Fc gamma receptors: Their evolution, genomic architecture, genetic variation, and impact on human disease.

Fc gamma receptors (FcγRs) are a family of receptors that bind IgG antibodies and interface at the junction of humoral and innate immunity. Precise regulation of receptor expression provides the necessary balance to achieve healthy immune homeostasis by establishing an appropriate immune threshold to limit autoimmunity but respond effectively to infection. The underlying genetics of the FCGR gene family are central to achieving this immune threshold by regulating affinity for IgG, signaling efficacy, and receptor expression. The FCGR gene locus was duplicated during evolution, retaining very high homology and resulting in a genomic region that is technically difficult to study. Here, we review the recent evolution of the gene family in mammals, its complexity and variation through copy number variation and single-nucleotide polymorphism, and impact of these on disease incidence, resolution, and therapeutic antibody efficacy. We also discuss the progress and limitations of current approaches to study the region and emphasize how new genomics technologies will likely resolve much of the current confusion in the field. This will lead to definitive conclusions on the impact of genetic variation within the FCGR gene locus on immune function and disease.

HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy.

BACKGROUND: Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. METHODS: We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. RESULTS: We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. CONCLUSION: Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Dual dean entrainment with volume ratio modulation for efficient droplet co-encapsulation: extreme single-cell indexing.

The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ∼1 million cells per hour, while increasing assay signal-to-noise with cell multiplet rates of ∼2.5% and capturing ∼70% of cells. The method, called Pirouette coupling, extends our capacity to investigate biological systems.

Transfer learning efficiently maps bone marrow cell types from mouse to human using single-cell RNA sequencing.

Biomedical research often involves conducting experiments on model organisms in the anticipation that the biology learnt will transfer to humans. Previous comparative studies of mouse and human tissues were limited by the use of bulk-cell material. Here we show that transfer learning-the branch of machine learning that concerns passing information from one domain to another-can be used to efficiently map bone marrow biology between species, using data obtained from single-cell RNA sequencing. We first trained a multiclass logistic regression model to recognize different cell types in mouse bone marrow achieving equivalent performance to more complex artificial neural networks. Furthermore, it was able to identify individual human bone marrow cells with 83% overall accuracy. However, some human cell types were not easily identified, indicating important differences in biology. When re-training the mouse classifier using data from human, less than 10 human cells of a given type were needed to accurately learn its representation. In some cases, human cell identities could be inferred directly from the mouse classifier via zero-shot learning. These results show how simple machine learning models can be used to reconstruct complex biology from limited data, with broad implications for biomedical research.

Twice-told tales: Self-repetition decreases observer assessments of performer authenticity.

People often engage in self-repetition-repeating the same story, joke, or presentation across different audiences. While behaving consistently has generally been found to enhance perceptions of authenticity, 10 studies demonstrate that performers who are revealed to be self-repeating are perceived as less authentic. We find convergent evidence that this effect is driven by observers' implicit assumption that social interactions are unique. Self-repetitions violate this assumption, leading observers to judge performers as inauthentic because they are thought to be falsely presenting their performance as unique when it is not. We demonstrate this effect across multiple contexts (politics, entrepreneurship, tour guiding, and comedy), finding that observer awareness of self-repetition decreases perceived authenticity even in situations in which it is normative to repeat a performance and in which repetition is required. The decrease in authenticity is eliminated only when performers overtly acknowledge self-repetition, as performers are no longer viewed as falsely presenting themselves. Moreover, performers who fail to acknowledge their self-repetition are penalized similarly to those who explicitly lie that the performance is unique-an unacknowledged self-repetition is thus seen as a lie by omission. Finally, we recorded repeated job interview responses and found that observers who were unaware of the self-repetition could not discern tangible differences between unrepeated and repeated responses. However, when observers believed that they were viewing a self-repetition, they judged the interviewees as less authentic. Together, our findings provide insight into how people assess the authenticity of self-presentational behaviors and the implicit assumptions that influence social judgments. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Regulation of the Bone Vascular Network is Sexually Dimorphic.

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High-resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone-derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone-derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.

Lanthanides compete with calcium for binding to cadherins and inhibit cadherin-mediated cell adhesion.

Lanthanides are rare-earth metals with a broad range of applications in biological research and medicine. In addition to their unique magnetic and spectroscopic properties, lanthanides are also effective mimics of calcium and can stimulate or inhibit the function of calcium-binding proteins. Cadherins are a large family of calcium-binding proteins that facilitate cell adhesion and play key roles in embryo development, tissue homeostasis and tumour metastasis. However, whether lanthanides can bind cadherins and functionally replace calcium binding has not been comprehensively explored. In this study, we investigated the effect of lanthanide binding on cadherin structure and function using terbium, which is a commonly used lanthanide for protein spectroscopy and a proposed anti-cancer agent. We demonstrate that terbium can compete with calcium for binding to calcium-binding sites in cadherins. Terbium binding to cadherins abolished their cell adhesive activity and rendered cadherins sensitive to proteolysis by trypsin. Molecular dynamics simulations indicate that replacement of calcium by terbium results in structural rearrangements and increases the flexibility of the cadherin ectodomain. These changes in structure and dynamics are likely to underlie the inability of lanthanide-bound cadherins to support cell adhesion. Taken together, our findings further knowledge on lanthanide interactions with calcium-binding proteins and provide new insight into the influence of metal chemistry on cadherin structure, dynamics and function.

A single cycle influenza virus coated in H7 haemagglutinin generates neutralizing antibody responses to haemagglutinin and neuraminidase glycoproteins and protection from heterotypic challenge.

A non-replicating form of pseudotyped influenza virus, inactivated by suppression of the haemagglutinin signal sequence (S-FLU), can act as a broadly protective vaccine. S-FLU can infect for a single round only, and induces heterotypic protection predominantly through activation of cross-reactive T cells in the lung. Unlike the licensed live attenuated virus, it cannot reassort a pandemic haemagglutinin (HA) into seasonal influenza. Here we present data on four new forms of S-FLU coated with H7 HAs from either A/Anhui/1/2013, A/Shanghai/1/2013, A/Netherlands/219/2003 or A/New York/107/2003 strains of H7 virus. We show that intranasal vaccination induced a strong local CD8 T cell response and protected against heterosubtypic X31 (H3N2) virus and highly virulent PR8 (H1N1), but not influenza B virus. Intranasal vaccination also induced a strong neutralizing antibody response to the encoded neuraminidase. If given at higher dose in the periphery with intraperitoneal administration, H7 S-FLU induced a specific neutralizing antibody response to H7 HA coating the particle. Polyvalent intraperitoneal vaccination with mixed H7 S-FLU induced a broadly neutralizing antibody response to all four H7 strains. S-FLU is a versatile vaccine candidate that could be rapidly mobilized ahead of a new pandemic threat.

Stem Cell Differentiation as a Non-Markov Stochastic Process.

Pluripotent stem cells can self-renew in culture and differentiate along all somatic lineages in vivo. While much is known about the molecular basis of pluripotency, the mechanisms of differentiation remain unclear. Here, we profile individual mouse embryonic stem cells as they progress along the neuronal lineage. We observe that cells pass from the pluripotent state to the neuronal state via an intermediate epiblast-like state. However, analysis of the rate at which cells enter and exit these observed cell states using a hidden Markov model indicates the presence of a chain of unobserved molecular states that each cell transits through stochastically in sequence. This chain of hidden states allows individual cells to record their position on the differentiation trajectory, thereby encoding a simple form of cellular memory. We suggest a statistical mechanics interpretation of these results that distinguishes between functionally distinct cellular "macrostates" and functionally similar molecular "microstates" and propose a model of stem cell differentiation as a non-Markov stochastic process.

Nanog Fluctuations in Embryonic Stem Cells Highlight the Problem of Measurement in Cell Biology.

A number of important pluripotency regulators, including the transcription factor Nanog, are observed to fluctuate stochastically in individual embryonic stem cells. By transiently priming cells for commitment to different lineages, these fluctuations are thought to be important to the maintenance of, and exit from, pluripotency. However, because temporal changes in intracellular protein abundances cannot be measured directly in live cells, fluctuations are typically assessed using genetically engineered reporter cell lines that produce a fluorescent signal as a proxy for protein expression. Here, using a combination of mathematical modeling and experiment, we show that there are unforeseen ways in which widely used reporter strategies can systematically disturb the dynamics they are intended to monitor, sometimes giving profoundly misleading results. In the case of Nanog, we show how genetic reporters can compromise the behavior of important pluripotency-sustaining positive feedback loops, and induce a bifurcation in the underlying dynamics that gives rise to heterogeneous Nanog expression patterns in reporter cell lines that are not representative of the wild-type. These findings help explain the range of published observations of Nanog variability and highlight the problem of measurement in live cells.

The need to belong motivates demand for authentic objects.

The present studies examine how demand for certain types of authentic objects is related to a more fundamental need to form social connections with others. Specifically, Experiment 1 demonstrates that manipulating the need to belong leads to greater valuation of celebrity memorabilia. Experiment 2 provides converging evidence by demonstrating that individual differences in the need to belong moderate the relationship between beliefs in essence transfer (i.e., contagion) and valuation. This paper lends insight into the underlying motives behind demand for authentic objects and, more broadly, reinforces the compensatory role of consumption in satisfying core psychological needs.

The regulatory role of invariant NKT cells in tumor immunity.

Invariant natural killer T (iNKT) cells are a unique population of T lymphocytes, which lie at the interface between the innate and adaptive immune systems, and are important mediators of immune responses and tumor surveillance. iNKT cells recognize lipid antigens in a CD1d-dependent manner; their subsequent activation results in a rapid and specific downstream response, which enhances both innate and adaptive immunity. The capacity of iNKT cells to modify the immune microenvironment influences the ability of the host to control tumor growth, making them an important population to be harnessed in the clinic for the development of anticancer therapeutics. Indeed, the identification of strong iNKT-cell agonists, such as α-galactosylceramide (α-GalCer) and its analogues, has led to the development of synthetic lipids that have shown potential in vaccination and treatment against cancers. In this Masters of Immunology article, we discuss these latest findings and summarize the major discoveries in iNKT-cell biology, which have enabled the design of potent strategies for immune-mediated tumor destruction.

CD1d-dependent endogenous and exogenous lipid antigen presentation.

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipids in the context of CD1d molecules, and through the activation and maturation of dendritic cells and B cells, can significantly enhance priming of antigen-specific T and B cell responses. Recent findings have provided important insights into the recognition of several novel endogenous lipids by iNKT cells, and into the mechanisms controlling their generation and loading onto CD1d molecules. In this review we discuss these latest findings and describe the role of autophagy in iNKT cell development and activation.

Are artworks more like people than artifacts? Individual concepts and their extensions.

This paper examines people's reasoning about identity continuity (i.e., how people decide that a particular object is the same object over time) and its relation to previous research on how people value one-of-a-kind artifacts, such as artwork. We propose that judgments about the continuity of artworks are related to judgments about the continuity of individual persons because art objects are seen as physical extensions of their creators. We report a reanalysis of previous data and the results of two new empirical studies that test this hypothesis. The first study demonstrates that the mere categorization of an object as "art" versus "a tool" changes people's intuitions about the persistence of those objects over time. In a second study, we examine some conditions that may lead artworks to be thought of as different from other artifacts. These observations inform both current understanding of what makes some objects one-of-a-kind as well as broader questions regarding how people intuitively think about the persistence of human agents.

Modelling of human low frequency sound localization acuity demonstrates dominance of spatial variation of interaural time difference and suggests uniform just-noticeable differences in interaural time difference.

Sound source localization is critical to animal survival and for identification of auditory objects. We investigated the acuity with which humans localize low frequency, pure tone sounds using timing differences between the ears. These small differences in time, known as interaural time differences or ITDs, are identified in a manner that allows localization acuity of around 1° at the midline. Acuity, a relative measure of localization ability, displays a non-linear variation as sound sources are positioned more laterally. All species studied localize sounds best at the midline and progressively worse as the sound is located out towards the side. To understand why sound localization displays this variation with azimuthal angle, we took a first-principles, systemic, analytical approach to model localization acuity. We calculated how ITDs vary with sound frequency, head size and sound source location for humans. This allowed us to model ITD variation for previously published experimental acuity data and determine the distribution of just-noticeable differences in ITD. Our results suggest that the best-fit model is one whereby just-noticeable differences in ITDs are identified with uniform or close to uniform sensitivity across the physiological range. We discuss how our results have several implications for neural ITD processing in different species as well as development of the auditory system.

Acute myeloid leukemia creates an arginase-dependent immunosuppressive microenvironment.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and the second most common frequent leukemia of childhood. Patients may present with lymphopenia or pancytopenia at diagnosis. We investigated the mechanisms by which AML causes pancytopenia and suppresses patients' immune response. This study identified for the first time that AML blasts alter the immune microenvironment through enhanced arginine metabolism. Arginase II is expressed and released from AML blasts and is present at high concentrations in the plasma of patients with AML, resulting in suppression of T-cell proliferation. We extended these results by demonstrating an arginase-dependent ability of AML blasts to polarize surrounding monocytes into a suppressive M2-like phenotype in vitro and in engrafted nonobese diabetic-severe combined immunodeficiency mice. In addition, AML blasts can suppress the proliferation and differentiation of murine granulocyte-monocyte progenitors and human CD34(+) progenitors. Finally, the study showed that the immunosuppressive activity of AML blasts can be modulated through small-molecule inhibitors of arginase and inducible nitric oxide synthase, suggesting a novel therapeutic target in AML. The results strongly support the hypothesis that AML creates an immunosuppressive microenvironment that contributes to the pancytopenia observed at diagnosis.

HLA-DP, HLA-DQ, and HLA-DR have different requirements for invariant chain and HLA-DM.

The MHC is central to the adaptive immune response. The human MHC class II is encoded by three different isotypes, HLA-DR, -DQ, and -DP, each being highly polymorphic. In contrast to HLA-DR, the intracellular assembly and trafficking of HLA-DP molecules have not been studied extensively. However, different HLA-DP variants can be either protective or risk factors for infectious diseases (e.g. hepatitis B), immune dysfunction (e.g. berylliosis), and autoimmunity (e.g. myasthenia gravis). Here, we establish a system to analyze the chaperone requirements for HLA-DP and to compare the assembly and trafficking of HLA-DP, -DQ, and -DR directly. Unlike HLA-DR1, HLA-DQ5 and HLA-DP4 can form SDS-stable dimers supported by invariant chain (Ii) in the absence of HLA-DM. Uniquely, HLA-DP also forms dimers in the presence of HLA-DM alone. In model antigen-presenting cells, SDS-stable HLA-DP complexes are resistant to treatments that prevent formation of SDS-stable HLA-DR complexes. The unexpected properties of HLA-DP molecules may help explain why they bind to a more restricted range of peptides than other human MHC class II proteins and frequently present viral peptides.

Evidence for seismogenic fracture of silicic magma.

It has long been assumed that seismogenic faulting is confined to cool, brittle rocks, with a temperature upper limit of approximately 600 degrees C (ref. 1). This thinking underpins our understanding of volcanic earthquakes, which are assumed to occur in cold rocks surrounding moving magma. However, the recent discovery of abundant brittle-ductile fault textures in silicic lavas has led to the counter-intuitive hypothesis that seismic events may be triggered by fracture and faulting within the erupting magma itself. This hypothesis is supported by recent observations of growing lava domes, where microearthquake swarms have coincided with the emplacement of gouge-covered lava spines, leading to models of seismogenic stick-slip along shallow shear zones in the magma. But can fracturing or faulting in high-temperature, eruptible magma really generate measurable seismic events? Here we deform high-temperature silica-rich magmas under simulated volcanic conditions in order to test the hypothesis that high-temperature magma fracture is seismogenic. The acoustic emissions recorded during experiments show that seismogenic rupture may occur in both crystal-rich and crystal-free silicic magmas at eruptive temperatures, extending the range of known conditions for seismogenic faulting.