Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

The current state and future of T-cell exhaustion research.

'Exhaustion' is a term used to describe a state of native and redirected T-cell hypo-responsiveness resulting from persistent antigen exposure during chronic viral infections or cancer. Although a well-established phenotype across mice and humans, exhaustion at the molecular level remains poorly defined and inconsistent across the literature. This is, in part, due to an overreliance on surface receptors to define these cells and explain exhaustive behaviours, an incomplete understanding of how exhaustion arises, and a lack of clarity over whether exhaustion is the same across contexts, e.g. chronic viral infections versus cancer. With the development of systems-based genetic approaches such as single-cell RNA-seq and CRISPR screens applied to in vivo data, we are moving closer to a consensus view of exhaustion, although understanding how it arises remains challenging given the difficulty in manipulating the in vivo setting. Accordingly, producing and studying exhausted T-cells ex vivo are burgeoning, allowing experiments to be conducted at scale up and with high throughput. Here, we first review what is currently known about T-cell exhaustion and how it's being studied. We then discuss how improvements in their method of isolation/production and examining the impact of different microenvironmental signals and cell interactions have now become an active area of research. Finally, we discuss what the future holds for the analysis of this physiological condition and, given the diversity of ways in which exhausted cells are now being generated, propose the adoption of a unified approach to clearly defining exhaustion using a set of metabolic-, epigenetic-, transcriptional-, and activation-based phenotypic markers, that we call 'M.E.T.A'.

Antigen discrimination by T cells relies on size-constrained microvillar contact.

T cells use finger-like protrusions called 'microvilli' to interrogate their targets, but why they do so is unknown. To form contacts, T cells must overcome the highly charged, barrier-like layer of large molecules forming a target cell's glycocalyx. Here, T cells are observed to use microvilli to breach a model glycocalyx barrier, forming numerous small (<0.5 µm diameter) contacts each of which is stabilized by the small adhesive protein CD2 expressed by the T cell, and excludes large proteins including CD45, allowing sensitive, antigen dependent TCR signaling. In the absence of the glycocalyx or when microvillar contact-size is increased by enhancing CD2 expression, strong signaling occurs that is no longer antigen dependent. Our observations suggest that, modulated by the opposing effects of the target cell glycocalyx and small adhesive proteins, the use of microvilli equips T cells with the ability to effect discriminatory receptor signaling.

The kinase occupancy of T cell coreceptors reconsidered.

The sensitivity of the αß T cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8αß, which are expressed primarily by cells of the helper and cytotoxic T cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor/kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4/Lck interaction was stoichiometric (~100%) and that the CD8αß/Lck interaction was substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor/Lck codiffusion in situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to also show that stoichiometric coupling to Lck required an amphipathic α-helix present in CD4 but not CD8α. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 outcompeted CD8αß for Lck. In T cells, TCR signaling induced CD4/Lck oligomerization but did not affect the high levels of CD4/Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting.

Offshore wind energy development: Research priorities for sound and vibration effects on fishes and aquatic invertebrates.

There are substantial knowledge gaps regarding both the bioacoustics and the responses of animals to sounds associated with pre-construction, construction, and operations of offshore wind (OSW) energy development. A workgroup of the 2020 State of the Science Workshop on Wildlife and Offshore Wind Energy identified studies for the next five years to help stakeholders better understand potential cumulative biological impacts of sound and vibration to fishes and aquatic invertebrates as the OSW industry develops. The workgroup identified seven short-term priorities that include a mix of primary research and coordination efforts. Key research needs include the examination of animal displacement and other behavioral responses to sound, as well as hearing sensitivity studies related to particle motion, substrate vibration, and sound pressure. Other needs include: identification of priority taxa on which to focus research; standardization of methods; development of a long-term highly instrumented field site; and examination of sound mitigation options for fishes and aquatic invertebrates. Effective assessment of potential cumulative impacts of sound and vibration on fishes and aquatic invertebrates is currently precluded by these and other knowledge gaps. However, filling critical gaps in knowledge will improve our understanding of possible sound-related impacts of OSW energy development to populations and ecosystems.

Different individual-level responses of great black-backed gulls (Larus marinus) to shifting local prey availability.

To grow, survive and reproduce under anthropogenic-induced changes, individuals must respond quickly and favourably to the surrounding environment. A species that feeds on a wide variety of prey types (i.e. generalist diet) may be comprised of generalist individuals, specialist individuals that feed on different prey types, or a combination of the two. If individuals within a population respond differently to an environmental change, population-level responses may not be detectable. By tracking foraging movements of great black-backed gulls (Larus marinus), a generalist species, we compared group-level and individual-level responses to an increase in prey biomass (capelin; Mallotus villosus) during the breeding season in coastal Newfoundland, Canada. As hypothesized, shifts in prey availability resulted in significantly different individual responses in foraging behaviour and space use, which was not detectable when data from individuals were combined. Some individuals maintained similar foraging areas, foraging trip characteristics (e.g., trip length, duration) and habitat use with increased capelin availability, while others shifted foraging areas and habitats resulting in either increased or decreased trip characteristics. We show that individual specialization can be non-contextual in some gulls, whereby these individuals continuously use the same feeding strategy despite significant change in prey availability conditions. Findings also indicate high response diversity among individuals to shifting prey conditions that a population- or group-level study would not have detected, emphasizing the importance of examining individual-level strategies for future diet and foraging studies on generalist species.

Trapping or slowing the diffusion of T cell receptors at close contacts initiates T cell signaling.

T cell activation is initiated by T cell receptor (TCR) phosphorylation. This requires the local depletion of large receptor-type phosphatases from "close contacts" formed when T cells interact with surfaces presenting agonistic TCR ligands, but exactly how the ligands potentiate signaling is unclear. It has been proposed that TCR ligands could enhance receptor phosphorylation and signaling just by holding TCRs in phosphatase-depleted close contacts, but this has not been directly tested. We devised simple methods to move the TCR in and out of close contacts formed by T cells interacting with supported lipid bilayers (SLBs) and to slow the receptor's diffusion in the contacts, using a series of anti-CD3ε Fab- and ligand-based adducts of the receptor. TCRs engaging a Fab extended with the large extracellular region of CD45 were excluded from contacts and produced no signaling. Conversely, allowing the extended Fab to become tethered to the SLB trapped the TCR in the close contacts, leading to very strong signaling. Importantly, attaching untethered anti-CD3ε Fab or peptide/MHC ligands, each of which were largely inactive in solution but both of which reduced TCR diffusion in close contacts approximately fivefold, also initiated signaling during cell/SLB contact. Our findings indicate that holding TCRs in close contacts or simply slowing their diffusion in phosphatase-depleted regions of the cell surface suffices to initiate signaling, effects we could reproduce in single-particle stochastic simulations. Our study shows that the TCR is preconfigured for signaling in a way that allows it to be triggered by ligands acting simply as receptor "traps."

Large metallicity variations in the Galactic interstellar medium.

The interstellar medium (ISM) comprises gases at different temperatures and densities, including ionized, atomic and molecular species, and dust particles1. The neutral ISM is dominated by neutral hydrogen2 and has ionization fractions of up to eight per cent3. The concentration of chemical elements heavier than helium (metallicity) spans orders of magnitudes in Galactic stars4, because they formed at different times. However, the gas in the vicinity of the Sun is assumed to be well mixed and to have a solar metallicity in traditional chemical evolution models5. The ISM chemical abundances can be accurately measured with ultraviolet absorption-line spectroscopy. However, the effects of dust depletion6-9-which removes part of the metals from the observable gaseous phase and incorporates it into solid grains-have prevented, until recently, a deeper investigation of the ISM metallicity. Here we report the dust-corrected metallicity of the neutral ISM measured towards 25 stars in our Galaxy. We find large variations in metallicity over a factor of ten (with an average of 55 ± 7 per cent solar metallicity and a standard deviation of 0.28 dex), including many regions of low metallicity, down to about 17 per cent solar metallicity and possibly below. Pristine gas falling onto the Galactic disk in the form of high-velocity clouds can cause the observed chemical inhomogeneities on scales of tens of parsecs. Our results suggest that this low-metallicity accreting gas does not efficiently mix into the ISM, which may help us understand metallicity deviations in nearby coeval stars.

Aggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells.

To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.

HLA-E-restricted, Gag-specific CD8+ T cells can suppress HIV-1 infection, offering vaccine opportunities.

Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)-restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1-specific, HLA-E-restricted T cells have not been observed in HIV-1-infected individuals. Here, HLA-E-restricted, HIV-1-specific CD8 + T cells were primed in vitro. These T cell clones and allogeneic CD8 + T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4 + T cells in vitro. Vaccine induction of efficacious HLA-E-restricted HIV-1-specific T cells should therefore be possible.

Soft Polydimethylsiloxane-Supported Lipid Bilayers for Studying T Cell Interactions.

Much of what we know about the early stages of T cell activation has been obtained from studies of T cells interacting with glass-supported lipid bilayers that favor imaging but are orders of magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing "soft bilayers" with physiological levels of mechanical resistance (Young's modulus of 4 kPa). Comparisons of T cell behavior on soft and glass-supported bilayers revealed that whereas late stages of T cell activation are thought to be substrate-stiffness dependent, early calcium signaling was unaffected by substrate rigidity, implying that early steps in T cell receptor triggering are not mechanosensitive. The exclusion of large receptor-type phosphatases was observed on the soft bilayers, however, even though it is yet to be demonstrated at authentic cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

Isotopic Discrimination (δ15N, δ13C) in Captive and Wild Common Murres (Uria aalge) and Atlantic Puffins (Fratercula arctica).

Studying the diet of consumers using stable isotopes provides insight into the foraging ecology of individuals and species. To accurately reconstruct the integrated diet of animals using stable isotope values, we must quantify diet-tissue discrimination factors (DTDFs), or the way in which stable isotopes in prey are incorporated into the tissues of consumers. To quantify DTDFs, controlled experiments are needed, whereby consumers are fed a constant diet. However, relatively few controlled-diet studies have been conducted for seabirds. In this study, captive adult Atlantic puffins (Fratercula arctica) and common murres (Uria aalge) were fed a two-source diet of capelin (Mallotus villosus) and Atlantic silverside (Menidia menidia) to determine the DTDFs for the cellular component of blood and plasma for both δ15N and δ13C. The DTDFs for the cellular component (Δ15N: 2.80±0.28; Δ13C: 1.21±0.22) and plasma (Δ15N: 1.72±1.03; Δ13C: -0.18±0.56) of puffins were similar to those for the cellular component (Δ15N: 2.91±0.18; Δ13C: 1.09±0.23) and plasma (Δ15N: 2.18±0.77; Δ13C: -0.70±0.18) of murres. We reconstructed the diet of wild murres and puffins breeding on the northeastern coast of Newfoundland using previously published DTDFs and estimated DTDFs from our feeding experiment. Reconstructed dietary proportions supported a priori knowledge of diet, although outputs were sensitive to the DTDF used. Despite the similarity of our DTDFs for puffins and murres, along with the similarity of our DTDFs with those of other seabird species, our sensitivity analysis revealed considerable differences among resultant dietary contributions from mixing models, further highlighting the importance of using species- and tissue-specific DTDFs to enhance knowledge in the foraging ecology of seabirds using stable isotopes.

The Costs of Close Contacts: Visualizing the Energy Landscape of Cell Contacts at the Nanoscale.

Cell-cell contacts often underpin signaling between cells. For immunology, the binding of a T cell receptor to an antigen-presenting pMHC initiates downstream signaling and an immune response. Although this contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm, many, often contradictory observations have been made regarding the influence of the contact on parameters such as the binding kinetics, spatial distribution, and diffusion of signaling proteins within the contact. Understanding the basic physical constraints on probes inside this crowded environment will help inform studies on binding kinetics and dynamics of signaling of relevant proteins in the synapse. By tracking quantum dots of different dimensions for extended periods of time, we have shown that it is possible to obtain the probability of a molecule entering the contact, the change in its diffusion upon entry, and the impact of spatial heterogeneity of adhesion protein density in the contact. By analyzing the contacts formed by a T cell interacting with adhesion proteins anchored to a supported lipid bilayer, we find that probes are excluded from contact entry in a size-dependent manner for gap-to-probe differences of 4.1 nm. We also observed probes being trapped inside the contact and a decrease in diffusion of up to 85% in dense adhesion protein contacts. This approach provides new, to our knowledge, insights into the nature of cell-cell contacts, revealing that cell contacts are highly heterogeneous because of topography- and protein-density-related processes. These effects are likely to profoundly influence signaling between cells.

Diet-tissue discrimination factors (δ15 N and δ13 C values) for blood components in Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins.

RATIONALE: Analysis of the stable isotope ratios of carbon and nitrogen (δ13 C and δ15 N values) is increasingly being used to gain insight into predator trophic ecology, which requires accurate diet-tissue discrimination factors (DTDFs), or the isotopic difference between prey and predator. Accurate DTDFs must be calculated from predators consuming an isotopically constant diet over time in controlled feeding experiments, but these studies have received little attention to date, especially among seabird species. METHODS: In this study, aquarium-housed Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins were fed a single-prey source diet (capelin Mallotus villosus) for eight weeks. Stable isotope ratios (δ13 C and δ15 N values) of penguin blood (cellular component and plasma) and capelin were measured using mass spectrometry and then used to calculate DTDFs for both components of penguin blood by comparison with prey values. RESULTS: The DTDFs for plasma were -0.63 ± 0.49 (mean ± SD) and -0.27 ± 0.22 for δ13 C values, and 2.60 ± 0.50 and 2.78 ± 0.22 for δ15 N values for Magellanic and southern rockhopper penguins, respectively, while the DTDFs for the cellular component were 1.22 ± 0.03 and 1.26 ± 0.03 for δ13 C values, and 2.54 ± 0.07 and 2.43 ± 0.17 for δ15 N values. CONCLUSIONS: We compare our DTDFs with published values from blood components of penguins and discuss the effects that lipid extraction, sample storage, and diet have on the DTDFs of penguin blood components. This study provides accurate DTDFs of blood components for two seabird species of conservation concern, and is one of the first to provide plasma DTDFs for penguins, which are underrepresented in the seabird literature.

Reconstitution of immune cell interactions in free-standing membranes.

The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. Therapeutic control of immune responses therefore relies on thorough elucidation of the molecular processes occurring at these interfaces. However, the detailed investigation of each component's contribution to the formation and regulation of the contact is hampered by the complexities of cell composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially in the use of advanced imaging technology. One approach that circumvents these problems is to establish in vitro systems that faithfully mimic immune cell interactions, but allow complexity to be 'dialled-in' as needed. Here, we present an in vitro system that makes use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system presented here is expected to be widely applicable.

The hidden mass and large spatial extent of a post-starburst galaxy outflow.

Outflowing winds of multiphase plasma have been proposed to regulate the buildup of galaxies, but key aspects of these outflows have not been probed with observations. By using ultraviolet absorption spectroscopy, we show that "warm-hot" plasma at 10(5.5) kelvin contains 10 to 150 times more mass than the cold gas in a post-starburst galaxy wind. This wind extends to distances > 68 kiloparsecs, and at least some portion of it will escape. Moreover, the kinematical correlation of the cold and warm-hot phases indicates that the warm-hot plasma is related to the interaction of the cold matter with a hotter (unseen) phase at >>10(6) kelvin. Such multiphase winds can remove substantial masses and alter the evolution of post-starburst galaxies.