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This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG

Dendritic cells (DCs) play a key role in the initiation of an immune response and are known as “professional” APCs because of their ability to activate naive T cells. A widely used method to generate DCs in vitro is to culture bone marrow (BM) cells or blood monocytes in the presence of GM-CSF and IL-4. In this study, we show that a small population of NK cells residing in the BM of RAG−/−, but not RAG−/− γc chain−/− mice, remain in the DC culture and is the source of IFN-γ produced after stimulation with LPS. These cells, which may represent early promoters of LPS-induced responses, have to be taken into account when interpreting experiments using BM-derived DCs.

Dendritic cells (DCs) play a key role in the initiation of an immune response and are known as “professional” antigen-presenting cells because of their ability to activate naïve T cells. A widely used method to generate DCs in vitro is to culture bone marrow cells, or blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Here we show that a small population of NK cells residing in the bone marrow of RAG(−/−), but not RAG(−/−) γ(c) chain(−/−) mice, remain in the DC culture and is the source of IFN-γ produced after stimulation with Lipopolysaccharide (LPS). These cells, which may represent early promoters of LPS-induced responses, have to be taken into account when interpreting experiments using bone marrow-derived DCs.

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite–host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action., eLife digest Malaria is a deadly disease caused by a parasite transmitted by mosquitoes. The parasite infects red blood cells, causing fever with flu-like symptoms. In some people, particularly pregnant women and children, the disease may be very serious and even lead to death. An effective malaria vaccine is urgently needed because malaria parasites are developing resistance to current drugs. People living in areas where malaria is common develop specific proteins called antibodies that protect them from malaria. Learning more about how the antibodies achieve this, could help to develop better vaccines. Scientists already know some antibodies bind to the malaria parasites and prevent them from entering red blood cells. Some vaccines have been based on these antibodies. Other antibodies bind to infected cells flagging them for destruction by cells of the immune system. Immune cells called natural killer cells can eliminate viruses or cancer cells this way, but it was not clear if they could also eliminate malaria parasite-infected red blood cells. Now, Arora et al. show that natural killer cells can selectively destroy malaria-infected red blood cells flagged with antibodies from people who live in areas where malaria is common. In laboratory experiments, natural killer cells from US volunteers, who were never exposed to malaria, did not kill normal or malaria-infected red blood cells. Adding antibodies collected from malaria-resistant volunteers from Africa allowed these natural killer cells from unexposed people to selectively seek out and destroy malaria-infected cells and leave uninfected red blood cells intact. Arora et al. also found that the antibodies from the malaria-resistant volunteers bound to parasite proteins on the surface of infected blood cells. The experiments suggest that vaccines designed to stimulate the production of antibodies to malaria proteins that are displayed on infected red blood cells, could destroy the parasite in infected people and help prevent disease and save lives.

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite–host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.

Natural killer (NK) cells are regulated through the coordinated functions of activating and inhibitory receptors. These receptors can act during the initial engagement of an NK cell with a target cell, or in subsequent NK cell engagements to maintain tolerance. Notably, each individual possesses a sizable minority-population of NK cells that are devoid of inhibitory receptors that recognize the surrounding MHC class I (ie, self-MHC). Since these NK cells cannot perform conventional inhibition, they are rendered less responsive through the process of NK cell education (also known as licensing) in order to reduce the likelihood of auto-reactivity. This review will delineate current views on NK cell education, clarify various misconceptions about NK cell education, and, lastly, discuss the relevance of NK cell education in anti-cancer therapies.

ATP-mediated activation of the purinergic receptor P2X7 elicits morphological changes and proinflammatory responses in macrophages. These changes include rapid shedding of microvesicles (MV) and the nonconventional secretion of cytokines, such as IL-1β and IL-18 following priming. In this study, we demonstrate the activation potential of P2X7-induced MV isolated from nonprimed murine macrophages. Cotreatment of nonprimed macrophages with ATP and calcium ionophore induced a rapid release of MV that were predominantly 0.5–1 μm in size. Exposure of primary murine bone marrow-derived macrophages to these MV resulted in costimulatory receptor upregulation and TNF-α secretion. Cell homogenates or supernatants cleared of MV did not activate macrophages. MV-mediated activation was p38 MAPK and NF-κB dependent, and partially dependent on TLR4 activity, but was high-mobility group box 1 independent. Biochemical fractionation of the MV demonstrated that the phospholipid fraction, not the protein fraction, mediated macrophage activation through a TLR4-dependent process. P2X7 activation is known to induce calcium-independent phospholipase A2, calcium-dependent phospholipase A2, and phospholipase D activities, but inhibition of these enzymes did not inhibit MV generation or shedding. However, blocking phospholipase D activity resulted in release of MV incapable of activating recipient macrophages. These data demonstrate a novel mechanism of macrophage activation resulting from exposure to MV from nonprimed macrophages, and identifies phospholipids in these MV as the biologically active component. We suggest that phospholipids delivered by MV may be mediators of sterile inflammation in a number of diseases.

Binding of NK cell inhibitory receptors to MHC class I (MHC-I) confers increased responsiveness to NK cells by a process known as NK cell licensing/education. Reduced MHC-I expression or a lack of inhibitory receptors for MHC-I results in diminished NK cell responsiveness. In this study, we evaluated the effect of human and mouse NK cell licensing on early stages of natural cytotoxicity. Unlicensed NK cells did not form as many stable conjugates with target cells. The reduction of NK cell conjugation to target cells was not attributed to altered β2 integrin LFA-1 properties but was instead due to reduced inside-out signaling to LFA-1 by activating receptors. For those unlicensed NK cells that did form conjugates, LFA-1–dependent granule polarization was similar to that in licensed NK cells. Thus, licensing controls signals as proximal as inside-out signaling by activating receptors but not integrin outside-in signaling for granule polarization.

Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a hyporesponsive state. The molecular basis of the mechanisms underlying the generation and maintenance of this hyporesponsive condition are unknown, thus an easy and reproducible mechanism able to induce hyporesponsiveness on human NK cells would be very useful to gain understanding of this process. Human NK cells treated with ionomycin lose their ability to degranulate and secrete IFN-γ in response to a variety of stimuli, but IL-2 stimulation can compensate these defects. Apart from reductions in the expression of CD11 a/CD18, no great changes were observed in the activating and inhibitory receptors expressed by these NK cells, however their transcriptional signature is different to that described for other hyporesponsive lymphocytes., This work was supported by grants from the Fondo de Investigación Sanitaria (PI11/00298 and PI08/1701), MINECO (SAF2014-58752-R) (to HTR), a JAE Predoc studentship from the CSIC and an EMBO Short term fellowship (to GRC) and the Intramural Research Program at the National Institutes of Health, National Institute of Allergy and Infectious Disease.

Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and limits inflammation and injury in response to many pulmonary insults. To test the hypothesis that EC-SOD has an important role in bacterial infections, wild-type and EC-SOD knockout (KO) mice were infected with Escherichia coli to induce pneumonia. Although mice in the EC-SOD KO group demonstrated greater pulmonary inflammation than did wild-type mice, there was less clearance of bacteria from their lungs after infection. Macrophages and neutrophils express EC-SOD; however, its function and subcellular localization in these inflammatory cells is unclear. In the present study, immunogold electron microscopy revealed EC-SOD in membrane-bound vesicles of phagocytes. These findings suggest that inflammatory cell EC-SOD may have a role in antibacterial defense. To test this hypothesis, phagocytes from wild-type and EC-SOD KO mice were evaluated. Although macrophages lacking EC-SOD produced more reactive oxygen species than did cells expressing EC-SOD after stimulation, they demonstrated significantly impaired phagocytosis and killing of bacteria. Overall, this suggests that EC-SOD facilitates clearance of bacteria and limits inflammation in response to infection by promoting bacterial phagocytosis.

This study explores the mechanism by which a bacterial toxin induces the secretion of a proinflammatory cytokine from innate immune cells. CDC are exotoxins secreted by many Gram-positive bacteria that bind cholesterol and oligomerize to form pores in eukaryotic cell membranes. We demonstrate that CDC TLO induces caspase-1 cleavage and the rapid release of IL-1β from LPS-primed murine BMDM. IL-1β secretion depends on functional toxin pore formation, as free cholesterol, which prevents TLO binding to cell membranes, blocks the cytokine release. Secretion of the mature forms of IL-1β and caspase-1 occurs only at lower TLO doses, whereas at a higher concentration, cells release the biologically inactive proforms. IL-1β release at a low TLO dose requires potassium efflux, calcium influx, and the activities of calcium-independent PLA2, caspase-1, and cathepsin B. Additionally, mature IL-1β release induced by a low TLO dose is dependent on the NLRP3 inflammasome, and pro-IL-1β release induced by a high TLO dose occurs independently of NLRP3. These results further elucidate a mechanism of CDC-induced IL-1β release and suggest a novel, immune evasion strategy in which IL-1β-containing macrophages might release primarily inactive cytokine following exposure to high doses of these toxins.