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MR1-restricted T cells have been implicated in microbial infections, sterile inflammation, wound healing and cancer. Similar to other antigen presentation molecules, evidence supports multiple, complementary MR1 antigen presentation pathways. To investigate ligand exchange pathways for MR1, we used MR1 monomers and tetramers loaded with 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) to deliver the antigen. Using MR1-deficient cells reconstituted with wild-type MR1 or MR1 molecules that cannot bind 5-OP-RU, we show that presentation of monomer-delivered 5-OP-RU is dependent on cellular MR1 and requires the transfer of ligand from the soluble molecule onto MR1 expressed by the antigen presenting cell. This mode of antigen delivery strengthens the evidence for post-ER ligand exchange pathways for MR1, which could represent an important avenue by which MR1 acquires antigens derived from endocytosed pathogens. Delivering MR1 ligand in the context of an MR1 monomer demonstrates transfer of this ligand onto cellular MR1. The stabilization of an inherently unstable ligand in the monomer has implications for antigen delivery and possibly vaccine design. [ABSTRACT FROM AUTHOR]

Membrane-binding proteins often associate with lipid membranes through a singular binding interface which is generally modeled as a two-state system: bound or unbound. However, even a single interface can engage with more than one mode of binding since a variety of interactions can contribute to the binding event. Unfortunately, the ability to clearly delineate the different binding modes of a singular binding interface has been elusive with existing models. Here, we present a study on milk fat globule EGF factor 8 (MFG-E8), which belongs to a class of proteins that identifies and binds phosphatidylserine (PS). These proteins detect membrane dysregulation implicated in exposed PS in apoptosis and malignant cells. In order to elucidate the factors affecting the binding of MFG-E8, we used a model system consisting of a series of lipid vesicles with varying PS mole fraction to identify the sensitivity of MFG-E8's binding affinity to changes in electrostatics using a tryptophan fluorescence spectral shift assay. Using a newly developed model, we experimentally identified three binding modes, each associated with a different number of PS lipids, with its cooperativity for binding being enhanced by the availability of negatively charged lipids. X-ray reflectivity experiments additionally suggest that MFG-E8's binding modes are influenced by membrane packing. The protocols established for elucidating MFG-E8's interaction with lipid membranes under different membrane conditions can be applied to the study of other membrane-binding proteins that target specific membrane attributes, such as fluidity and electrostatics, and help elucidate these membrane targeting mechanisms and their subsequent binding events. [ABSTRACT FROM AUTHOR]

γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background1–5. γδ T cells can sense conserved cell stress signals prevalent in transformed cells2,3, although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells—the most abundant subset of human γδ T cells4—recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6–8), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1–BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.A combination of genome-wide CRISPR screens in target cancer cells identifies pathways that regulate γδ T cell killing and BTN3A cell surface expression. [ABSTRACT FROM AUTHOR]

MR1-restricted T (MR1T) cells recognize microbial small molecule metabolites presented on the MHC Class I-like molecule MR1 and have been implicated in early effector responses to microbial infection. As a result, there is considerable interest in identifying chemical properties of metabolite ligands that permit recognition by MR1T cells, for consideration in therapeutic or vaccine applications. Here, we made chemical modifications to known MR1 ligands to evaluate the effect on MR1T cell activation. Specifically, we modified 6,7-dimethyl-8-d-ribityllumazine (DMRL) to generate 6,7-dimethyl-8-d-ribityldeazalumazine (DZ), and then further derivatized DZ to determine the requirements for retaining MR1 surface stabilization and agonistic properties. Interestingly, the IFN-γ response toward DZ varied widely across a panel of T cell receptor (TCR)-diverse MR1T cell clones; while one clone was agnostic toward the modification, most displayed either an enhancement or depletion of IFN-γ production when compared with its response to DMRL. To gain insight into a putative mechanism behind this phenomenon, we used in silico molecular docking techniques for DMRL and its derivatives and performed molecular dynamics simulations of the complexes. In assessing the dynamics of each ligand in the MR1 pocket, we found that DMRL and DZ exhibit differential dynamics of both the ribityl moiety and the aromatic backbone, which may contribute to ligand recognition. Together, our results support an emerging hypothesis for flexibility in MR1:ligand-MR1T TCR interactions and enable further exploration of the relationship between MR1:ligand structures and MR1T cell recognition for downstream applications targeting MR1T cells. [ABSTRACT FROM AUTHOR]

Background: Healthcare provider burnout is considered a factor in quality of care, yet little is known about the consistency and magnitude of this relationship. This meta-analysis examined relationships between provider burnout (emotional exhaustion, depersonalization, and reduced personal accomplishment) and the quality (perceived quality, patient satisfaction) and safety of healthcare.Methods: Publications were identified through targeted literature searches in Ovid MEDLINE, PsycINFO, Web of Science, CINAHL, and ProQuest Dissertations & Theses through March of 2015. Two coders extracted data to calculate effect sizes and potential moderators. We calculated Pearson's r for all independent relationships between burnout and quality measures, using a random effects model. Data were assessed for potential impact of study rigor, outliers, and publication bias.Results: Eighty-two studies including 210,669 healthcare providers were included. Statistically significant negative relationships emerged between burnout and quality (r = -0.26, 95 % CI [-0.29, -0.23]) and safety (r = -0.23, 95 % CI [-0.28, -0.17]). In both cases, the negative relationship implied that greater burnout among healthcare providers was associated with poorer-quality healthcare and reduced safety for patients. Moderators for the quality relationship included dimension of burnout, unit of analysis, and quality data source. Moderators for the relationship between burnout and safety were safety indicator type, population, and country. Rigor of the study was not a significant moderator.Discussion: This is the first study to systematically, quantitatively analyze the links between healthcare provider burnout and healthcare quality and safety across disciplines. Provider burnout shows consistent negative relationships with perceived quality (including patient satisfaction), quality indicators, and perceptions of safety. Though the effects are small to medium, the findings highlight the importance of effective burnout interventions for healthcare providers. Moderator analyses suggest contextual factors to consider for future study. [ABSTRACT FROM AUTHOR]

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity. [ABSTRACT FROM AUTHOR]

Nano-TiO is an engineered nanomaterial whose production and use are increasing rapidly. Hence, aquatic habitats are at risk for nano-TiO contamination due to potential inputs from urban and suburban runoff and domestic wastewater. Nano-TiO has been shown to be toxic to a wide range of aquatic organisms, but little is known about the effects of nano-TiO on benthic microbial communities. This study used artificial stream mesocosms to assess the effects of a single addition of nano-TiO (P25 at a final concentration of 1 mg l) on the abundance, activity, and community composition of sediment-associated bacterial communities. The addition of nano-TiO resulted in a rapid (within 1 day) decrease in bacterial abundance in artificial stream sediments, but bacterial abundance returned to control levels within 3 weeks. Pyrosequencing of partial 16S rRNA genes did not indicate any significant changes in the relative abundance of any bacterial taxa with nano-TiO treatment, indicating that nano-TiO was toxic to a broad range of bacterial taxa and that recovery of the bacterial communities was not driven by changes in community composition. Addition of nano-TiO also resulted in short-term increases in respiration rates and denitrification enzyme activity, with both returning to control levels within 3 weeks. The results of this study demonstrate that single-pulse additions of nano-TiO to aquatic habitats have the potential to significantly affect the abundance and activity of benthic microbial communities and suggest that interactions of TiO nanoparticles with environmental matrices may limit the duration of their toxicity. [ABSTRACT FROM AUTHOR]

The human MHC class I (MHC-I) chain-related genes A and B ( MICA and MICB) encode stress-induced glycoproteins, ligands for the activating receptor NKG2D. They display an unusually high degree of polymorphism, next only to that of classical MHC-I. The functional relevance and selective pressure behind this peculiar polymorphism, which is quite distinct from that of classical MHC-I, remain largely unknown. This study increases the repertoire of allelic sequences determined for the MIC genes of non-human primates. Sequencing (mainly exons 2, 3, 4, 5) MIC genes of 72 Macaca fascicularis (Mafa), 63 Pan troglodytes (Patr), and 18 Gorilla gorilla (Gogo) individuals led to the identification of 35, 14, and 3 new alleles, respectively. Additionally, we confirm the existence of three independent MIC genes in M. fascicularis, i.e., Mafa- MICA, Mafa- MICB, and Mafa- MICB/A, the latter being a hybrid of Mafa- MICB and Mafa- MICA. By multiple sequence alignment and phylogenetic analysis, we further demonstrate that the present day MIC genes most likely derive from a single human MICB-like ancestral gene. [ABSTRACT FROM AUTHOR]

It remains unclear whether γδ T cell antigen receptors (TCRs) detect antigens in a way similar to antibodies or αβ TCRs. Here we show that reactivity between the G8 and KN6 γδ TCRs and the major histocompatibility complex class Ib molecule T22 could be recapitulated, with retention of wild-type ligand affinity, in an αβ TCR after grafting of a G8 or KN6 complementarity-determining region 3-δ (CDR3δ) loop in place of the CDR3α loop of an αβ TCR. We also found that a shared sequence motif in CDR3δ loops of all T22-reactive γδ TCRs bound T22 in energetically distinct ways, and that T10d, which bound G8 with weak affinity, was converted into a high-affinity ligand by a single point mutation. Our results demonstrate unprecedented autonomy of a single CDR3 loop in antigen recognition. [ABSTRACT FROM AUTHOR]

Patr-AL is a recently described gene found only in the common chimpanzee, but closely related in structure to the highly polymorphic Patr-A and HLA-A genes of the chimpanzee and human MHCs, respectively. Unlike Patr-A and HLA-A, the Patr-AL gene has little polymorphism and is not fixed in the chimpanzee genome. To determine whether Patr-AL is located in the MHC or elsewhere, we compared segregation of the Patr-AL gene with segregation of Patr-A and -B alleles in chimpanzee families. The results demonstrate that Patr-AL is an MHC class I gene present on different MHC haplotypes as defined by their combination of Patr-A and B alleles. [ABSTRACT FROM AUTHOR]

HLA-B and C are related class I genes which are believed to have arisen by duplication of a common ancestor. Previous study showed the presence of orthologues for both HLA-B and C in African apes but only for HLA-B in Asian apes. These observations suggested that the primate C locus evolved subsequent to the divergence of the Pongidae and Hominidae. From an analysis of orangutan Tengku two HLA-C-like alleles ( Popy C*0101 and Popy C*0201) were defined as well as three HLA-B-like ( Popy-B) alleles. By contrast, no Popy-C alleles were obtained from orangutan Hati, although three Popy-B alleles were defined. Thus an HLA-C-like locus exists in the orangutan (as well as a duplicated B locus), implying that the primate C locus evolved prior to the divergence of the Pongidae and Hominidae and is at least 12–13 million years old. Uncertain is whether all orangutan MHC haplotypes contain a C locus, as the failure to find C alleles in some individuals could be due to a mispairing of HLA-C-specific primers with certain Popy-C alleles. These results raise the possibilities that other primate species have a C locus and that the regulation of natural killer cells by C allotypes evolved earlier in primate evolution than has been thought. [ABSTRACT FROM AUTHOR]

Little is known regarding the rates at which natural selection can modify or retain antigen presenting alleles at the major histocompatibility complex (MHC). Discovery of identical [1101 base pairs (bp)] coding regions at the MHC class I C locus in Pan troglodytes and Pan paniscus, chimpanzee species that diverged ∼2.3 million years ago, now indicates that a class I allotype can survive for at least this period. Remarkable conservation was also reflected in the (1799 bp) introns where a maximum of only six substitutions distinguished five alleles (three from P. troglodytes and two from P. paniscus) that encoded the identical heavy chain allotype. Analysis of a more distantly related human allele, HLA-Cw * 0702, corroborated that intron variation was non-uniform along the gene. Thus we provide a clear reference frame for the lifetime of an MHC class I allotype, a direct estimate of allelic substitution rates, and evidence for an unusual evolution of MHC class I introns. [ABSTRACT FROM AUTHOR]