Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Characterization of Adaptive-like γδ T Cells in Ugandan Infants during Primary Cytomegalovirus Infection

Gamma-delta (γδ) T cells are unconventional T cells that help control cytomegalovirus (CMV) infection in adults. γδ T cells develop early in gestation, and a fetal public γδ T cell receptor (TCR) clonotype is detected in congenital CMV infections. However, age-dependent γδ T cell responses to primary CMV infection are not well-understood. Flow cytometry and TCR sequencing was used to comprehensively characterize γδ T cell responses to CMV infection in a cohort of 32 infants followed prospectively from birth. Peripheral blood γδ T cell frequencies increased during infancy, and were higher among CMV-infected infants relative to uninfected. Clustering analyses revealed associations between CMV infection and activation marker expression on adaptive-like Vδ1 and Vδ3, but not innate-like Vγ9Vδ2 γδ T cell subsets. Frequencies of NKG2C⁺CD57⁺ γδ T cells were temporally associated with the quantity of CMV shed in saliva by infants with primary infection. The public γδ TCR clonotype was only detected in CMV-infected infants <120 days old and at lower frequencies than previously described in fetal infections. Our findings support the notion that CMV infection drives age-dependent expansions of specific γδ T cell populations, and provide insight for novel strategies to prevent CMV transmission and disease.     

Targeting the PI3K/Akt signaling pathway in pancreatic β‐cells to enhance their survival and function: An emerging therapeutic strategy for type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of the insulin‐producing β‐cells within the pancreas. Islet transplantation represents one cure; however, during islet preparation and post transplantation significant amounts of β‐cell death occur. Therefore, prevention and cure of T1D is dependent upon the preservation of β‐cell function and the prevention of β‐cell death. Phosphoinositide 3‐kinase (PI3K)/Akt signaling represents a promising therapeutic target for T1D due to its pronounced effects on cellular survival, proliferation, and metabolism. A growing amount of evidence indicates that PI3K/Akt signaling is a critical determinant of β‐cell mass and function. Modulation of the PI3K/Akt pathway, directly (via the use of highly specific protein and peptide‐based biologics, excretory/secretory products of parasitic worms, and complex constituents of plant extracts) or indirectly (through microRNA interactions) can regulate the β‐cell processes to ultimately determine the fate of β‐cell mass. An important consideration is the identification of the specific PI3K/Akt pathway modulators that enhance β‐cell function and prevent β‐cell death without inducing excessive β‐cell proliferation, which may carry carcinogenic side effects. Among potential PI3K/Akt pathway agonists, we have identified a novel parasite‐derived protein, termed FhHDM‐1 (Fasciola hepatica helminth defense molecule 1), which efficiently stimulates the PI3K/Akt pathway in β‐cells to enhance function and prevent death without concomitantly inducing proliferation unlike several other identified stimulators of PI3K/Akt signaling . As such, FhHDM‐1 will inform the design of biologics aimed at targeting the PI3K/Akt pathway to prevent/ameliorate not only T1D but also T2D, which is now widely recognized as an inflammatory disease characterized by β‐cell dysfunction and death. This review will explore the modulation of the PI3K/Akt signaling pathway as a novel strategy to enhance β‐cell function and survival.     

The Unknown Unknowns: Recovering Gamma-Delta T Cells for Control of Human Immunodeficiency Virus (HIV)

Recent advances in γδ T cell biology have focused on the unique attributes of these cells and their role in regulating innate and adaptive immunity, promoting tissue homeostasis, and providing resistance to various disorders. Numerous bacterial and viral pathogens, including human immunodeficiency virus-1 (HIV), greatly alter the composition of γδ T cells in vivo. Despite the effectiveness of antiretroviral therapy (ART) in controlling HIV and restoring health in those affected, γδ T cells are dramatically impacted during HIV infection and fail to reconstitute to normal levels in HIV-infected individuals during ART for reasons that are not clearly understood. Importantly, their role in controlling HIV infection, and the implications of their failure to rebound during ART are also largely unknown and understudied. Here, we review important aspects of human γδ T cell biology, the effector and immunomodulatory properties of these cells, their prevalence and function in HIV, and their immunotherapeutic potential.     

In Acute Dengue Infection,High TIM-3 Expression May Contribute to the Impairment of IFNγ Production by Circulating Vδ2 T Cells

γδ T cells are innate cells able to quickly eliminate pathogens or infected/tumoral cells by their antiviral and adjuvancy activities. The role of γδ T cells during Dengue Viral Infection (DENV) infection is not fully elucidated. Nevertheless, human primary γδ T cells have been shown to kill in vitro DENV-infected cells, thus highlighting their possible antiviral function. The aim of this work was to characterize the phenotype and function of Vδ2 T cells in DENV patients. Fifteen DENV patients were enrolled for this study and peripheral blood mononuclear cells (PBMC) were used to analyze Vδ2-T-cell frequency, differentiation profile, activation/exhaustion status, and functionality by multiparametric flow cytometry. Our data demonstrated that DENV infection was able to significantly reduce Vδ2-T-cell frequency and to increase their activation (CD38 and HLA-DR) and exhaustion markers (PD-1 and TIM-3). Furthermore, Vδ2 T cells showed a reduced capability to produce IFN-γ after phosphoantigenic stimulation that can be associated to TIM-3 expression. Several studies are needed to depict the possible clinical impact of γδ-T-cell impairment on disease severity and to define the antiviral and immunoregulatory activities of γδ T cells in the first phases of infection.     

Reducing the γ′-Particle Size in CMSX-4 for Membrane Development

Colloidal emulsions for lipophilic drugs can be fabricated using premix membrane emulsification. The state of the art is the application of membranes made from, for example, polycarbonate or polyester, which, however, are prone to fouling and cause waste, due to the low number of cycles. With the use of metallic membranes made from the nickel based single crystalline superalloy CMSX-4, these key disadvantages are eliminated. However, instead, the pore size and the resulting droplet size distribution need to be adjusted and improved. This can be realized by tailoring the size of the γ′-particles, which is controllable by the time and temperature used during precipitation heat treatment and the quenching method after homogenization heat treatment. Therefore, we utilized different heat treatment protocols, varying the cooling rate (water quenching and air cooling) after homogenization heat treatment and the holding time and temperature during precipitation heat treatment. Then, we investigated the γ/γ′-microstructure, including the γ′-morphology and γ′-particle size. We show that water quenching has a significant impact on the γ/γ′-microstructure and often leads to irregular-shaped and poorly aligned γ′-particles after precipitation heat treatment. In comparison, air cooling, followed by a subsequent precipitation heat treatment, results in well-aligned and cubic shaped γ′-particles and is, therefore, favorable for membrane fabrication. A reduction in precipitation temperature leads to morphology changes to the γ′-particles. A reduction of the holding time during precipitation heat treatment diminishes the γ′-particle growth, resulting in smaller γ′-particles. Additionally, a suitable heat treatment protocol for membrane fabrication was identified with a γ′-edge length of 224 ± 52 nm and well-aligned, cubic shaped γ′-particles.     

γδ T Cells in Emerging Viral Infection: An Overview

New emerging viruses belonging to the Coronaviridae, Flaviviridae, and Filoviridae families are serious threats to public health and represent a global concern. The surveillance to monitor the emergence of new viruses and their transmission is an important target for public health authorities. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an excellent example of a pathogen able to cause a pandemic. In a few months, SARS-CoV-2 has spread globally from China, and it has become a world health problem. Gammadelta (γδ) T cell are sentinels of innate immunity and are able to protect the host from viral infections. They enrich many tissues, such as the skin, intestines, and lungs where they can sense and fight the microbes, thus contributing to the protective immune response. γδ T cells perform their direct antiviral activity by cytolytic and non-cytolytic mechanisms against a wide range of viruses, and they are able to orchestrate the cellular interplay between innate and acquired immunity. For their pleiotropic features, γδ T cells have been proposed as a target for immunotherapies in both cancer and viral infections. In this review, we analyzed the role of γδ T cells in emerging viral infections to define the profile of the response and to better depict their role in the host protection.     

SARS-CoV-2 Infects Human ACE2-Negative Endothelial Cells through an αvβ3 Integrin-Mediated Endocytosis Even in the Presence of Vaccine-Elicited Neutralizing Antibodies

Integrins represent a gateway of entry for many viruses and the Arg-Gly-Asp (RGD) motif is the smallest sequence necessary for proteins to bind integrins. All Severe Acute Respiratory Syndrome Virus type 2 (SARS-CoV-2) lineages own an RGD motif (aa 403–405) in their receptor binding domain (RBD). We recently showed that SARS-CoV-2 gains access into primary human lung microvascular endothelial cells (HL-mECs) lacking Angiotensin-converting enzyme 2 (ACE2) expression through this conserved RGD motif. Following its entry, SARS-CoV-2 remodels cell phenotype and promotes angiogenesis in the absence of productive viral replication. Here, we highlight the α_vβ₃ integrin as the main molecule responsible for SARS-CoV-2 infection of HL-mECs via a clathrin-dependent endocytosis. Indeed, pretreatment of virus with α_vβ₃ integrin or pretreatment of cells with a monoclonal antibody against α_vβ₃ integrin was found to inhibit SARS-CoV-2 entry into HL-mECs. Surprisingly, the anti-Spike antibodies evoked by vaccination were neither able to impair Spike/integrin interaction nor to prevent SARS-CoV-2 entry into HL-mECs. Our data highlight the RGD motif in the Spike protein as a functional constraint aimed to maintain the interaction of the viral envelope with integrins. At the same time, our evidences call for the need of intervention strategies aimed to neutralize the SARS-CoV-2 integrin-mediated infection of ACE2-negative cells in the vaccine era.     

Pilot Study on Interferon-γ-producing T Cell Subsets after the Protective Vaccination with Radiation-attenuated Cercaria of Schistosoma japonicum in the Miniature Pig Model

CLAWN miniature pig has been shown to serve as a suitable host for the experimental infection of Schistosoma japonicum. In this study, we found that radiation-attenuated cercaria (RAC) vaccine gave CLAWN miniature pigs protective immunity against subsequent challenge infection with S. japonicum cercaria. To characterize the protective immune response of the pig model vaccinated by attenuated cercaria, flow cytometric analysis of the reactive T cell subsets was performed. The intracellular interferon (IFN)-γ and the cell surface markers revealed the peripheral blood CD3+ T-lymphocytes produced significant amounts of IFN-γ during the immunization period and after the challenge infection. CD4+ αβ-T cells as well as CD4+/CD8α^mid double positive and/or CD8α^high αβ-T cells were the major IFN-γ-producing CD3+ T cells. On the contrary, γδ T cells did not produce intracellular IFN-γ. Our results suggested that RAC-vaccinated miniature pigs showed effective protective immunity through the activation of αβ T cells bearing antigen specific T-cell receptors but not through the activation of γδ T cells.     

Molecular genetics of β-thalassemia: A narrative review

β-thalassemia is a hereditary hematological disease caused by over 350 mutations in the β-globin gene (HBB). Identifying the genetic variants affecting fetal hemoglobin (HbF) production combined with the α-globin genotype provides some prediction of disease severity for β-thalassemia. However, the generation of an additive composite genetic risk score predicts prognosis, and guide management requires a larger panel of genetic modifiers yet to be discovered.Presently, using data from prior clinical trials guides the design of further research and academic studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene therapy approaches.Genetic studies have successfully characterized the causal variants and pathways involved in HbF regulation, providing novel therapeutic targets for HbF reactivation. In addition to these HBB mutation-independent strategies involving HbF synthesis de-repression, the expanding genome editing toolkit provides increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin restoration for personalized treatment of hemoglobinopathies. Allogeneic hematopoietic stem cell transplantation was, until very recently, the curative option available for patients with transfusion-dependent β-thalassemia. Gene therapy currently represents a novel therapeutic promise after many years of extensive preclinical research to optimize gene transfer protocols.We summarize the current state of developments in the molecular genetics of β-thalassemia over the last decade, including the mechanisms associated with ineffective erythropoiesis, which have also provided valid therapeutic targets, some of which have been shown as a proof-of-concept.     

PDGF-D−PDGFRβ signaling enhances IL-15–mediated human natural killer cell survival

The axis of platelet-derived growth factor (PDGF) and PDGF receptor-beta (PDGFRβ) plays prominent roles in cell growth and motility. In addition, PDGF-D enhances human natural killer (NK) cell effector functions when binding to the NKp44 receptor. Here, we report an additional but previously unknown role of PDGF-D, whereby it mediates interleukin-15 (IL-15)–induced human NK cell survival but not effector functions via its binding to PDGFRβ but independent of its binding to NKp44. Resting NK cells express no PDGFRβ and only a low level of PDGF-D, but both are significantly up-regulated by IL-15, via the nuclear factor κB signaling pathway, to promote cell survival in an autocrine manner. Both ectopic and IL-15–induced expression of PDGFRβ improves NK cell survival in response to treatment with PDGF-D. Our results suggest that the PDGF-D−PDGFRβ signaling pathway is a mechanism by which IL-15 selectively regulates the survival of human NK cells without modulating their effector functions.

Natural killer (NK) cells—a distinct lymphocyte subset in the circulation—play critical roles in antiviral and antitumor immunity (1). One advantage of NK cells is that they recognize “nonself” cells without being activated by specific antigens, allowing a more rapid response than with T cells. This broad cytotoxicity and rapid killing make NK cells ideal for cancer immunotherapy (2). Of note, chimeric-antigen-receptor (CAR)-NK cells have several therapeutic advantages over CAR-T cells (2–4). However, NK cells’ shorter lifespan may limit their clinical efficacy. A better understanding of the mechanisms that regulate NK cell survival might therefore improve their clinical application for cancer immunotherapy.Platelet-derived growth factor (PDGF) is one of the main growth factors that regulate cell growth and division (5). The PDGF family consists of PDGF-A, PDGF-B, PDGF-C, and PDGF-D (5). These ligands bind to two tyrosine kinase receptors, PDGFRα and PDGFRβ (5). Upon activation by PDGFs, PDGF receptors dimerize and undergo autophosphorylation on tyrosine residues in the intracellular domain, which mediates the binding of cofactors and subsequently activates signal transduction, including Ras/Raf/MEK/Erk mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase/protein kinase B (P13K/AKT) (5). PDGFs play prominent roles in cell differentiation, cell growth, cell transformation, and cell migration (5). A recent study showed that PDGF-D is a ligand of NKp44, one of the natural cytotoxicity receptors expressed by activated human NK cells (6). PDGF-D binding to NKp44 prompted NK cells to secrete interferon (IFN)-γ and tumor necrosis factor (TNF)-α, arresting the growth of tumor cells (6). However, little is known about the role of PDGFR signaling in NK cell immunity.In this study, we report a previously unknown role of PDGF-D: regulation of interleukin 15 (IL-15)–mediated cell survival—not effector functions in human NK cells—that is dependent on PDGFRβ but independent of NKp44. Our findings suggest that the PDGF-D−PDGFRβ signaling pathway is a mechanism by which IL-15 selectively regulates the survival of human NK cells but not their effector functions.     

Revisiting the Role of γδ T Cells in Anti-CMV Immune Response after Transplantation

Gamma delta (γδ) T cells form an unconventional subset of T lymphocytes that express a T cell receptor (TCR) consisting of γ and δ chains. Unlike conventional αβ T cells, γδ T cells share the immune signature of both the innate and the adaptive immunity. These features allow γδ T cells to act in front-line defense against infections and tumors, rendering them an attractive target for immunotherapy. The role of γδ T cells in the immune response to cytomegalovirus (CMV) has been the focus of intense research for several years, particularly in the context of transplantation, as CMV reactivation remains a major cause of transplant-related morbidity and mortality. Therefore, a better understanding of the mechanisms that underlie CMV immune responses could enable the design of novel γδ T cell-based therapeutic approaches. In this regard, the advent of next-generation sequencing (NGS) and single-cell TCR sequencing have allowed in-depth characterization of CMV-induced TCR repertoire changes. In this review, we try to shed light on recent findings addressing the adaptive role of γδ T cells in CMV immunosurveillance and revisit CMV-induced TCR reshaping in the era of NGS. Finally, we will demonstrate the favorable and unfavorable effects of CMV reactive γδ T cells post-transplantation.     

Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β₂-adrenergic receptors (β₂AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β₂AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β₂AR actions favorable for treating obstructive lung disease.

Most G protein–coupled receptors (GPCRs) are now recognized as multisignal transducers (1, 2). Early concepts of agonist–receptor interactions were based on the idea that there was a single “active” receptor conformation induced by the binding of any agonist, resulting in an interaction with the heterotrimeric G protein and a universal, singular signal. Generally, the α-subunit of the G protein, upon its dissociation, was considered the primary activator (or inhibitor) of the effector, resulting in the intracellular signal. Subsequently, it became clear that multiple signaling outcomes from activation of a given GPCR can occur from a single agonist due to specific molecular determinants of the receptor triggering independent mechanisms (3–5). As these multiple functions were being identified, it was apparent that agonists with different structures could act at a given receptor to preferentially activate one signal with minimal engagement of others, a property later termed signal biasing (6–8). Biased agonists, then, could represent important advantages over nonbiased agonists due to this signal selectivity, activating a specified therapeutic pathway while minimally evoking unnecessary or deleterious signaling. The pathway selectivity of biased agonists is thought to be established by the stabilization of specific conformation(s) of the agonist–receptor complex via a set of interactions that differ from those of unbiased (also called balanced) agonists (9–12). While it is conceivable that small modifications of established cognate agonists might yield such specialized signaling, significant deviation from common agonist structures may be necessary to meet this goal (13).The signals/functions of a given GPCR that might be sought for selective activation are defined by the cell type, disease, and desired final physiologic function. In asthma and chronic obstructive pulmonary disease (COPD), active human airway smooth muscle (HASM) cellular contraction limits airflow, representing a major cause of morbidity and mortality. β₂-adrenergic receptors (β₂ARs) expressed on HASM cells are the targets for binding of therapeutically administered β-agonists, which relax the cells via a cyclic adenosine monophosphate–mediated mechanism (14). β-agonists are used for treating acute bronchospasm as well as for long-term prevention. However, the HASM bronchodilator response to acute β-agonist is attenuated by receptor desensitization (15), with typical treatments of humans, or isolated HASM cells, leading to a loss of receptor function over time (16–18), clinically termed tachyphylaxis.Agonist-promoted desensitization of β₂AR (and other GPCRs) is due to partial uncoupling of the receptor to the G protein, which is initiated by phosphorylation of intracellular Ser/Thr residues of the receptor by G protein–coupled receptor kinases (GRKs) (19, 20). The GRK-phosphorylated β₂AR recruits β-arrestin1 or β-arrestin2 to these receptors, with subsequent interactions that appear to compete with the receptor for its binding to the Gα subunit, thus attenuating the intracellular response (11, 21). Such competition has been strongly inferred for the β₂AR (22, 23) and is compelling for rhodopsin–arrestin interactions (24). In addition, β-arrestin binding to GPCRs can initiate receptor internalization and other events such as receptor activation of ERK1/2 (25) through its multiprotein adapter functions. Thus β-arrestin engagement can be considered an early “second signal” of the β₂AR as well as a desensitization initiator for attenuating the Gs signal. An agonist that is biased toward Gαs coupling (cAMP production and airway smooth muscle [ASM] relaxation) and away from β-arrestin binding (desensitization) would be desirable in treating obstructive lung diseases, since efficacy would not be attenuated acutely, nor would tachyphylaxis be experienced from extended treatment. While biased agonists favoring either G protein or β-arrestin (6) signaling have been described for some GPCRs (such as μ-opioid and type 1 angiotensin II receptors), Gαs biasing has not been apparent from most studies with catecholamine-like compounds for the β₂AR. Thus, we have little information as to whether the two β₂AR pathways can be differentially activated in a selective manner by an efficacious agonist, nor is it apparent from a structural standpoint what strategy might be employed to design agonists biased in this manner for this receptor.In order to find this type of biasing for the β₂AR, we screened a 40-million-compound scaffold ranking (SR) library that was agnostic to known β₂AR agonist structures. We found a scaffold in which substitutions of certain R groups led to individual compounds that are apparently Gαs-biased agonists for β₂AR with no apparent engagement of β-arrestin in model systems. Additional studies in HASM cells revealed a lack of tachyphylaxis of the relaxation effect by the lead compound compared with the most widely utilized β₂AR agonist, albuterol. The structure of this biased agonist is very different from that of catecholamine-like agonists. To ascertain the mechanism that may underlie this biased activity, we used structural modeling and molecular simulations and studied homologous compounds with different R groups and receptor mutagenesis to predict the interaction sites with the activated β₂AR. Such studies uncovered distinct structural characteristics that may be responsible for the biasing effect.     

Prolonged antigen survival and cytosolic export in cross-presenting human γδ T cells

Human blood Vγ9Vδ2 T cells respond to signals from microbes and tumors and subsequently differentiate into professional antigen-presenting cells (γδ T-APCs) for induction of CD4⁺ and CD8⁺ T cell responses. γδ T-APCs readily take up and degrade exogenous soluble protein for peptide loading on MHC I, in a process termed antigen cross-presentation. The mechanisms underlying antigen cross-presentation are ill-defined, most notably in human dendritic cells (DCs), and no study has addressed this process in γδ T-APCs. Here we show that intracellular protein degradation and endosomal acidification were significantly delayed in γδ T-APCs compared with human monocyte-derived DCs (moDCs). Such conditions are known to favor antigen cross-presentation. In both γδ T-APCs and moDCs, internalized antigen was transported across insulin-regulated aminopeptidase (IRAP)–positive early and late endosomes; however, and in contrast to various human DC subsets, γδ T-APCs efficiently translocated soluble antigen into the cytosol for processing via the cytosolic proteasome-dependent cross-presentation pathway. Of note, γδ T-APCs cross-presented influenza antigen derived from virus-infected cells and from free virus particles. The robust cross-presentation capability appears to be a hallmark of γδ T-APCs and underscores their potential application in cellular immunotherapy.     

Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

Comparative Evaluation of the Foot-and-Mouth Disease Virus Permissive LF-BK αVβ6 Cell Line for Senecavirus A Research

Senecavirus A (SVA) is a member of the family Picornaviridae and enzootic in domestic swine. SVA can induce vesicular lesions that are clinically indistinguishable from Foot-and-mouth disease, a major cause of global trade barriers and agricultural productivity losses worldwide. The LF-BK α_Vβ₆ cell line is a porcine-derived cell line transformed to stably express an α_Vβ₆ bovine integrin and primarily used for enhanced propagation of Foot-and-mouth disease virus (FMDV). Due to the high biosecurity requirements for working with FMDV, SVA has been considered as a surrogate virus to test and evaluate new technologies and countermeasures. Herein we conducted a series of comparative evaluation in vitro studies between SVA and FMDV using the LF-BK α_Vβ₆ cell line. These include utilization of LF-BK α_Vβ₆ cells for field virus isolation, production of high virus titers, and evaluating serological reactivity and virus susceptibility to porcine type I interferons. These four methodologies utilizing LF-BK α_Vβ₆ cells were applicable to research with SVA and results support the current use of SVA as a surrogate for FMDV.