-dependent cell-mediated cytotoxicity (ADCC). We have generated the new technology “Pin”, which enables the arming of modified monoclonal antibodies (mAbs) onto the CD16a of ex vivo expanded NK (eNK) cells.MethodsEx vivo eNK were prepared from umbilical cord blood cells and expanded using interleukin (IL)-2/IL-15 and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid feeder cells. mAbs were engineered with four substitutions called Pin mutations to increase their affinity to CD16a. eNK were incubated with anti-CD20 or anti-CD19 Pin-mAbs to generate “armed” eNK and were used to assess effector functions in vitro on cancer cell lines, lymphoma patient cells and in vivo.Results CD16a/Pin-mAb interaction is stable for several days and Pin-mAb eNK inherit the mAb specificity and exclusively induce ADCC against targets expressing the cognate antigen. Hence, Pin-mAbs confer long-term selectivity to eNK, which allows specific elimination of the target cells in several in vivo mouse models. Finally, we showed that it is possible to arm eNK with at least two Pin-mAbs simultaneously, to increase efficacy against heterogenous cancer cell populations.Conclusions The Pin technology provides an off-the-shelf NK cell therapy platform to generate CAR-like NK cells, without genetic modifications, that easily target multiple tumor antigens.

ObjectivesTo evaluate extracellular vesicles levels in a cohort of SARS-CoV-2’s patients hospitalized in an intensive care unit with and without COVID-19 associated thromboembolic events.MethodsIn this study, we aim to assess endothelial and platelet membrane-derived extracellular vesicles levels in a cohort of SARS-CoV-2 patients with and without COVID-19-associated thromboembolic events who were hospitalized in an intensive care unit. Annexin-V positive extracellular vesicles levels were prospectively assessed by flow cytometry in one hundred twenty-three critically ill adults diagnosed with acute respiratory distress syndrome associated with a SARS-CoV-2 infection, ten adults diagnosed for moderate SARS-CoV-2 infection and 25 healthy volunteers.ResultsOn our critically ill patients, thirty-four patients (27.6%) had a thromboembolic event, Fifty-three (43%) died. Endothelial and platelet membrane-derived extracellular vesicles were drastically increased in SARS-CoV-2 patients hospitalized in the ICU compared to healthy volunteers. Moreover a slighty higher small/large ratio for platelets membrane-derived extracellular vesicles in patients was linked to thrombo-embolic events.ConclusionA comparison between total annexin-V positive extracellular vesicles levels in severe and moderate SARS-CoV-2 infection and healthy controls showed a significant increase in patients with severe infection and their sizes could be considered as biomarkers of SARS-CoV-2 associated thrombo-embolic events.

Abstract Space weather phenomena can threaten space technologies. A hazard among these is the population of relativistic electrons in the Van Allen radiation belts. To reduce the threat, artificial processes can be introduced by transmitting very-low-frequency (VLF) waves into the belts. The resulting wave-particle interactions may deplete these harmful electrons. However, when transmitting VLF waves in space plasma, the antenna, plasma, and waves interact in a manner that is not well-understood. We conducted a series of VLF transmission experiments in the radiation belts and measured the power and radiation impedance under various frequencies and conditions. The results demonstrate the critical role played by the plasma-antenna-wave interaction around high-voltage space antennae and open the possibility to transmit high power in space. The physical insight obtained in this study can provide guidance to future high-power space-borne VLF transmitter developments, laboratory whistler-mode wave injection experiments, and the interpretation of various astrophysical and optical phenomena.

Abstract Very low frequency (VLF) waves (about 3–30 kHz) in the Earth’s magnetosphere interact strongly with energetic electrons and are a key element in controlling dynamics of the Van Allen radiation belts. Bistatic very low frequency (VLF) transmission experiments have recently been conducted in the magnetosphere using the high-power VLF transmitter on the Air Force Research Laboratory’s Demonstration and Science Experiments (DSX) spacecraft and an electric field receiver onboard the Japan Aerospace Exploration Agency’s Arase (ERG) spacecraft. On 4 September 2019, the spacecraft came within 410 km of each other and were in geomagnetic alignment. During this time, VLF signals were successfully transmitted from DSX to Arase, marking the first successful reception of a space-to-space VLF signal. Arase measurements were consistent with field-aligned propagation as expected from linear cold plasma theory. Details of the transmission event and comparison to VLF propagation model predictions are presented. The capability to directly inject VLF waves into near-Earth space provides a new way to study the dynamics of the radiation belts, ushering in a new era of space experimentation. Graphical Abstract

Abstract Background In the context of acute respiratory distress syndrome (ARDS), the response to lung recruitment maneuvers (LRMs) varies considerably from one patient to another and so is difficult to predict. The aim of the study was to determine whether or not the recruitment-to-inflation (R/I) ratio could differentiate between patients according to the change in lung mechanics during the LRM. Methods We evaluated the changes in gas exchange and respiratory mechanics induced by a stepwise LRM at a constant driving pressure of 15 cmH2O during pressure-controlled ventilation. We assessed lung recruitability by measuring the R/I ratio. Patients were dichotomized with regard to the median R/I ratio. Results We included 30 patients with moderate-to-severe ARDS and a median [interquartile range] R/I ratio of 0.62 [0.42–0.83]. After the LRM, patients with high recruitability (R/I ratio ≥ 0.62) presented an improvement in the PaO2/FiO2 ratio, due to significant increase in respiratory system compliance (33 [27–42] vs. 42 [35–60] mL/cmH2O; p

Abstract Sphingolipid long chain bases (LCBs) are building blocks of sphingolipids and can serve as signalling molecules, but also have antimicrobial activity and were effective in reducing growth of a range of human pathogens. In plants, LCBs are linked to cell death processes and the regulation of defence reactions against pathogens, but their role in directly influencing growth of plant-interacting microorganisms has received little attention. Therefore, we tested the major plant LCB phytosphingosine in in vitro tests with the plant pathogenic fungi Verticillium longisporum, Fusarium graminearum and Sclerotinia sclerotiorum, the plant symbiotic fungal endophyte Serendipita indica, the bacterial pathogens Pseudomonas syringae pv. tomato (Pst), Agrobacterium tumefaciens, and the related beneficial strain Rhizobium radiobacter. Phytosphingosine inhibited growth of these organisms at micromolar concentrations. Among the fungal pathogens, S. sclerotiorum was the most, and F. graminearum was the least sensitive. 15.9 μg/mL phytosphingosine effectively killed 95% of the three bacterial species. Plant disease symptoms and growth of Pst were also inhibited by phytosphingosine when co-infiltrated into Arabidopsis leaves, with no visible negative effect on host tissue. Taken together, we demonstrate that the plant LCB phytosphingosine inhibits growth of plant-interacting microorganisms. We discuss the potential of elevated LCB levels to enhance plant pathogen resistance.

Sphingolipid long-chain bases (LCBs) are building blocks for membrane-localized sphingolipids, and are involved in signal transduction pathways in plants. Elevated LCB levels are associated with the induction of programmed cell death and pathogen-derived toxin-induced cell death. Therefore, levels of free LCBs can determine survival of plant cells. To elucidate the contribution of metabolic pathways regulating high LCB levels, we applied the deuterium-labeled LCB D-erythro-sphinganine-d7 (D7-d18:0), the first LCB in sphingolipid biosynthesis, to Arabidopsis leaves and quantified labeled LCBs, LCB phosphates (LCB-Ps), and 14 abundant ceramide (Cer) species over time. We show that LCB D7-d18:0 is rapidly converted into the LCBs d18:0P, t18:0, and t18:0P. Deuterium-labeled ceramides were less abundant, but increased over time, with the highest levels detected for Cer(d18:0/16:0), Cer(d18:0/24:0), Cer(t18:0/16:0), and Cer(t18:0/22:0). A more than 50-fold increase of LCB-P levels after leaf incubation in LCB D7-d18:0 indicated that degradation of LCBs via LCB-Ps is important, and we hypothesized that LCB-P degradation could be a rate-limiting step to reduce high levels of LCBs. To functionally test this hypothesis, we constructed a transgenic line with dihydrosphingosine-1-phosphate lyase 1 (DPL1) under control of an inducible promotor. Higher expression of DPL1 significantly reduced elevated LCB-P and LCB levels induced by Fumonisin B1, and rendered plants more resistant against this fungal toxin. Taken together, we provide quantitative data on the contribution of major enzymatic pathways to reduce high LCB levels, which can trigger cell death. Specifically, we provide functional evidence that DPL1 can be a rate-limiting step in regulating high LCB levels.

Antiviral monoclonal antibodies (mAbs) can generate protective immunity through Fc-FcγRs interactions. We previously showed a role for immune complexes (ICs) in the enhancement of antiviral T-cell responses through FcγR-mediated activation of dendritic cells (DCs). Here we addressed how mAb therapy in retrovirus-infected mice affects the activation of neutrophils and inflammatory monocytes, two FcγR-expressing innate effector cells rapidly recruited to sites of infection. We found that both cell-types activated in vitro by viral ICs secreted chemokines able to recruit monocytes and neutrophils themselves. Moreover, inflammatory cytokines potentiated chemokines and cytokines release by IC-activated cells and induced FcγRIV upregulation. Similarly, infection and mAb-treatment upregulated FcγRIV on neutrophils and inflammatory monocytes and enhanced their cytokines/chemokines secretion. Notably, upon antibody therapy neutrophils and inflammatory monocytes displayed distinct functional activation states and sequentially modulated the antiviral immune response by secreting Th1-type polarizing cytokines and chemokines, which occurred in a FcγRIV-dependent manner. Consistently, FcγRIV- blocking in mAb-treated, infected mice led to reduced immune protection. Our work provides new findings on the immunomodulatory role of neutrophils and monocytes in the enhancement of immune responses upon antiviral mAb therapy.

Monoclonal antibodies (mAbs) are now considered as a therapeutic approach to prevent and treat severe viral infections. Using a mouse retroviral model, we showed that mAbs induce protective immunity (vaccinal effects). Here, we investigated the role of natural killer (NK) cells on this effect. NK cells are effector cells that are crucial to control viral propagation upon mAb treatment. However, their immunomodulatory activity during antiviral mAb immunotherapies has been little studied. Our data reveal that the mAb treatment of infected mice preserves the functional activation of NK cells. Importantly, functional NK cells play an essential role in preventing immune dysfunction and inducing antiviral protective immunity upon mAb therapy. Thus, NK cell depletion in mAb-treated, viral-infected mice leads to the upregulation of molecules involved in immunosuppressive pathways (i.e., PD-1, PD-L1 and CD39) on dendritic cells and T cells. NK cell depletion also abrogates the vaccinal effects induced by mAb therapy. Our data also reveal a role for IFNγ-producing NK cells in the enhancement of the B-cell responses through the potentiation of the B-cell helper properties of neutrophils. These findings suggest that preserved NK cell functions and counts might be required for achieving mAb-induced protective immunity. They open new prospects for improving antiviral immunotherapies.

Monoclonal antibodies (mAbs), which currently constitute the main class of biotherapeutics, are now recognized as major medical tools that are increasingly being considered to fight severe viral infections. Indeed, the number of antiviral mAbs developed in recent years has grown exponentially. Although their direct effects on viral blunting have been studied in detail, their potential immunomodulatory actions have been overlooked until recently. The ability of antiviral mAbs to modulate antiviral immune responses in infected organisms has recently been revealed. More specifically, upon recognition of their cognate antigens, mAbs form immune complexes (ICs) that can be recognized by the Fc receptors expressed on different immune cells of infected individuals. This binding may be followed by the modulation of the host immune responses. Harnessing this immunomodulatory property may facilitate improvements in the therapeutic potential of antiviral mAbs. This review focuses on the role of ICs formed with different viral determinants and mAbs in the induction of antiviral immune responses in the context of both passive immunotherapies and vaccination strategies. Potential deleterious effects of ICs on the host immune response are also discussed.

Abstract In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin–proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub‐complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high‐resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub‐complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon‐γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes.

Protein complexes are the main molecular machines that support all major cellular pathways and their in-depth characterization are essential to understand their functions. Determining the stoichiometry of the different subunits of a protein complex still remains challenging. Recently, many label-free quantitative proteomic approaches have been developed to study the composition of protein complexes. It is therefore of great interest to evaluate these different methods in a stoichiometry oriented objective. Here we compare the ability of four absolute quantitative label-free methods currently used in proteomic studies to determine the stoichiometry of a well-characterized protein complex, the 26S proteasome.