G6b-B antibody-based cis-acting platelet receptor inhibitors (CAPRIs) as a new family of anti-thrombotic therapeutics.

Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet therapies elicit bleeding, raising the question whether the thrombotic activity of platelets can be targeted separately. In this study, we describe a novel approach of inhibiting platelet activation through the use of bispecific single-chain variable fragments (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that harness the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor G6b-B (G6B) to suppress immunoreceptor tyrosine-based (ITAM)-containing receptor-mediated platelet activation. CAPRI-mediated hetero-clustering of G6B with either the ITAM-containing GPVI-FcR γ-chain complex or FcγRIIA (CD32A) inhibited collagen- or immune complex-induced platelet aggregation. G6B-GPVI CAPRIs strongly and specifically inhibited thrombus formation on collagen under arterial shear, whereas G6B-CD32A CAPRI strongly and specifically inhibited thrombus formation to heparin-induced thrombocytopenia, vaccine-induced thrombotic thrombocytopenia and antiphospholipid syndrome complexes on Von Willebrand Factor-coated surfaces and photochemical-injured endothelial cells under arterial shear. Our findings provide proof-of-concept that CAPRIs are highly effective at inhibiting ITAM receptor-mediated platelet activation, laying the foundation for a novel family of anti-thrombotic therapeutics with potentially improved efficacy and fewer bleeding outcomes compared with current anti-platelet therapies. .

Standardized high-dimensional spectral cytometry protocol and panels for whole blood immune phenotyping in clinical and translational studies.

Flow cytometry is the method of choice for immunophenotyping in the context of clinical, translational, and systems immunology studies. Among the latter, the Milieu Intérieur (MI) project aims at defining the boundaries of a healthy immune response to identify determinants of immune response variation. MI used immunophenotyping of a 1000 healthy donor cohort by flow cytometry as a principal outcome for immune variance at steady state. New generation spectral cytometers now enable high-dimensional immune cell characterization from small sample volumes. Therefore, for the MI 10-year follow up study, we have developed two high-dimensional spectral flow cytometry panels for deep characterization of innate and adaptive whole blood immune cells (35 and 34 fluorescent markers, respectively). We have standardized the protocol for sample handling, staining, acquisition, and data analysis. This approach enables the reproducible quantification of over 182 immune cell phenotypes at a single site. We have applied the protocol to discern minor differences between healthy and patient samples and validated its value for application in immunomonitoring studies. Our protocol is currently used for characterization of the impact of age and environmental factors on peripheral blood immune phenotypes of >400 donors from the initial MI cohort.

Isolation methods determine human neutrophil responses after stimulation.

Studying neutrophils is challenging due to their limited lifespan, inability to proliferate, and resistance to genetic manipulation. Neutrophils can sense various cues, making them susceptible to activation by blood collection techniques, storage conditions, RBC lysis, and the isolation procedure itself. Here we assessed the impact of the five most used methods for neutrophil isolation on neutrophil yield, purity, activation status and responsiveness. We monitored surface markers, reactive oxygen species production, and DNA release as a surrogate for neutrophil extracellular trap (NET) formation. Our results show that neutrophils isolated by negative immunomagnetic selection and density gradient methods, without RBC lysis, resembled untouched neutrophils in whole blood. They were also less activated and more responsive to milder stimuli in functional assays compared to neutrophils obtained using density gradients requiring RBC lysis. Our study highlights the importance of selecting the appropriate method for studying neutrophils, and underscores the need for standardizing isolation protocols to facilitate neutrophil subset characterization and inter-study comparisons.

A human immune system (HIS) mouse model that dissociates roles for mouse and human FcR+ cells during antibody-mediated immune responses.

Human immune system (HIS) mice provide a model to study human immune responses in vivo. Currently available HIS mouse models may harbor mouse Fc Receptor (FcR)-expressing cells that exert potent effector functions following administration of human Ig. Previous studies showed that the ablation of the murine FcR gamma chain (FcR-γ) results in loss of antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vivo. We created a new FcR-γ-deficient HIS mouse model to compare host (mouse) versus graft (human) effects underlying antibody-mediated immune responses in vivo. FcR-γ-deficient HIS recipients lack expression and function of mouse activating FcRs and can be stably and robustly reconstituted with human immune cells. By screening blood B-cell depletion by rituximab Ig variants, we found that human FcγRs-mediated IgG1 effects, whereas mouse activating FcγRs were dominant in IgG4 effects. Complement played a role as an IgG1 variant (IgG1 K322A) lacking complement binding activity was largely ineffective. Finally, we provide evidence that FcγRIIIA on human NK cells could mediate complement-independent B-cell depletion by IgG1 K322A. We anticipate that our FcR-γ-deficient HIS model will help clarify mechanisms of action of exogenous administered human antibodies in vivo.

IgG Subclass-Dependent Pulmonary Antigen Retention during Acute IgG-Dependent Systemic Anaphylaxis in Mice.

Mouse models of active systemic anaphylaxis rely predominantly on IgG Abs forming IgG-allergen immune complexes that induce IgG receptor-expressing neutrophils and monocytes/macrophages to release potent mediators, leading to systemic effects. Whether anaphylaxis initiates locally or systemically remains unknown. In this study, we aimed at identifying the anatomical location of IgG-allergen immune complexes during anaphylaxis. Active systemic anaphylaxis was induced following immunization with BSA and i.v. challenge with fluorescently labeled BSA. Ag retention across different organs was examined using whole-body fluorescence imaging, comparing immunized and naive animals. Various mouse models and in vivo deletion strategies were employed to determine the contribution of IgG receptors, complement component C1q, myeloid cell types, and anaphylaxis mediators. We found that following challenge, Ag diffused systemically, but specifically accumulated in the lungs of mice sensitized to that Ag, where it formed large Ab-dependent aggregates in the vasculature. Ag retention in the lungs did not rely on IgG receptors, C1q, neutrophils, or macrophages. IgG2a-mediated, but neither IgG1- nor IgG2b-mediated, passive systemic anaphylaxis led to Ag retention in the lung. Neutrophils and monocytes significantly accumulated in the lungs after challenge and captured high amounts of Ag, which led to downmodulation of surface IgG receptors and triggered their activation. Thus, within minutes of systemic injection in sensitized mice, Ag formed aggregates in the lung and liver vasculature, but accumulated specifically and dose-dependently in the lung. Neutrophils and monocytes recruited to the lung captured Ag and became activated. However, Ag aggregation in the lung vasculature was not necessary for anaphylaxis induction.

The role of neutrophils in antibody-driven autoimmune cytopenias.

Autoimmune cytopenias are a consequence of autoantibodies that target blood cell lineages and mark them for their accelerated destruction, mostly through phagocytosis by monocytes and macrophages and complement activation. Neutrophils, although equipped with Fc and complement receptors and effector mechanisms that are critical in other autoimmune conditions, remained long overlooked. Recent reports, however, propose a new and possibly critical role of neutrophils. In this review, we gathered available evidence on the contribution of neutrophils to the development, onset, and consequences of autoantibody-dependent cytopenias.

Specificity of mouse and human Fcgamma receptors and their polymorphic variants for IgG subclasses of different species.

IgG is the predominant antibody class generated during infections and used for the generation of therapeutic antibodies. Antibodies are mainly characterized in or generated from animal models that support particular infections, respond to particular antigens or allow the generation of hybridomas. Due to the availability of numerous transgenic mouse models and the ease of performing bioassays with human blood cells in vitro, most antibodies from species other than mice and humans are tested in vitro using human cells and/or in vivo using mice. In this process, it is expected, but not yet systematically documented, that IgG from these species interact with human or mouse IgG receptors (FcγRs). In this study, we undertook a systematic assessment of binding specificities of IgG from various species to the families of mouse and human FcγRs including their polymorphic variants. Our results document the specific binding patterns for each of these IgG (sub)classes, reveal possible caveats of antibody-based immunoassays, and will be a useful reference for the transition from one animal model to preclinical mouse models or human cell-based bioassays.

Neutrophil-specific gain-of-function mutations in Nlrp3 promote development of cryopyrin-associated periodic syndrome.

Gain-of-function mutations in NLRP3 are responsible for a spectrum of autoinflammatory diseases collectively referred to as "cryopyrin-associated periodic syndromes" (CAPS). Treatment of CAPS patients with IL-1-targeted therapies is effective, confirming a central pathogenic role for IL-1ß. However, the specific myeloid cell population(s) exhibiting inflammasome activity and sustained IL-1ß production in CAPS remains elusive. Previous reports suggested an important role for mast cells (MCs) in this process. Here, we report that, in mice, gain-of-function mutations in Nlrp3 restricted to neutrophils, and to a lesser extent macrophages/dendritic cells, but not MCs, are sufficient to trigger severe CAPS. Furthermore, in patients with clinically established CAPS, we show that skin-infiltrating neutrophils represent a substantial biological source of IL-1ß. Together, our data indicate that neutrophils, rather than MCs, can represent the main cellular drivers of CAPS pathology.

Platelet FcγRIIA-induced serotonin release exacerbates the severity of transfusion-related acute lung injury in mice.

Transfusion-related acute lung injury (TRALI) remains a major cause of transfusion-related fatalities. The mechanism of human antibody-mediated TRALI, especially the involvement of the Fcγ receptors, is not clearly established. Contrary to mice, human platelets are unique in their expression of the FcγRIIA/CD32A receptor, suggesting that our understanding of the pathogenesis of antibody-mediated TRALI is partial, as the current murine models incompletely recapitulate the human immunology. We evaluated the role of FcγRIIA/CD32A in TRALI using a humanized mouse model expressing the FcγRIIA/CD32A receptor. When challenged with a recombinant chimeric human immunoglobulin G1/mouse anti-major histocompatibility complex class I monoclonal antibody, these mice exhibited exacerbated alveolar edema and higher mortality compared with wild-type (WT) mice. Unlike in WT mice, monocytes/macrophages in CD32A+ mice were accessory for TRALI initiation, indicating the decisive contribution of another cell type. Platelet activation was dramatically increased in CD32A+ animals, resulting in their increased consumption and massive release of their granule contents. Platelet depletion prevented the exacerbation of TRALI in CD32A+ mice but did not affect TRALI in WT animals. By blocking platelet serotonin uptake with fluoxetine, we showed that the severity of TRALI in CD32A+ mice resulted from the serotonin released by the activated platelets. Furthermore, inhibition of 5-hydroxytryptamine 2A serotonin receptor with sarpogrelate, before or after the induction of TRALI, abolished the aggravation of lung edema in CD32A+ mice. Our findings show that platelet FcγRIIA/CD32A activation exacerbates antibody-mediated TRALI and provide a rationale for designing prophylactic and therapeutic strategies targeting the serotonin pathway to attenuate TRALI in patients.

Cofilin1-driven actin dynamics controls migration of thymocytes and is essential for positive selection in the thymus.

Actin dynamics is essential for T-cell development. We show here that cofilin1 is the key molecule for controlling actin filament turnover in this process. Mice with specific depletion of cofilin1 in thymocytes showed increased steady-state levels of actin filaments, and associated alterations in the pattern of thymocyte migration and adhesion. Our data suggest that cofilin1 is controlling oscillatory F-actin changes, a parameter that influences the migration pattern in a 3-D environment. In a collagen matrix, cofilin1 controls the speed and resting intervals of migrating thymocytes. Cofilin1 was not involved in thymocyte proliferation, cell survival, apoptosis or surface receptor trafficking. However, in cofilin1 mutant mice, impaired adhesion and migration resulted in a specific block of thymocyte differentiation from CD4/CD8 double-positive thymocytes towards CD4 and CD8 single-positive cells. Our data suggest that tuning of the dwelling time of thymocytes in the thymic niches is tightly controlled by cofilin1 and essential for positive selection during T-cell differentiation. We describe a novel role of cofilin1 in the physiological context of migration-dependent cell differentiation.

Human IgA binds a diverse array of commensal bacteria.

In humans, several grams of IgA are secreted every day in the intestinal lumen. While only one IgA isotype exists in mice, humans secrete IgA1 and IgA2, whose respective relations with the microbiota remain elusive. We compared the binding patterns of both polyclonal IgA subclasses to commensals and glycan arrays and determined the reactivity profile of native human monoclonal IgA antibodies. While most commensals are dually targeted by IgA1 and IgA2 in the small intestine, IgA1+IgA2+ and IgA1-IgA2+ bacteria coexist in the colon lumen, where Bacteroidetes is preferentially targeted by IgA2. We also observed that galactose-α terminated glycans are almost exclusively recognized by IgA2. Although bearing signs of affinity maturation, gut-derived IgA monoclonal antibodies are cross-reactive in the sense that they bind to multiple bacterial targets. Private anticarbohydrate-binding patterns, observed at clonal level as well, could explain these apparently opposing features of IgA, being at the same time cross-reactive and selective in its interactions with the microbiota.

Expression, Role, and Regulation of Neutrophil Fcγ Receptors.

Neutrophils are best known for their critical role in host defense, for which they utilize multiple innate immune mechanisms, including microbe-associated pattern recognition, phagocytosis, production of reactive oxygen species, and the release of potent proteases, mediators, antimicrobials, and neutrophil extracellular traps. Beyond their well-established contribution to innate immunity, neutrophils were more recently reported to interact with various other cell types, including cells from the adaptive immune system, thereby enabling neutrophils to tune the overall immune response of the host. Neutrophils express different receptors for IgG antibodies (Fcγ receptors), which facilitate the engulfment of IgG-opsonized microbes and trigger cell activation upon cross-linking of several receptors. Indeed, FcγRs (via IgG antibodies) confer neutrophils with a key feature of the adaptive immunity: an antigen-specific cell response. This review summarizes the expression and function of FcγRs on human neutrophils in health and disease and how they are affected by polymorphisms in the FCGR loci. Additionally, we will discuss the role of neutrophils in providing help to marginal zone B cells for the production of antibodies, which in turn may trigger neutrophil effector functions when engaging FcγRs.

An IgG-induced neutrophil activation pathway contributes to human drug-induced anaphylaxis.

Anaphylaxis is a systemic acute hypersensitivity reaction that is considered to depend on allergen-specific immunoglobulin E (IgE) antibodies and histamine release by mast cells and basophils. Nevertheless, allergen-specific IgG antibodies have been proposed to contribute when the allergen is an abundant circulating large molecule, e.g., after infusions of therapeutic antibodies or dextran. Data from animal models demonstrate a pathway involving platelet-activating factor (PAF) release by monocytes/macrophages and neutrophils activated via their Fc gamma receptors (FcγRs). We hypothesized that such a pathway may also apply to small drugs and could be responsible for non-IgE-mediated anaphylaxis and influence anaphylaxis severity in humans. We prospectively conducted a multicentric study of 86 patients with suspected anaphylaxis to neuromuscular-blocking agents (NMBAs) during general anesthesia and 86 matched controls. We found that concentrations of anti-NMBA IgG and markers of FcγR activation, PAF release, and neutrophil activation correlated with anaphylaxis severity. Neutrophils underwent degranulation and NETosis early after anaphylaxis onset, and plasma-purified anti-NMBA IgG triggered neutrophil activation ex vivo in the presence of NMBA. Neutrophil activation could also be observed in patients lacking evidence of classical IgE-dependent anaphylaxis. This study supports the existence of an IgG-neutrophil pathway in human NMBA-induced anaphylaxis, which may aggravate anaphylaxis in combination with the IgE pathway or underlie anaphylaxis in the absence of specific IgE. These results reconcile clinical and experimental data on the role of antibody classes in anaphylaxis and could inform diagnostic approaches to NMBA-induced acute hypersensitivity reactions.

Mouse Models and Tools for the in vivo Study of Neutrophils.

Neutrophils are the most abundant leukocytes in human blood and critical actors of the immune system. Many neutrophil functions and facets of their activity in vivo were revealed by studying genetically modified mice or by tracking fluorescent neutrophils in animals using imaging approaches. Assessing the roles of neutrophils can be challenging, especially when exact molecular pathways are questioned or disease states are interrogated that alter normal neutrophil homeostasis. This review discusses the main in vivo models for the study of neutrophils, their advantages and limitations. The side-by-side comparison underlines the necessity to carefully choose the right model(s) to answer a given scientific question, and exhibit caveats that need to be taken into account when designing experimental procedures. Collectively, this review suggests that at least two models should be employed to legitimately conclude on neutrophil functions.

Publisher Correction: Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors.

In the version of this article initially published, the name of one author was incorrect (James P. Santo). The correct name is James P. Di Santo. The error has been corrected in the HTML and PDF versions of the article.