Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation.

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

Immunoglobulins and serum proteins impair anti-tumor NK cell effector functions in malignant ascites.

Introduction: Malignant ascites indicates ovarian cancer progression and predicts poor clinical outcome. Various ascites components induce an immunosuppressive crosstalk between tumor and immune cells, which is poorly understood. In our previous study, imbalanced electrolytes, particularly high sodium content in malignant ascites, have been identified as a main immunosuppressive mechanism that impaired NK and T-cell activity. Methods: In the present study, we explored the role of high concentrations of ascites proteins and immunoglobulins on antitumoral NK effector functions. To this end, a coculture system consisting of healthy donor NK cells and ovarian cancer cells was used. The anti-EGFR antibody Cetuximab was added to induce antibody-dependent cellular cytotoxicity (ADCC). NK activity was assessed in the presence of different patient ascites samples and immunoglobulins that were isolated from ascites. Results: Overall high protein concentration in ascites impaired NK cell degranulation, conjugation to tumor cells, and intracellular calcium signaling. Immunoglobulins isolated from ascites samples competitively interfered with NK ADCC and inhibited the conjugation to target cells. Furthermore, downregulation of regulatory surface markers CD16 and DNAM-1 on NK cells was prevented by ascites-derived immunoglobulins during NK cell activation. Conclusion: Our data show that high protein concentrations in biological fluids are able to suppress antitumoral activity of NK cells independent from the mechanism mediated by imbalanced electrolytes. The competitive interference between immunoglobulins of ascites and specific therapeutic antibodies could diminish the efficacy of antibody-based therapies and should be considered in antibody-based immunotherapies.

HIV vaccines induce CD8+ T cells with low antigen receptor sensitivity.

Current HIV vaccines designed to stimulate CD8+ T cells have failed to induce immunologic control upon infection. The functions of vaccine-induced HIV-specific CD8+ T cells were investigated here in detail. Cytotoxic capacity was significantly lower than in HIV controllers and was not a consequence of low frequency or unaccumulated functional cytotoxic proteins. Low cytotoxic capacity was attributable to impaired degranulation in response to the low antigen levels present on HIV-infected targets. The vaccine-induced T cell receptor (TCR) repertoire was polyclonal and transduction of these TCRs conferred the same reduced functions. These results define a mechanism accounting for poor antiviral activity induced by these vaccines and suggest that an effective CD8+ T cell response may require a vaccination strategy that drives further TCR clonal selection.

The Alleviating Effect of Lagerstroemia indica Flower Extract on Stretch Marks through Regulation of Mast Cells.

Striae distensae (SD) or stretch marks are common linear scars of atrophic skin with disintegrating extracellular matrix (ECM) structures. Although fibroblasts contribute to the construction of ECM structure in SD, some studies have reported that mast cell degranulation causes the disruption of ECM in early SD lesions. Lagerstroemia indica flower (LIF) has traditionally been used in India as a diuretic. However, little is known about the effect and molecular action of Lagerstroemia indica flower extract (LIFE) on alleviating SD. This study evaluated the effects of LIFE on mast cell degranulation and the synthesis of ECM components in fibroblasts. LIFE inhibits the adhesion of rat basophilic leukemia (RBL) cells, RBL-2H3 on fibronectin (FN) and the expression of integrin, a receptor for FN, thereby reducing focal adhesion kinase (FAK) phosphorylation. In addition, LIFE attenuated the allergen-induced granules and cytokine interleukin 3 (IL-3) through the adhesion with FN. Moreover, the conditioned medium (CM) of activated mast cells decreases the synthesis of ECM components, and LIFE restores the abnormal expressions induced by activated mast cells. These results demonstrate that LIFE suppresses FN-induced mast cell activation and promotes the synthesis of ECM components in fibroblast, which indicates that LIFE may be a useful cosmetic agent for SD treatment.

Regulation of Trafficking and Signaling of the High Affinity IgE Receptor by FcεRIß and the Potential Impact of FcεRIß Splicing in Allergic Inflammation.

Mast cells are tissue-resident immune cells that function in both innate and adaptive immunity through the release of both preformed granule-stored mediators, and newly generated proinflammatory mediators that contribute to the generation of both the early and late phases of the allergic inflammatory response. Although mast cells can be activated by a vast array of mediators to contribute to homeostasis and pathophysiology in diverse settings and contexts, in this review, we will focus on the canonical setting of IgE-mediated activation and allergic inflammation. IgE-dependent activation of mast cells occurs through the high affinity IgE receptor, FcεRI, which is a multimeric receptor complex that, once crosslinked by antigen, triggers a cascade of signaling to generate a robust response in mast cells. Here, we discuss FcεRI structure and function, and describe established and emerging roles of the ß subunit of FcεRI (FcεRIß) in regulating mast cell function and FcεRI trafficking and signaling. We discuss current approaches to target IgE and FcεRI signaling and emerging approaches that could target FcεRIß specifically. We examine how alternative splicing of FcεRIß alters protein function and how manipulation of splicing could be employed as a therapeutic approach. Targeting FcεRI directly and/or IgE binding to FcεRI are promising approaches to therapeutics for allergic inflammation. The characteristic role of FcεRIß in both trafficking and signaling of the FcεRI receptor complex, the specificity to IgE-mediated activation pathways, and the preferential expression in mast cells and basophils, makes FcεRIß an excellent, but challenging, candidate for therapeutic strategies in allergy and asthma, if targeting can be realized.

SLAMF7 selectively favors degranulation to promote cytotoxicity in human NK cells.

NK cells play an important role in immunity by recognizing and eliminating cells undergoing infection or malignant transformation. This role is dependent on the ability of NK cells to lyse targets cells in a perforin-dependent mechanism and by secreting inflammatory cytokines. Both effector functions are controlled by several cell surface receptors. The Signaling Lymphocyte Activation Molecule (SLAM) family of receptors plays an essential role in regulating NK cell activation. Several studies have demonstrated that SLAMF7 regulates NK cell activation. However, the molecular and cellular mechanisms by which SLAMF7 influences NK effector functions are unknown. Here, we present evidence that physiological ligation of SLAMF7 in human NK cells enhances the lysis of target cells expressing SLAMF7. This effect was dependent on the ability of SLAMF7 to promote NK cell degranulation rather than cytotoxic granule polarization or cell adhesion. Moreover, SLAMF7-dependent NK cell degranulation was predominantly dependent on PLC-γ when compared to PI3K. These data provide novel information on the cellular mechanism by which SLAMF7 regulates human NK cell activation. Finally, this study supports a model for NK cell activation where activated receptors contribute by regulating specific discrete cellular events rather than multiple cellular processes.

E-Cadherin restricts mast cell degranulation in mice.

Crosslinking of FcεRI-bound IgE triggers the release of a large number of biologically active, potentially anaphylactic compounds by mast cells. FcεRI activation ought to be well-controlled to restrict adverse activation. As mast cells are embedded in tissues, adhesion molecules may contribute to limiting premature activation. Here, we report that E-Cadherin serves that purpose. Having confirmed that cultured mast cells express E-Cadherin, a mast-cell-specific E-Cadherin deficiency, Mcpt5-Cre E-Cdhfl/fl mice, was used to analyze mast cell degranulation in vitro and in vivo. Cultured peritoneal mast cells from Mcpt5-Cre E-Cdhfl/fl mice were normal with respect to many parameters but showed much-enhanced degranulation in three independent assays. Soluble E-Cadherin reduced the degranulation of control cells. The release of some newly synthesized inflammatory cytokines was decreased by E-Cadherin deficiency. Compared to controls, Mcpt5-Cre E-Cdhfl/fl mice reacted much stronger to IgE-dependent stimuli, developing anaphylactic shock. We suggest E-Cadherin-mediated tissue interactions restrict mast cell degranulation to prevent their precocious activation.

Enhanced Calcium Signal Induces NK Cell Degranulation but Inhibits Its Cytotoxic Activity.

Although the mechanism of NK cell activation is still unclear, the strict calcium dependence remains the hallmark for lytic granule secretion. A plethora of studies claiming that impaired Ca2+ signaling leads to severely defective cytotoxic granule exocytosis accompanied by weak target cell lysis has been published. However, there has been little discussion about the effect of induced calcium signal on NK cell cytotoxicity. In our study, we observed that small-molecule inhibitor UNC1999, which suppresses global H3K27 trimethylation (H3K27me3) of human NK cells, induced a PKD2-dependent calcium signal. Enhanced calcium entry led to unbalanced vesicle release, which resulted into fewer target cells acquiring lytic granules and subsequently being killed. Further analyses revealed that the ability of conjugate formation, lytic synapse formation, and granule polarization were normal in NK cells treated with UNC1999. Cumulatively, these data indicated that induced calcium signal exclusively enhances unbalanced degranulation that further inhibits their cytotoxic activity in human NK cells.

IL-33 priming amplifies ATP-mediated mast cell cytokine production.

Inflammatory responses are required to block pathogen infection but can also lead to hypersensitivity and chronic inflammation. Barrier tissues actively release IL-33, ATP, and other alarmins during cell stress, helping identify pathogenic stimuli. However, it is unclear how these signals are integrated. Mast cells are critical initiators of allergic inflammation and respond to IL-33 and ATP. We found that mouse mast cells had a 3-6-fold increase in ATP-induced cytokine production when pre-treated with IL-33. This effect was observed at ATP concentrations < 100 µM and required < 30-minute IL-33 exposure. ATP-induced degranulation was not enhanced by pretreatment nor was the response to several pathogen molecules. Mechanistic studies implicated the P2X7 receptor and calcineurin/NFAT pathway in the enhanced ATP response. Finally, we found that IL-33 + ATP co-stimulation enhanced peritoneal eosinophil and macrophage recruitment. These results support the hypothesis that alarmins collaborate to surpass a threshold necessary to initiate an inflammatory response.

Neutrophil-Dependent Immunity During Pulmonary Infections and Inflammations.

Rapid recruitment of neutrophils to an inflamed site is one of the hallmarks of an effective host defense mechanism. The main pathway through which this happens is by the innate immune response. Neutrophils, which play an important part in innate immune defense, migrate into lungs through the modulation actions of chemokines to execute a variety of pro-inflammatory functions. Despite the importance of chemokines in host immunity, little has been discussed on their roles in host immunity. A holistic understanding of neutrophil recruitment, pattern recognition pathways, the roles of chemokines and the pathophysiological roles of neutrophils in host immunity may allow for new approaches in the treatment of infectious and inflammatory disease of the lung. Herein, this review aims at highlighting some of the developments in lung neutrophil-immunity by focusing on the functions and roles of CXC/CC chemokines and pattern recognition receptors in neutrophil immunity during pulmonary inflammations. The pathophysiological roles of neutrophils in COVID-19 and thromboembolism have also been summarized. We finally summarized various neutrophil biomarkers that can be utilized as prognostic molecules in pulmonary inflammations and discussed various neutrophil-targeted therapies for neutrophil-driven pulmonary inflammatory diseases.

Ex vivo expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells through different receptor-ligand interactions.

BACKGROUND: Recently, allogeneic natural killer (NK) cells have gained considerable attention as promising immunotherapeutic tools due to their unique biological functions and characteristics. Although many NK expansion strategies have been reported previously, a deeper understanding of cryopreserved allogeneic NK cells is needed for specific therapeutic approaches. METHODS: We isolated CD3-CD56+ primary natural killer (pNK) cells from healthy donors and expanded them ex vivo using a GMP-compliant method without any feeder to generate large volumes of therapeutic pNK cells and cryopreserved stocks. After validation for high purity and activating phenotypes, we performed RNA sequencing of the expanded and cryopreserved pNK cells. The pNK cells were used against various cancer cell lines in 7-AAD/CFSE cytotoxicity assay. For in vivo efficacy study, NSG mice bearing subcutaneous cisplatin-resistant A2780cis xenografts were treated with our pNK cells or cisplatin. Antitumor efficacy was assessed by measuring tumor volume and weight. RESULTS: Compared to the pNK cells before expansion, pNK cells after expansion showed 2855 upregulated genes, including genes related to NK cell activation, cytotoxicity, chemokines, anti-apoptosis, and proliferation. Additionally, the pNK cells showed potent cytolytic activity against various cancer cell lines. Interestingly, our activated pNK cells showed a marked increase in NKp44 (1064-fold), CD40L (12,018-fold), and CCR5 (49-fold), and did not express the programmed cell death protein 1(PD-1). We also demonstrated the in vitro and in vivo efficacies of pNK cells against cisplatin-resistant A2780cis ovarian cancer cells having a high programmed death-ligand 1(PD-L1) and low HLA-C expression. CONCLUSIONS: Taken together, our study provides the first comprehensive genome wide analysis of ex vivo-expanded cryopreserved pNK cells. It also indicates the potential use of expanded and cryopreserved pNK cells as a highly promising immunotherapy for anti-cancer drug resistant patients.

[Elucidation of Mast Cell Activation Mechanism Mediated by Purinergic Signaling].

Mast cells (MCs) are immune cells that are distributed in all tissues throughout the body, and their cytoplasm is rich in granules containing histamine and tryptase. When MCs recognize antigens through IgE bound to FcεRI, they release these mediators by degranulation. Because degranulation induces various type I allergic reactions, such as anaphylactic shock and hay fever, elucidation of the control mechanism of degranulation is important to the development of a therapeutic strategy for allergic diseases. It is known that the antigen-induced degranulation response is fine-tuned by various humoral factors via the activation of G protein-coupled receptors. We found that extracellular ATP enhanced antigen-dependent and -independent MC degranulation via activation of ionotropic P2X4 receptors. P2X4 receptor activation itself had no effect on MC degranulation, but significantly enhanced antigen-triggered degranulation. Stimulation of the P2X4 receptor potentiated the FcεRI-mediated tyrosine kinase signaling cascade. In addition to antigen-induced responses, P2X4 receptor signaling also affected antigen-independent MC responses. Thus, co-stimulation of ATP and Gi-coupled receptor agonists, such as prostaglandin E2 (PGE2) and adenosine, resulted in synergistic degranulation. The significance of P2X4 receptor signaling in allergic and inflammatory responses in vivo was confirmed by impaired responses of antigen-induced passive anaphylaxis and PGE2-induced increases in vascular permeability in P2rx4 knockout mice compared to that of wild-type mice. These results suggest that the P2X4 receptor is a potential therapeutic target for both antigen-dependent and -independent allergic reactions.

Eosinophils and Neutrophils Eliminate Migrating Strongyloides ratti Larvae at the Site of Infection in the Context of Extracellular DNA Trap Formation.

Parasitic nematodes such as hookworms actively penetrate the skin of their hosts, encountering skin-resident innate immune cells that represent the host´s first line of defense. Here we use Strongyloides ratti as a model for an intestinal helminth parasite with tissue migrating stages. We show that interception and killing of migrating larvae in mice during a 1st infection occurred predominantly in skin and muscle tissue before larvae migrated via lung and head tissue to the intestine. Inhibition of larval migration was even more efficient in immune mice during a 2nd infection where larvae barely left the site of entry i.e. the foot. Using cell-deficient mice we show that interception in the tissue was predominantly mediated by neutrophils and eosinophils while basophils and mast cells were dispensable in vivo. Likewise, neutrophils and eosinophils inhibited S. ratti L3 motility in vitro in the context of ETosis. Thereby eosinophils were strictly dependent on the presence of anti-S. ratti antibodies while neutrophils inhibited L3 motility as such. Also, MPO and MMP-9 were released by neutrophils in response to L3 alone, but immune plasma further stimulated MPO release in an antibody-dependent manner. In summary, our findings highlight the central role of the skin as first line of defense against helminth parasites in both, innate and adaptive immunity.

TLR2 Regulates Mast Cell IL-6 and IL-13 Production During Listeria monocytogenes Infection.

Listeria monocytogenes (L.m) is efficiently controlled by several cells of the innate immunity, including the Mast Cell (MC). MC is activated by L.m inducing its degranulation, cytokine production and microbicidal mechanisms. TLR2 is required for the optimal control of L.m infection by different cells of the immune system. However, little is known about the MC receptors involved in recognizing this bacterium and whether these interactions mediate MC activation. In this study, we analyzed whether TLR2 is involved in mediating different MC activation responses during L.m infection. We found that despite MC were infected with L.m, they were able to clear the bacterial load. In addition, MC degranulated and produced ROS, TNF-α, IL-1ß, IL-6, IL-13 and MCP-1 in response to bacterial infection. Interestingly, L.m induced the activation of signaling proteins: ERK, p38 and NF-κB. When TLR2 was blocked, L.m endocytosis, bactericidal activity, ROS production and mast cell degranulation were not affected. Interestingly, only IL-6 and IL-13 production were affected when TLR2 was inhibited in response to L.m infection. Furthermore, p38 activation depended on TLR2, but not ERK or NF-κB activation. These results indicate that TLR2 mediates only some MC activation pathways during L.m infection, mainly those related to IL-6 and IL-13 production.

IgE-Induced Mast Cell Activation Is Suppressed by Dihydromyricetin through the Inhibition of NF-κB Signaling Pathway.

Mast cells play a crucial role in the pathogenesis of type 1 allergic reactions by binding to IgE and allergen complexes and initiating the degranulation process, releasing pro-inflammatory mediators. Recently, research has focused on finding a stable and effective anti-allergy compound to prevent or treat anaphylaxis. Dihydromyricetin (DHM) is a flavonoid compound with several pharmacological properties, including free radical scavenging, antithrombotic, anticancer, and anti-inflammatory activities. In this study, we investigated the anti-allergic inflammatory effects and the underlying molecular mechanism of DHM in the DNP-IgE-sensitized human mast cell line, KU812. The cytokine levels and mast cell degranulation assays were determined by enzyme-linked immunosorbent assay (ELISA). The possible mechanism of the DHM-mediated anti-allergic signaling pathway was analyzed by western blotting. It was found that treatment with DHM suppressed the levels of inflammatory cytokines TNF-α and IL-6 in DNP-IgE-sensitized KU812 cells. The anti-allergic inflammatory properties of DHM were mediated by inhibition of NF-κB activation. In addition, DHM suppressed the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and mast cell-derived tryptase production. Our study shows that DHM could mitigate mast cell activation in allergic diseases.

Positive and negative roles of lipids in mast cells and allergic responses.

Mast cells are a central immune cell population that are crucial in allergic responses. They secrete granule contents and cytokines and produce a panel of lipid mediators in response to FcεRI-dependent or independent stimuli. Leukotrienes and prostaglandins derived from ω6 arachidonic acid, or specialized pro-resolving lipid mediators derived from ω3 eicosapentaenoic and docosahexaenoic acids, exert pleiotropic effects on various cells in the tissue microenvironment, thereby positively or negatively regulating allergic responses. Mast cells also express the inhibitory receptors CD300a and CD300f, which recognize structural lipids. CD300a or CD300f binding to externalized phosphatidylserine or extracellular ceramides, respectively, inhibits FcεRI-mediated mast cell activation. The inhibitory CD300-lipid axis downregulates IgE-driven, mast cell-dependent type I hypersensitivity through different mechanisms. Herein, we provide an overview of our current understanding of the biological roles of lipids in mast cell-dependent allergic responses.

Safranal Alleviated OVA-Induced Asthma Model and Inhibits Mast Cell Activation.

Introduction: Asthma is a chronic and recurring airway disease, which related to mast cell activation. Many compounds derived from Chinese herbal medicine has promising effects on stabilizing mast cells and decreasing inflammatory mediator production. Safranal, one of the active compounds from Crocus sativus, shows many anti-inflammatory properties. In this study, we evaluated the effect of safranal in ovalbumin (OVA)-induced asthma model. Furthermore, we investigate the effectiveness of safranal on stabilizing mast cell and inhibiting the production of inflammatory mediators in passive systemic anaphylaxis (PSA) model. Methods: OVA-induced asthma and PSA model were used to evaluate the effect of safranal in vivo. Lung tissues were collected for H&E, TB, IHC, and PAS staining. ELISA were used to determine level of IgE and chemokines (IL-4, IL-5, TNF-α, and IFN-γ). RNA sequencing was used to uncovers genes that safranal regulate. Bone marrow-derived mast cells (BMMCs) were used to investigate the inhibitory effect and mechanism of safranal. Cytokine production (IL-6, TNF-α, and LTC4) and NF-κB and MAPKs signaling pathway were assessed. Results: Safranal reduced the level of serum IgE, the number of mast cells in lung tissue were decreased and Th1/Th2 cytokine levels were normalized in OVA-induced asthma model. Furthermore, safranal inhibited BMMCs degranulation and inhibited the production of LTC4, IL-6, and TNF-α. Safranal inhibits NF-κB and MAPKs pathway protein phosphorylation and decreases NF-κB p65, AP-1 nuclear translocation. In the PSA model, safranal reduced the levels of histamine and LTC4 in serum. Conclusions: Safranal alleviates OVA-induced asthma, inhibits mast cell activation and PSA reaction. The possible mechanism occurs through the inhibition of the MAPKs and NF-κB pathways.

Bispecific NKG2D-CD3 and NKG2D-CD16 Fusion Proteins as Novel Treatment Option in Advanced Soft Tissue Sarcomas.

Soft tissue sarcoma (STS) constitutes a rare group of heterogeneous malignancies. Effective treatment options for most subtypes of STS are still limited. As a result, especially in metastatic disease, prognosis is still dismal. The ligands for the activating immunoreceptor NKG2D (NKG2DL) are commonly expressed in STS, but generally absent in healthy tissues. This provides the rationale for utilization of NKG2DL as targets for immunotherapeutic approaches. We here report on the preclinical characterization of bispecific fusion proteins (BFP) consisting of the extracellular domain of the NKG2D receptor fused to Fab-fragments directed against CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) for treatment of STS. After characterization of NKG2DL expression patterns on various STS cell lines, we demonstrated that both NKG2D-CD16 and NKG2D-CD3 induce profound T and NK cell reactivity as revealed by analysis of activation, degranulation and secretion of IFNγ as well as granule associated proteins, resulting in potent target cell lysis. In addition, the stimulatory capacity of the constructs to induce T and NK cell activation was analyzed in heavily pretreated STS patients and found to be comparable to healthy donors. Our results emphasize the potential of NKG2D-CD3 and NKG2D-CD16 BFP to target STS even in an advanced disease.