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Tables S1, S2 Figures S1-S7

Supplemental Table 4. NK cells were negatively enriched from pooled spleens of 3 WT and 3 Cd47-/- littermate mice and cultured with/out thrombospondin-1 (2nM) for 24 hours in RPMI (without FCS) + IL-15 (2.5 ng/ml). Golgi-stop was added at last 4 hours of culture. FcRs were blocked with rat and rabbit serum and cell surface CD47 and NK1.1 were stained with Aqua Live/Dead. Cells were then fixed, permeabilized and i.c. stained for Ki-67, IL-2, IFNγ, TNFα and GzmB. Values indicate the MFI of indicated proteins.

Elevated CD47 expression in some cancers is associated with decreased survival and limited clearance by phagocytes expressing the CD47 counterreceptor SIRPα. In contrast, elevated CD47 mRNA expression in human melanomas was associated with improved survival. Gene-expression data were analyzed to determine a potential mechanism for this apparent protective function and suggested that high CD47 expression increases recruitment of natural killer (NK) cells into the tumor microenvironment. The CD47 ligand thrombospondin-1 inhibited NK cell proliferation and CD69 expression in vitro. Cd47−/− NK cells correspondingly displayed augmented effector phenotypes, indicating an inhibitory function of CD47 on NK cells. Treating human NK cells with a CD47 antibody that blocks thrombospondin-1 binding abrogated its inhibitory effect on NK cell proliferation. Similarly, treating wild-type mice with a CD47 antibody that blocks thrombospondin-1 binding delayed B16 melanoma growth, associating with increased NK cell recruitment and increased granzyme B and interferon-γ levels in intratumoral NK but not CD8+ T cells. However, B16 melanomas grew faster in Cd47−/− than in wild-type mice. Melanoma-bearing Cd47−/− mice exhibited decreased splenic NK cell numbers, with impaired effector protein expression and elevated exhaustion markers. Proapoptotic gene expression in Cd47−/− NK cells was associated with stress-mediated increases in mitochondrial proton leak, reactive oxygen species, and apoptosis. Global gene-expression profiling in NK cells from tumor-bearing mice identified CD47-dependent transcriptional responses that regulate systemic NK activation and exhaustion. Therefore, CD47 positively and negatively regulates NK cell function, and therapeutic antibodies that block inhibitory CD47 signaling can enhance NK immune surveillance of melanomas.

Supplemental Figure S2. CD47 blockade enhances T cell mediated cancer cell killing without affecting target cell proliferation.

Supplementary Table 1. Th1 vs. Th2 Cytokine Trend. Supplementary Table 2. Effect of L-NAME on Radiation-induced Cytokine Expression.

LCC9 (A) or ZR-75-1 (B) cells were transfected with control siRNA, GRP78 siRNA, or GRP78 cDNA for 24 hours and then plated in an ACEA E-plate in the presence of vehicle or 100 nM 4-OHT; cell index was measured by electrical impedance every 12 hours for 72 hours. n=3, *p

A. Heat map of lipid metabolites in LCC1 and LCC9 cells transfected with control or GRP78 siRNA and treated with or without TAM. n=6. B. Relative levels of oleic ethanolamide and palmitoyl ethanolamide in LCC1 and LCC9 cells treated with vehicle, TAM, GRP78 siRNA or GRP78 siRNA+TAM. n=6. C. LCC9 cells were treated with 10 nM -1 microM oleic ethanolamide (OEA), and/or various doses (vehicle, 10 nM, 100 nM, 1000 nM) TAM for 6 days. Relative cell density was determined by crystal violet assay. n=4, *p

RT-PCR gene expression of SCD1 (A), FASN (B), ACC (C), and CPT1A (D) was measured in LCC1 and LCC9 breast cancer cells transfected with control or GRP78 siRNA for 72 hours and normalized with HRPT housekeeping gene expression. n=3; *p

The unfolded protein response is an endoplasmic reticulum stress pathway mediated by the protein chaperone glucose regulated-protein 78 (GRP78). Metabolic analysis of breast cancer cells shows that GRP78 silencing increases the intracellular concentrations of essential polyunsaturated fats, including linoleic acid. Accumulation of fatty acids is due to an inhibition of mitochondrial fatty acid transport, resulting in a reduction of fatty acid oxidation. These data suggest a novel role of GRP78-mediating cellular metabolism. We validated the effect of GRP78-regulated metabolite changes by treating tumor-bearing mice with tamoxifen and/or linoleic acid. Tumors treated with linoleic acid plus tamoxifen exhibited reduced tumor area and tumor weight. Inhibition of either GRP78 or linoleic acid treatment increased MCP-1 serum levels, decreased CD47 expression, and increased macrophage infiltration, suggesting a novel role for GRP78 in regulating innate immunity. GRP78 control of fatty acid oxidation may represent a new homeostatic function for GRP78. Cancer Res; 76(19); 5657–70. ©2016 AACR.

A. LCC9 breast cancer cells were transfected with control siRNA, GRP78 siRNA, control pcDNA, or GRP78 cDNA for 72 hours and CD47, calreticulin, and HMGB1 protein expression measured by Western blot hybridization. B. LCC9 xenografts from control mice or mice treated with TAM, GRP78M, or GRP78M+TAM were stained for CD47 and visualized at 20X. n=6; *p

Supplemental Figure Legend. Legend for Supplemental Figures S1-S5.

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47+ EVs. Mass spectrometry was employed here to identify potential mechanisms by which CD47 regulates the trafficking of specific RNAs to EVs. Specific interactions of CD47 and its cytoplasmic adapter ubiquilin-1 with components of the exportin-1/Ran nuclear export complex were identified and confirmed by coimmunoprecipitation. Exportin-1 is known to regulate nuclear to cytoplasmic trafficking of 5’-7-methylguanosine (m7G)-modified microRNAs and mRNAs that interact with its cargo protein EIF4E. Interaction with CD47 was inhibited following alkylation of exportin-1 at Cys528 by its covalent inhibitor leptomycin B. Leptomycin B increased levels of m7G-modified RNAs, and their association with exportin-1 in EVs released from wild type but not CD47-deficient cells. In addition to perturbing nuclear to cytoplasmic transport, transcriptomic analyses of EVs released by wild type and CD47-deficient Jurkat T cells revealed a global CD47-dependent enrichment of m7G-modified microRNAs and mRNAs in EVs released by CD47-deficient cells. Correspondingly, decreasing CD47 expression in wild type cells or treatment with thrombospondin-1 enhanced levels of specific m7G-modified RNAs released in EVs, and re-expressing CD47 in CD47-deficient T cells decreased their levels. Therefore, CD47 signaling limits the trafficking of m7G-modified RNAs to EVs through physical interactions with the exportin-1/Ran transport complex.

In The Longing for Total Revolution, Bernard Yack claims not to account for totalitarianism but simply to unearth a new, specifically modern mindset. Still, the problem of totalitarianism, and what...

Traditionally, Roman temples and shrines in Britain have been contextualised in relation to wider ‘Roman’ religious practices. Until recently, considerations of architectural form and named deities have dominated discussions. The wider turn in archaeological discourse recognising ritual in everyday contexts has highlighted the importance of lived experience and landscape practice in shaping belief. Here we reflect on the implications of such ideas when approaching ritual practice at Roman temples, using a recently excavated example from Wiltshire, southern Britain, as a case study. The exceptional artefactual assemblages from the site demonstrate the importance of local and regional landscape practices and belief in shaping ritual practice in a sacred space. In addition, geophysical survey and analysis of Portable Antiquities Scheme (PAS) finds suggests that those occupying the landscape had long-term access to wealth. Deposition in the temple itself indicates the continuing importance attached to prehistoric objects in the Roman period, but also to the adoption of new votive practices of miniaturisation, mutilation and sacrifice. These rituals, although part of wider grammars of religious behaviour, had their roots in specific local contexts. Our detailed analyses provide a picture of a temple dedicated to a previously unknown local god, Bregneus, framed against that of an active community involved in farming, iron processing, quarrying, hunting and woodland management.

CD47 is a ubiquitously expressed cell surface glycoprotein that functions as a signaling receptor for thrombospondin-1 and as the counter-receptor for signal regulatory protein-α (SIRPα). Engaging SIRPα on macrophages inhibits phagocytosis, and CD47 thereby serves as a physiological marker of self. However, elevated CD47 expression on some cancer cells also protects tumors from innate immune surveillance and limits adaptive antitumor immunity via inhibitory SIRPα signaling in antigen-presenting cells. CD47 also mediates inhibitory thrombospondin-1 signaling in vascular cells, T cells, and NK cells, and blocking inhibitory CD47 signaling on cytotoxic T cells directly increases tumor cell killing. Therefore, CD47 functions as an innate and adaptive immune checkpoint. These findings have led to the development of antibodies and other therapeutic approaches to block CD47 functions in the tumor microenvironment. Preclinical studies in mice demonstrated that blocking CD47 can limit the growth of hematologic malignancies and solid tumors and enhance the efficacy of conventional chemotherapy, radiation therapy, and some targeted cancer therapies. Humanized CD47 antibodies are showing promise in early clinical trials, but side effects related to enhanced phagocytic clearance of circulating blood cells remain a concern. Approaches to circumvent these include antibody preloading strategies and development of antibodies that recognize tumor-specific epitopes of CD47, SIRPα antibodies, and bivalent antibodies that restrict CD47 blockade to specific tumor cells. Preclinical and clinical development of antibodies and related biologics that inhibit CD47/SIRPα signaling are reviewed, including strategies to combine these agents with various conventional and targeted therapeutics to improve patient outcome for various cancers.

Numerous age-dependent alterations at the molecular, cellular, tissue and organ systems levels underlie the pathophysiology of aging. Herein, the focus is upon the secreted protein thrombospondin-1 (TSP1) as a promoter of aging and age-related diseases. TSP1 has several physiological functions in youth, including promoting neural synapse formation, mediating responses to ischemic and genotoxic stress, minimizing hemorrhage, limiting angiogenesis, and supporting wound healing. These acute functions of TSP1 generally require only transient expression of the protein. However, accumulating basic and clinical data reinforce the view that chronic diseases of aging are associated with accumulation of TSP1 in the extracellular matrix, which is a significant maladaptive contributor to the aging process. Identification of the relevant cell types that chronically produce and respond to TSP1 and the molecular mechanisms that mediate the resulting maladaptive responses could direct the development of therapeutic agents to delay or revert age-associated maladies.

Comprehensive social cognition training programs have been effective to improve social cognition in people with chronic schizophrenia, although there is insufficient quality evidence for recent-onset psychosis. The aim of this study was to assess the effects of Social Cognition and Interaction Training (SCIT) in a sample of recent-onset schizophrenia outpatients. Sixteen participants who had their first psychotic episode for less than 2 years were randomly allocated to the SCIT group during 20 weeks (weekly sessions) or to a psychoeducation group and completed baseline and post-training assessment for cognitive biases, social cognition, clinical symptoms and functioning. Permutation-based analysis revealed improvements in overall functioning (P = 0.036) and blame score (P = 0.070) in the SCIT group compared to the psychoeducation intervention, with large effect sizes (d = 1.438 and d = 1.204, respectively). There were also large effect sizes for hostility, emotion recognition, social perception, positive and total symptoms (d = 0.833-1.158). These results suggest that SCIT may be an effective tool to improve attributional biases and functional outcomes in recent-onset schizophrenia outpatients. Future controlled trials with larger sample size and follow-up assessments should be developed to further understand effective intervention outcomes for this population.

T cells and endothelial cells engage in bidirectional communication that regulates angiogenesis and T cell transmigration. Extracellular vesicles (EVs) mediate intercellular communication by the transfer of bioactive molecules including RNAs. EVs produced by a given cell type are heterogeneous in their RNA content, but it is unclear how specific EV surface markers relate to their functional effects on target cells. Our previous work established that Jurkat T cell EVs bearing CD63, MHC-I, or CD47 surface markers contain distinct noncoding RNA populations. The present study reveals that CD63+ and MHC-I+ EVs from CD47-deficient Jurkat T cells are enriched in small non-coding RNAs relative to EVs from wild-type Jurkat T cells. CD47-deficient Jurkat T cells secrete more CD63+ and MHC-I+ EVs, but MHC-I+ EVs are selectively taken up more by human umbilical vein endothelial cells. Transcriptomics analysis of endothelial cells treated with CD63+ or MHC-I+ EVs showed surface marker- and CD47-dependent changes in gene expression in the target cells. Gene set enrichment analysis identified CD47-dependent, and surface marker-dependent effects of T cell EVs on VEGF and inflammatory signaling, cell cycle, and lipid and cholesterol metabolism. Thus, subsets of T cell EVs differentially regulate endothelial cell metabolism and inflammatory and angiogenic responses.

CD47, also known as integrin-associated protein, is a constitutively and ubiquitously expressed transmembrane receptor. CD47 is conserved across amniotes including mammals, reptiles, and birds. Expression is increased in many cancers and, in non-malignant cells, by stress and with aging. The up-regulation of CD47 expression is generally epigenetic, whereas gene amplification occurs with low frequency in some cancers. CD47 is a high affinity signaling receptor for the secreted protein thrombospondin-1 (THBS1) and the counter-receptor for signal regulatory protein-α (SIRPA, SIRPα) and SIRPγ (SIRPG). CD47 interaction with SIRPα serves as a marker of self to innate immune cells and thereby protects cancer cells from phagocytic clearance. Consequently, higher CD47 correlates with a poor prognosis in some cancers, and therapeutic blockade can suppress tumor growth by enhancing innate antitumor immunity. CD47 expressed on cytotoxic T cells, dendritic cells, and NK cells mediates inhibitory THBS1 signaling that further limits antitumor immunity. CD47 laterally associates with several integrins and thereby regulates cell adhesion and migration. CD47 has additional lateral binding partners in specific cell types, and ligation of CD47 in some cases modulates their function. THBS1-CD47 signaling in non-malignant cells inhibits nitric oxide/cGMP, calcium, and VEGF signaling, mitochondrial homeostasis, stem cell maintenance, protective autophagy, and DNA damage response, and promotes NADPH oxidase activity. CD47 signaling is a physiological regulator of platelet activation, angiogenesis and blood flow. THBS1/CD47 signaling is frequently dysregulated in chronic diseases.

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47

International audience; The identification of thrombospondin-1 as an angiogenesis inhibitor in 1990 prompted interest in its role in cancer biology and potential as a therapeutic target. Decreased thrombospondin-1 mRNA and protein expression are associated with progression in several cancers, while expression by nonmalignant cells in the tumor microenvironment and circulating levels in cancer patients can be elevated. THBS1 is not a tumor suppressor gene, but the regulation of its expression in malignant cells by oncogenes and tumor suppressor genes mediates some of their effects on carcinogenesis, tumor progression, and metastasis. In addition to regulating angiogenesis and perfusion of the tumor vasculature, thrombospondin-1 limits antitumor immunity by CD47-dependent regulation of innate and adaptive immune cells. Conversely, thrombospondin-1 is a component of particles released by immune cells that mediate tumor cell killing. Thrombospondin-1 differentially regulates the sensitivity of malignant and nonmalignant cells to genotoxic stress caused by radiotherapy and chemotherapy. The diverse activities of thrombospondin-1 to regulate autophagy, senescence, stem cell maintenance, extracellular vesicle function, and metabolic responses to ischemic and genotoxic stress are mediated by several cell surface receptors and by regulating the functions of several secreted proteins. This review highlights progress in understanding thrombospondin-1 functions in cancer and the challenges that remain in harnessing its therapeutic potential.

Ischemia reperfusion (IR) injury is a process defined by the temporary loss of blood flow and tissue perfusion followed later by restoration of the same. Brief periods of IR can be tolerated with little permanent deficit, but sensitivity varies for different target cells and tissues. Ischemia reperfusion injuries have multiple causes including peripheral vascular disease and surgical interventions that disrupt soft tissue and organ perfusion as occurs in general and reconstructive surgery. Ischemia reperfusion injury is especially prominent in organ transplantation where substantial effort has been focused on protecting the transplanted organ from the consequences of IR. A number of factors mediate IR injury including the production of reactive oxygen species and inflammatory cell infiltration and activation. In the kidney, IR injury is a major cause of acute injury and secondary loss of renal function. Transplant-initiated renal IR is also a stimulus for innate and adaptive immune-mediated transplant dysfunction. The cell surface molecule CD47 negatively modulates cell and tissue responses to stress through limitation of specific homeostatic pathways and initiation of cell death pathways. Herein, a summary of the maladaptive activities of renal CD47 will be considered as well as the possible therapeutic benefit of interfering with CD47 to limit renal IR.

U-Pb age spectra of detrital zircons are widely used to estimate maximum depositional ages (MDA) for sedimentary successions of various age. Different methods have been proposed for calculating an MDA. The most common are based on calculated ages of either the youngest single grain (YSG), the youngest grain cluster composed of three or more grains that overlap at 2σ (YGC 2σ), or the youngest graphical peak (YPP). Many of these methods produce MDAs consistent with biostratigraphic age or the radiometric age of volcanic horizons within the same unit; however, several studies have shown that MDA estimates based on detrital zircon can be younger than the true depositional age, particularly in active tectonic settings, indicating that the methods should be applied with care for successions where independent depositional age control is lacking.In this contribution we present a compilation of 27 detrital zircon samples from Ordovician to Silurian strata from a part of the Trondheim Nappe Complex of the central Scandinavian Caledonides. The samples belong to six stratigraphically distinct units with independent age control from fossils, dated volcanic horizons or bracketing units of known age. These successions represent various marginal basins filled during the closing stages of the Iapetus Ocean in an overall active tectonic setting with detritus from both continental landmasses and Cambro-Ordovician island arcs. Shortly after deposition, the successions were folded and metamorphosed at up to greenschist facies during Taconian accretionary events and/or the Scandian continent-continent collision.We calculated MDAs by the three methods YSG, YGC 2σ and YPP for all samples based on 206Pb/ 238U ages, applying a rigorous discordance filter of 5% (most studies use 10%), in order to use the most reliable analyses possible. Our analysis shows that the YSG MDA is up to 36 m.y. younger than the known depositional age for 17 of the 27 samples, with up to six individual grains giving too young age estimates in some samples. Hence, YSG MDA obviously does not provide a reliable MDA estimate. Of the YGC 2σ (weighted mean age) estimates, six are still significantly younger than known depositional age; and an additional seven are younger but overlap with the known depositional age when considering the maximum error on the YGC 2σ estimate. The only method which provides an MDA estimate within the age of known deposition or older for all samples is the YPP method.Our results indicate that statistically robust estimates of MDA from detrital zircon data in such an active orogenic setting are provided only by the YPP method; both the YSG and the YGC 2σ methods provided unreliably young estimates even with a discordance filter of 5% (using a filter of only 10% makes the problem considerably worse). The spuriously young ages of up to six near-concordant grains in some samples is probably due to concealed lead loss, possibly caused by (fluid-assisted?) recrystallisation of zircon domains during regional greenschist-facies metamorphism shortly after deposition.

Targeted gene disruption in mice has provided valuable insights into the functions of matricellular proteins. Apart from missense and loss of function mutations that have been associated with inherited diseases, however, their functions in humans remain unclear. The availability of deep exome sequencing data from over 140,000 individuals in the Genome Aggregation Database provided an opportunity to examine intolerance to loss of function and missense mutations in human matricellular genes. The probability of loss-of-function intolerance (pLI) differed widely within members of the thrombospondin, CYR61/CTGF/NOV (CCN), tenascin, small integrin-binding ligand N-linked glycoproteins (SIBLING), and secreted protein, acidic and rich in cysteine (SPARC) gene families. Notably, pLI values in humans had limited correlation with viability of the corresponding homozygous null mice. Among the thrombospondins, only THBS1 was highly loss-intolerant (pLI = 1). In contrast, Thbs1 is not essential for viability in mice. Several known thrombospondin-1 receptors were similarly loss-intolerant, although thrombospondin-1 is not the exclusive ligand for some of these receptors. The frequencies of missense mutations in THBS1 and the gene encoding its signaling receptor CD47 indicated conservation of some residues implicated in specific receptor binding. Deficits in missense mutations were also observed for other thrombospondin genes and for SPARC, SPOCK1, SPOCK2, TNR, and DSPP. The intolerance of THBS1 to loss of function in humans and elevated pLI values for THBS2, SPARC, SPOCK1, TNR, and CCN1 support important functions for these matricellular protein genes in humans, some of which may relate to functions in reproduction or responding to environmental stresses.

This volume synthesises an extensive programme of geophysical survey of the Roman town at Aldborough, North Yorkshire, and a thorough investigation of all previous excavations of the site. The synt...