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Increased use of antiviral monoclonal antibodies (mAbs) for treatment and prophylaxis necessitates better understanding of their impact on endogenous immunity to vaccines and viruses. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presented an opportunity to study immunity in individuals who received antiviral mAbs and were subsequently immunized with vaccines encoding the mAb-targeted viral spike antigen. Here, we describe the impact of administration of an antibody combination, casirivimab plus imdevimab (CAS+IMD), on immune responses to subsequent SARS-CoV-2 vaccination in humans, nonhuman primates, and mice. The presence of CAS+IMD at the time of vaccination led to a specific diminishment of vaccine-elicited pseudovirus neutralizing antibody titers without overall dampening of spike protein-directed immune responses, including antibody, B cell, and T cell responses. The impact on pseudovirus neutralizing titers extended to other therapeutic anti-spike protein antibodies when used as either monotherapy or combination therapy. The specific reduction in pseudovirus neutralizing titers was the result of epitope masking, a phenomenon where specific epitopes are bound by high-affinity antibodies and blocked from B cell recognition. Encouragingly, this reduction in pseudovirus neutralizing titers was reversible with additional booster vaccination. Moreover, by assessing the antiviral immune response in SARS-CoV-2-infected individuals treated therapeutically with CAS+IMD, we demonstrated alteration of antiviral humoral immunity in those who had received mAb therapy, but only in those individuals who had yet to start mounting their natural immune response at the time of mAb treatment. Together, these data demonstrate that antiviral mAbs can alter endogenous humoral immunity during vaccination or infection.

Immunoglobulin E (IgE) is a key driver of type 1 hypersensitivity reactions and allergic disorders, which are globally increasing in number and severity. Although eliminating pathogenic IgE may be a powerful way to treat allergy, no therapeutic strategy reported to date can fully ablate IgE production. Interleukin-4 receptor α (IL-4Rα) signaling is required for IgE class switching, and IL-4Rα blockade gradually reduces, but does not eliminate, IgE. The persistence of IgE after IL-4Rα blockade may be due to long-lived IgE + plasma cells that maintain serological memory to allergens and thus may be susceptible to plasma cell-targeted therapeutics. We demonstrate that transient administration of a B cell maturation antigen x CD3 (BCMAxCD3) bispecific antibody markedly depletes IgE, as well as other immunoglobulins, by ablating long-lived plasma cells, although IgE and other immunoglobulins rapidly rebound after treatment. Concomitant IL-4Rα blockade specifically and durably prevents the reemergence of IgE by blocking IgE class switching while allowing the restoration of other immunoglobulins. Moreover, this combination treatment prevented anaphylaxis in mice. Together with additional cynomolgus monkey and human data, our studies demonstrate that allergic memory is primarily maintained by both non-IgE + memory B cells that require class switching and long-lived IgE + plasma cells. Our combination approach to durably eliminate pathogenic IgE has potential to benefit allergy in humans while preserving antibody-mediated immunity.

Deficiency in the adipose-derived hormone leptin or leptin receptor signaling causes class 3 obesity in individuals with genetic loss-of-function mutations in leptin or its receptor LEPR and metabolic and liver disease in individuals with hypoleptinemia secondary to lipoatrophy such as in individuals with generalized lipodystrophy. Therapies that restore leptin-LEPR signaling may resolve these metabolic sequelae. We developed a fully human monoclonal antibody (mAb), REGN4461 (mibavademab), that activates the human LEPR in the absence or presence of leptin. In obese leptin knockout mice, REGN4461 normalized body weight, food intake, blood glucose, and insulin sensitivity. In a mouse model of generalized lipodystrophy, REGN4461 alleviated hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, and hepatic steatosis. In a phase 1, randomized, double-blind, placebo-controlled two-part study, REGN4461 was well tolerated with an acceptable safety profile. Treatment of individuals with overweight or obesity with REGN4461 decreased body weight over 12 weeks in those with low circulating leptin concentrations (<8 ng/ml) but had no effect on body weight in individuals with higher baseline leptin. Furthermore, compassionate-use treatment of a single patient with atypical partial lipodystrophy and a history of undetectable leptin concentrations associated with neutralizing antibodies to metreleptin was associated with noteable improvements in circulating triglycerides and hepatic steatosis. Collectively, these translational data unveil an agonist LEPR mAb that may provide clinical benefit in disorders associated with relatively low leptin concentrations.

The interleukin-6 (IL-6) family cytokines signal through gp130 receptor homodimerization or heterodimerization with a second signaling receptor and play crucial roles in various cellular processes. We determined cryo-electron microscopy structures of five signaling complexes of this family, containing full receptor ectodomains bound to their respective ligands ciliary neurotrophic factor, cardiotrophin-like cytokine factor 1 (CLCF1), leukemia inhibitory factor, IL-27, and IL-6. Our structures collectively reveal similarities and differences in the assembly of these complexes. The acute bends at both signaling receptors in all complexes bring the membrane-proximal domains to a ~30 angstrom range but with distinct distances and orientations. We also reveal how CLCF1 engages its secretion chaperone cytokine receptor-like factor 1. Our data provide valuable insights for therapeutically targeting gp130-mediated signaling.

The common γ chain (γc; IL-2RG) is a subunit of the interleukin (IL) receptors for the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The lack of appropriate neutralizing antibodies recognizing IL-2RG has made it difficult to thoroughly interrogate the role of γc cytokines in inflammatory and autoimmune disease settings. Here, we generated a γc cytokine receptor antibody, REGN7257, to determine whether γc cytokines might be targeted for T cell-mediated disease prevention and treatment. Biochemical, structural, and in vitro analysis showed that REGN7257 binds with high affinity to IL-2RG and potently blocks signaling of all γc cytokines. In nonhuman primates, REGN7257 efficiently suppressed T cells without affecting granulocytes, platelets, or red blood cells. Using REGN7257, we showed that γc cytokines drive T cell-mediated disease in mouse models of graft-versus-host disease (GVHD) and multiple sclerosis by affecting multiple aspects of the pathogenic response. We found that our xenogeneic GVHD mouse model recapitulates hallmarks of acute and chronic GVHD, with T cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. Our findings indicate that γc cytokines contribute to GVHD and aplastic anemia pathology by promoting these characteristic features. By demonstrating that broad inhibition of γc cytokine signaling with REGN7257 protects from immune-mediated disorders, our data provide evidence of γc cytokines as key drivers of pathogenic T cell responses, offering a potential strategy for the management of T cell-mediated diseases.

Although many patients with diffuse large B cell lymphoma (DLBCL) may achieve a complete response to frontline chemoimmunotherapy, patients with relapsed/refractory disease typically have poor outcomes. Odronextamab, a CD20xCD3 bispecific antibody that provides "signal 1" through the activation of the T cell receptor/CD3 complex, has exhibited early, promising activity for patients with highly refractory DLBCL in phase 1 trials. However, not all patients achieve complete responses, and many relapse, thus representing a high unmet medical need. Here, we investigated whether adding a costimulatory "signal 2" by engaging CD28 receptors on T cells could augment odronextamab activity. We demonstrate that REGN5837, a bispecific antibody that cross-links CD22-expressing tumor cells with CD28-expressing T cells, enhances odronextamab by potentiating T cell activation and cytolytic function. In preclinical DLBCL studies using human immune system-reconstituted animals, REGN5837 promotes the antitumor activity of odronextamab and induces intratumoral expansion of reprogrammable T cells while skewing away from a dysfunctional state. Although REGN5837 monotherapy shows limited activity and no toxicity in primate studies, it augments T cell activation when dosed in combination with odronextamab. In addition, analysis of non-Hodgkin lymphoma clinical samples reveals an increase in CD28 + CD8 + T cells after odronextamab treatment, demonstrating the presence of a population that could potentially be targeted by REGN5837. Collectively, our data demonstrate that REGN5837 can markedly enhance the antitumor activity of odronextamab in preclinical NHL models, and the combination of these two bispecific antibodies may provide a chemotherapy-free approach for the treatment of DLBCL.

Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1 + RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1 + RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαβ variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting “VelociT” mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αβ T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I– and MHC-II–restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

Large-scale human exome sequencing can identify rare protein-coding variants with a large impact on complex traits such as body adiposity. We sequenced the exomes of 645,626 individuals from the United Kingdom, the United States, and Mexico and estimated associations of rare coding variants with body mass index (BMI). We identified 16 genes with an exome-wide significant association with BMI, including those encoding five brain-expressed G protein-coupled receptors ( CALCR , MC4R , GIPR , GPR151 , and GPR75 ). Protein-truncating variants in GPR75 were observed in ~4/10,000 sequenced individuals and were associated with 1.8 kilograms per square meter lower BMI and 54% lower odds of obesity in the heterozygous state. Knock out of Gpr75 in mice resulted in resistance to weight gain and improved glycemic control in a high-fat diet model. Inhibition of GPR75 may provide a therapeutic strategy for obesity., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Although radiotherapy has been used for over a century to locally control tumor growth, alone it rarely induces an abscopal response or systemic antitumor immunity capable of inhibiting distal tumors outside of the irradiation field. Results from recent studies suggest that combining immune checkpoint blockades to radiotherapy may enhance abscopal activity. However, the treatment conditions and underlying immune mechanisms that consistently drive an abscopal response during radiation therapy combinations remain unknown. Here, we analyzed the antitumor responses at primary and distal tumor sites, demonstrating that the timing of αPD-1 antibody administration relative to radiotherapy determined the potency of the induced abscopal response. Blockade of the PD-1 pathway after local tumor irradiation resulted in the expansion of polyfunctional intratumoral CD8 + T cells, a decrease in intratumoral dysfunctional CD8 + T cells, expansion of reprogrammable CD8 + T cells, and induction of potent abscopal responses. However, administration of αPD-1 before irradiation almost completely abrogated systemic immunity, which associated with increased radiosensitivity and death of CD8 + T cells. The subsequent reduction of polyfunctional effector CD8 + T cells at the irradiated tumor site generated a suboptimal systemic antitumor response and the loss of abscopal responses. Therefore, this report maximizes the potential synergy between radiotherapy and αPD-1 immunotherapy, information that will benefit clinical combinations of radiotherapy and immune checkpoint blockade., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Deficiency in interleukin-36R (IL-36R) antagonist caused by loss-of-function mutations in IL-36RN leads to DITRA (deficiency of IL-36 receptor antagonist), a rare inflammatory human disease that belongs to a subgroup of generalized pustular psoriasis (GPP). We report a functional genetic mouse model of DITRA with enhanced IL-36R signaling analogous to that observed in patients with DITRA, which provides new insight into our understanding of the IL-36 family of molecules in regulating barrier integrity across multiple tissues. Humanized DITRA-like mice displayed increased skin inflammation in a preclinical model of psoriasis, and in vivo blockade of IL-36R pathway using anti-human IL-36R antibody ameliorated imiquimod-induced skin pathology as both prophylactic and therapeutic treatments. Deeper characterization of the humanized DITRA-like mice revealed that deregulated IL-36R signaling promoted tissue pathology during intestinal injury and led to impairment in mucosal restoration in the repair phase of chronic dextran sulfate sodium (DSS)-induced colitis. Blockade of IL-36R pathway significantly ameliorated DSS-induced intestinal inflammation and rescued the inability of DITRA-like mice to recover from mucosal damage in vivo. Our results indicate a central role for IL-36 in regulating proinflammatory responses in the skin and epithelial barrier function in the intestine, suggesting a new therapeutic potential for targeting the IL-36R axis in psoriasis and at the later stages of intestinal pathology in inflammatory bowel disease., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

An urgent global quest for effective therapies to prevent and treat coronavirus disease 2019 (COVID-19) is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987 and REGN10933) that targets nonoverlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques, which may model mild disease, and golden hamsters, which may model more severe disease. We demonstrate that REGN-COV-2 can greatly reduce virus load in the lower and upper airways and decrease virus-induced pathological sequelae when administered prophylactically or therapeutically in rhesus macaques. Similarly, administration in hamsters limits weight loss and decreases lung titers and evidence of pneumonia in the lungs. Our results provide evidence of the therapeutic potential of this antibody cocktail., (Copyright © 2020, American Association for the Advancement of Science.)

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Antibodies targeting the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present a promising approach to combat the coronavirus disease 2019 (COVID-19) pandemic; however, concerns remain that mutations can yield antibody resistance. We investigated the development of resistance against four antibodies to the spike protein that potently neutralize SARS-CoV-2, individually as well as when combined into cocktails. These antibodies remain effective against spike variants that have arisen in the human population. However, novel spike mutants rapidly appeared after in vitro passaging in the presence of individual antibodies, resulting in loss of neutralization; such escape also occurred with combinations of antibodies binding diverse but overlapping regions of the spike protein. Escape mutants were not generated after treatment with a noncompeting antibody cocktail., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cancer risk is highly variable in carriers of the common TP53- R337H founder allele, possibly due to the influence of modifier genes. Whole-genome sequencing identified a variant in the tumor suppressor XAF1 (E134*/Glu134Ter/rs146752602) in a subset of R337H carriers. Haplotype-defining variants were verified in 203 patients with cancer, 582 relatives, and 42,438 newborns. The compound mutant haplotype was enriched in patients with cancer, conferring risk for sarcoma ( P = 0.003) and subsequent malignancies ( P = 0.006). Functional analyses demonstrated that wild-type XAF1 enhances transactivation of wild-type and hypomorphic TP53 variants, whereas XAF1 -E134* is markedly attenuated in this activity. We propose that cosegregation of XAF1- E134* and TP53- R337H mutations leads to a more aggressive cancer phenotype than TP53- R337H alone, with implications for genetic counseling and clinical management of hypomorphic TP53 mutant carriers., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Immunoglobulin E (IgE) plays an important role in allergic diseases. Nevertheless, the source of IgE serological memory remains controversial. We reexamined the mechanism of serological memory in allergy using a dual reporter system to track IgE + plasma cells in mice. Short-term allergen exposure resulted in the generation of IgE + plasma cells that resided mainly in secondary lymphoid organs and produced IgE that was unable to degranulate mast cells. In contrast, chronic allergen exposure led to the generation of long-lived IgE + plasma cells that were primarily derived from sequential class switching of IgG1, accumulated in the bone marrow, and produced IgE capable of inducing anaphylaxis. IgE + plasma cells were found in the bone marrow of human allergic, but not nonallergic donors, and allergen-specific IgE produced by these cells was able to induce mast cell degranulation when transferred to mice. These data demonstrate that long-lived IgE + bone marrow plasma cells arise during chronic allergen exposure and establish serological memory in both mice and humans., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide-major histocompatibility complexes ("signal 1"); activation is enhanced by engagement of a second "costimulatory" receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell ("signal 2"). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8 + T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8 + T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8 + T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1-Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Platelets are key regulators of vascular integrity; however, their role in anaphylaxis, a life-threatening systemic allergic reaction characterized by the loss of vascular integrity and vascular leakage, remains unknown. Anaphylaxis is a consequence of inappropriate cellular responses triggered by antibodies to generally harmless antigens, resulting in a massive mediator release and rapidly occurring organ dysfunction. Human platelets express receptors for immunoglobulin G (IgG) antibodies and can release potent mediators, yet their contribution to anaphylaxis has not been previously addressed in mouse models, probably because mice do not express IgG receptors on platelets. We investigated the contribution of platelets to IgG-dependent anaphylaxis in human IgG receptor-expressing mouse models and a cohort of patients suffering from drug-induced anaphylaxis. Platelet counts dropped immediately and markedly upon anaphylaxis induction only when they expressed the human IgG receptor FcγRIIA/CD32A. Platelet depletion attenuated anaphylaxis, whereas thrombocythemia substantially worsened its severity. FcγRIIA-expressing platelets were directly activated by IgG immune complexes in vivo and were sufficient to restore susceptibility to anaphylaxis in resistant mice. Serotonin released by activated platelets contributed to anaphylaxis severity. Data from a cohort of patients suffering from drug-induced anaphylaxis indicated that platelet activation was associated with anaphylaxis severity and was accompanied by a reduction in circulating platelet numbers. Our findings identify platelets as critical players in IgG-dependent anaphylaxis and provide a rationale for the design of platelet-targeting strategies to attenuate the severity of anaphylactic reactions., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by episodically exuberant heterotopic ossification (HO), whereby skeletal muscle is abnormally converted into misplaced, but histologically normal bone. This HO leads to progressive immobility with catastrophic consequences, including death by asphyxiation. FOP results from mutations in the intracellular domain of the type I BMP (bone morphogenetic protein) receptor ACVR1; the most common mutation alters arginine 206 to histidine (ACVR1(R206H)) and has been thought to drive inappropriate bone formation as a result of receptor hyperactivity. We unexpectedly found that this mutation rendered ACVR1 responsive to the activin family of ligands, which generally antagonize BMP signaling through ACVR1 but cannot normally induce bone formation. To test the implications of this finding in vivo, we engineered mice to carry the Acvr1(R206H) mutation. Because mice that constitutively express Acvr1[R206H] die perinatally, we generated a genetically humanized conditional-on knock-in model for this mutation. When Acvr1[R206H] expression was induced, mice developed HO resembling that of FOP; HO could also be triggered by activin A administration in this mouse model of FOP but not in wild-type controls. Finally, HO was blocked by broad-acting BMP blockers, as well as by a fully human antibody specific to activin A. Our results suggest that ACVR1(R206H) causes FOP by gaining responsiveness to the normally antagonistic ligand activin A, demonstrating that this ligand is necessary and sufficient for driving HO in a genetically accurate model of FOP; hence, our human antibody to activin A represents a potential therapeutic approach for FOP., (Copyright © 2015, American Association for the Advancement of Science.)

Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide-binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A- or Ric-8B-deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A(-/-) ES cells, which resulted from reduced steady-state amounts of Gα(i), Gα(q), and Gα(13) proteins to <5% of those of wild-type cells. The amounts of Gα(s) and total Gβ protein were partially reduced in Ric-8A(-/-) cells compared to those in wild-type cells, and only the amount of Gα(s) was reduced substantially in Ric-8B(-/-) cells. The abundances of mRNAs encoding the G protein α subunits were largely unchanged by loss of Ric-8A or Ric-8B. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous Gα(i) and Gα(q) were efficiently translated in Ric-8A(-/-) cells but integrated into endomembranes poorly; however, the reduced amounts of G protein α subunits that reached the membrane still bound to nascent Gβγ. Finally, Gα(i), Gα(q), and Gβ(1) proteins exhibited accelerated rates of degradation in Ric-8A(-/-) cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent Gα subunits with cellular membranes.