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Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.

The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.

Progestins, used in contraceptives and hormone replacement therapy, have been linked to breast cancer. Two teams working independently have now found a mechanistic basis for this association. Schramek et al. show in a mouse model that synthetic progestins can promote mammary tumour formation by inducing the osteoclast differentiation factor RANKL, which acts on mammary epithelial cells through the RANKL receptor RANK. Gonzalez-Suarez et al. find that inhibition of RANKL reduces tumorigenesis in hormone-induced as well as in other mouse mammary gland tumour models, suggesting a new therapeutic approach. One RANKL inhibitor (denosumab) is in clinical trials as a treatment for bone loss in post-menopausal osteoporosis and for the treatment of skeletal-related symptoms in metastatic bone disease. Progestins, used in contraceptives and hormone replacement therapy, have been linked to breast cancer. These authors provide a mechanistic basis for this association. They show in a mouse model that synthetic progestins can promote mammary tumour formation by inducing RANKL (receptor activator of NF-KB ligand), which acts on mammary epithelial cells through the RANKL receptor RANK. Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe1. The Women’s Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer2,3. In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the in vivo administration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the CD49fhi stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer.

Angiotensin -converting enzyme 2 (ACE2) is a regulator of the renin angiotensin system involved in acute lung failure, cardiovascular functions and severe acute respiratory syndrome (SARS) infections in mammals. A gene encoding a homologue to ACE2, termed collectrin (Tmem27), has been identified in immediate proximity to the ace2 locus. The in vivo function of collectrin was unclear. Here we report that targeted disruption of collectrin in mice results in a severe defect in renal amino acid uptake owing to downregulation of apical amino acid transporters in the kidney. Collectrin associates with multiple apical transporters and defines a novel group of renal amino acid transporters. Expression of collectrin in Xenopus oocytes and Madin-Darby canine kidney (MDCK) cells enhances amino acid transport by the transporter B(0)AT1. These data identify collectrin as a key regulator of renal amino acid uptake.

Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Here we show that angiotensin-converting enzyme 2 (ace2) maps to a defined quantitative trait locus (QTL) on the X chromosome in three different rat models of hypertension. In all hypertensive rat strains, ACE2 messenger RNA and protein expression were markedly reduced, suggesting that ace2 is a candidate gene for this QTL. Targeted disruption of ACE2 in mice results in a severe cardiac contractility defect, increased angiotensin II levels, and upregulation of hypoxia-induced genes in the heart. Genetic ablation of ACE on an ACE2 mutant background completely rescues the cardiac phenotype. But disruption of ACER, a Drosophila ACE2 homologue, results in a severe defect of heart morphogenesis. These genetic data for ACE2 show that it is an essential regulator of heart function in vivo.

Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, and members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains called Toll/IL-1R/plant R gene homology (TIR) domains. Intracellular signalling mechanisms mediated by TIRs are similar, with MyD88 (refs 5-8) and TRAF6 (refs 9, 10) having critical roles. Signal transduction between MyD88 and TRAF6 is known to involve the serine-threonine kinase IL-1 receptor-associated kinase 1 (IRAK-1) and two homologous proteins, IRAK-2 (ref. 12) and IRAK-M. However, the physiological functions of the IRAK molecules remain unclear, and gene-targeting studies have shown that IRAK-1 is only partially required for IL-1R and TLR signalling. Here we show by gene-targeting that IRAK-4, an IRAK molecule closely related to the Drosophila Pelle protein, is indispensable for the responses of animals and cultured cells to IL-1 and ligands that stimulate various TLRs. IRAK-4-deficient animals are completely resistant to a lethal dose of lipopolysaccharide (LPS). In addition, animals lacking IRAK-4 are severely impaired in their responses to viral and bacterial challenges. Our results indicate that IRAK-4 has an essential role in innate immunity.

The outcome of T-cell responses after T-cell encounter with specific antigens is modulated by co-stimulatory signals, which are required for both lymphocyte activation and development of adaptive immunity1,2,3. ICOS4,5, an inducible co-stimulator with homology to CD28, is expressed on activated, but not resting T cells, and shows T-cell co-stimulatory function in vitro. ICOS binds specifically to its counter-receptor B7RP-1 (refs 5,6,7), but not to B7-1 or B7-2. Here we provide in vivo genetic evidence that ICOS delivers a co-stimulatory signal that is essential both for efficient interaction between T and B cells and for normal antibody responses to T-cell-dependent antigens. To determine the physiological function of ICOS, we generated and characterized gene-targeted ICOS-deficient mice. In vivo, a lack of ICOS results in severely deficient T-cell-dependent B-cell responses. Germinal centre formation is impaired and immunoglobulin class switching, including production of allergy-mediating IgE, is defective. ICOS-deficient T cells primed in in vivo and restimulated in vitro with specific antigen produce only low levels of interleukin-4, but remain fully competent to produce interferon-γ.

Phosphoinositide-3-OH kinases (PI(3)Ks) constitute a family of evolutionarily conserved lipid kinases that regulate a vast array of fundamental cellular responses, including proliferation, transformation, differentiation and protection from apoptosis. PI(3)K-mediated activation of the cell survival kinase PKB/Akt, and negative regulation of PI(3)K signalling by the tumour suppressor PTEN (refs 3, 4) are key regulatory events in tumorigenesis. Thus, a model has arisen that PI(3)Ks promote development of cancers. Here we report that genetic inactivation of the p110gamma catalytic subunit of PI(3)Kgamma (ref. 8) leads to development of invasive colorectal adenocarcinomas in mice. In humans, p110gamma protein expression is lost in primary colorectal adenocarcinomas from patients and in colon cancer cell lines. Overexpression of wild-type or kinase-dead p110gamma in human colon cancer cells with mutations of the tumour suppressors APC and p53, or the oncogenes beta-catenin and Ki-ras, suppressed tumorigenesis. Thus, loss of p110gamma in mice leads to spontaneous, malignant epithelial tumours in the colorectum and p110gamma can block the growth of human colon cancer cells.

Bone remodelling and bone loss are controlled by a balance between the tumour necrosis factor family molecule osteoprotegerin ligand (OPGL) and its decoy receptor osteoprotegerin (OPG)1,2,3. In addition, OPGL regulates lymph node organogenesis, lymphocyte development and interactions between T cells and dendritic cells in the immune system3,4,5. The OPGL receptor, RANK, is expressed on chondrocytes, osteoclast precursors and mature osteoclasts4,6. OPGL expression in T cells is induced by antigen receptor engagement7, which suggests that activated T cells may influence bone metabolism through OPGL and RANK. Here we report that activated T cells can directly trigger osteoclastogenesis through OPGL. Systemic activation of T cells in vivo leads to an OPGL-mediated increase in osteoclastogenesis and bone loss. In a T-cell-dependent model of rat adjuvant arthritis characterized by severe joint inflammation, bone and cartilage destruction and crippling, blocking of OPGL through osteoprotegerin treatment at the onset of disease prevents bone and cartilage destruction but not inflammation. These results show that both systemic and local T-cell activation can lead to OPGL production and subsequent bone loss, and they provide a novel paradigm for T cells as regulators of bone physiology.

Distinct and evolutionarily conserved signal transduction cascades mediate survival or death in response to developmental and environmental cues. The stress-activated protein kinases, or Jun N-terminal kinases (SAPKs/JNKs)1,2, are activated in response to a variety of cellular stresses such as changes in osmolarity and metabolism, DNA damage, heat shock, ischaemia, or inflammatory cytokines3–6. Sek1 (JNKK/MKK4) is a direct activator of SAPKs/JNKs in response to environmental stresses or mitogenic factors7–9. Here we investigate the role of Sek1 in development and apoptosis by deleting sek1 in embryonic stem (ES) cells by homologous recombination. We provide genetic evidence that different stresses utilize distinct signalling pathways for SAPK/ JNK activation, sek1−/− /rag2−/− chimaeric mice have normal numbers of mature T cells but fewer immature CD4+CD8+ thymocytes. The sek1 mutation did not affect the induction of apoptosis in response to environmental stresses in ES and T cells: instead, sek1 protected thymocytes from CD95 (Fas)- and CD3-mediated apoptosis. These data indicate that SEK1 mediates survival signals in T-cell development.

The concept of tumour surveillance implies that specific and non-specific components of the immune system eliminate tumours in the early phase of malignancy. The immunological mechanisms that control growth of preneoplastic cells are, however, not known. T cells expressing gamma delta T-cell receptors (TCR) were first described as lymphocytes with reactivity against various tumour cells, which suggests that gamma delta T cells could mediate tumour surveillance. Here we show that TCRV gamma 1.1J gamma 4C gamma 4 transgenic mice are spontaneously resistant to acute T-cell leukaemias but cannot reject non-haematopoietic tumours. TCRV gamma 1.1J gamma 4C gamma 4+ hybridomas isolated from these mice react in vitro against almost all haematopoietic tumour cell lines tested. Recognition of tumour cells depends on the gamma delta TCR but is independent of major histocompatibility complex (MHC) class I, MHC class II, or TAP-2 peptide transporter expression. Ligand recognition is influenced by the murine Nromp gene, which confers resistance or susceptibility to tuberculosis, lepra and leishmaniasis. These data indicate that TCRV gamma 1.1+ T cells confer spontaneous immunity against haematopoietic tumours in vivo and link innate resistance to bacterial infections with tissue-specific tumour surveillance by gamma delta+ T cells.

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies2. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology3. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a a 'pill' that awakens the innate immune system to kill cancer metastases. © 2014 Macmillan Publishers Limited. Fil: Paolino, Magdalena. Institute Of Molecular Biotechnology, Vienna; Austria Fil: Choidas, Axel. Lead Discovery Center GmbH; Alemania Fil: Wallner, Stephanie. Medizinische Universitat Innsbruck; Austria Fil: Pranjic, Blanka. Institute Of Molecular Biotechnology, Vienna; Austria Fil: Uribesalgo, Iris. Institute Of Molecular Biotechnology, Vienna; Austria Fil: Loeser, Stefanie. Institute Of Molecular Biotechnology, Vienna; Austria Fil: Jamieson, Amanda M.. University Brown; Estados Unidos Fil: Langdon, Wallace Y.. University of Western Australia; Australia Fil: Ikeda, Fumiyo. Institute Of Molecular Biotechnology, Vienna; Austria Fil: Fededa, Juan Pablo. Institute Of Molecular Biotechnology, Vienna; Austria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Cronin, Shane J.. Institute Of Molecular Biotechnology, Vienna; Austria Fil: Nitsch, Roberto. Institute Of Molecular Biotechnology, Vienna; Austria Fil: Schultz-Fademrecht, Carsten. Lead Discovery Center GmbH; Alemania Fil: Eickhoff, Jan. Lead Discovery Center GmbH; Alemania Fil: Menninger, Sascha. Lead Discovery Center GmbH; Alemania Fil: Unger, Anke. Lead Discovery Center GmbH; Alemania Fil: Torka, Robert. Institute for Biochemistry Max-Planck; Alemania Fil: Gruber, Thomas. Medizinische Universitat Innsbruck; Austria Fil: Hinterleitner, Reinhard. Medizinische Universitat Innsbruck; Austria Fil: Baier, Gottfried. Medizinische Universitat Innsbruck; Austria Fil: Wolf, Dominik. University Hospital Bonn; Alemania. Medical University Innsbruck; Austria Fil: Ullrich, Axel. Institute for Biochemistry Max-Planck; Alemania Fil: Klebl, Bert M.. Lead Discovery Center GmbH; Alemania Fil: Penninger, Josef M.. Institute Of Molecular Biotechnology, Vienna; Austria

Receptor-activator of NF-kappa B ligand (TNFSF11, also known as RANKL, OPGL, TRANCE and ODF) and its tumour necrosis factor (TNF)-family receptor RANK are essential regulators of bone remodelling, lymph node organogenesis and formation of a lactating mammary gland(1-4). RANKL and RANK are also expressed in the central nervous system(5,6). However, the functional relevance of RANKL/RANK in the brain was entirely unknown. Here we report that RANKL and RANK have an essential role in the brain. In both mice and rats, central RANKL injections trigger severe fever. Using tissue-specific Nestin-Cre and GFAP-Cre rank(floxed) deleter mice, the function of RANK in the fever response was genetically mapped to astrocytes. Importantly, Nestin-Cre and GFAP-Cre rank(floxed) deleter mice are resistant to lipopolysaccharide-induced fever as well as fever in response to the key inflammatory cytokines IL-1 beta and TNF alpha. Mechanistically, RANKL activates brain regions involved in thermoregulation and induces fever via the COX2-PGE(2)/EP3R pathway. Moreover, female Nestin-Cre and GFAP-Cre rank(floxed) mice exhibit increased basal body temperatures, suggesting that RANKL and RANK control thermoregulation during normal female physiology. We also show that two children with RANK mutations exhibit impaired fever during pneumonia. These data identify an entirely novel and unexpected function for the key osteoclast differentiation factors RANKL/RANK in female thermoregulation and the central fever response in inflammation. Austrian Ministry of Sciences Austrian Academy of Sciences GEN-AU ( AustroMouse) European Commission European Research Council (ERC) European Commission Uehara Memorial Foundation Japan Foundation for Applied Enzymology Austrian Ministry for Science and Research Wiener Wissenschafts-, Forschungs- und Technologiefonds (WWTF) IMBA

Wound healing is essential for maintaining the integrity of multicellular organisms. In every species studied, disruption of an epithelial layer instantaneously generates endogenous electric fields, which have been proposed to be important in wound healing. The identity of signalling pathways that guide both cell migration to electric cues and electric-field-induced wound healing have not been elucidated at a genetic level. Here we show that electric fields, of a strength equal to those detected endogenously, direct cell migration during wound healing as a prime directional cue. Manipulation of endogenous wound electric fields affects wound healing in vivo. Electric stimulation triggers activation of Src and inositol-phospholipid signalling, which polarizes in the direction of cell migration. Notably, genetic disruption of phosphatidylinositol-3-OH kinase-gamma (PI(3)Kgamma) decreases electric-field-induced signalling and abolishes directed movements of healing epithelium in response to electric signals. Deletion of the tumour suppressor phosphatase and tensin homolog (PTEN) enhances signalling and electrotactic responses. These data identify genes essential for electrical-signal-induced wound healing and show that PI(3)Kgamma and PTEN control electrotaxis.

Drug hope for SARS The SARS (severe acute respiratory syndrome) epidemic of 2003 caused almost 800 deaths, many of them due to acute respiratory distress syndrome (ARDS) as a complication. There are no effective drugs available for treating ARDS, but new work in mice suggests that ACE2 (angiotensin-converting enzyme 2) might be an option. ACE2 can protect mice from lung injury in an ARDS-like syndrome, whereas other components of the renin–angiotensin system for controlling blood pressure and salt balance actually make the condition worse. ACE2 is expressed in the healthy lung but downregulated by lung injury and it was shown recently (Nature 426, 450–454; 2003) to be a receptor for the SARS coronavirus. Supplementary information The online version of this article (doi:10.1038/nature03712) contains supplementary material, which is available to authorized users., Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, is a devastating clinical syndrome with a high mortality rate (30–60%) (refs 1–3). Predisposing factors for ARDS are diverse1,3 and include sepsis, aspiration, pneumonias and infections with the severe acute respiratory syndrome (SARS) coronavirus4,5. At present, there are no effective drugs for improving the clinical outcome of ARDS1,2,3. Angiotensin-converting enzyme (ACE) and ACE2 are homologues with different key functions in the renin–angiotensin system6,7,8. ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 inactivates angiotensin II and is a negative regulator of the system. ACE2 has also recently been identified as a potential SARS virus receptor and is expressed in lungs9,10. Here we report that ACE2 and the angiotensin II type 2 receptor (AT2) protect mice from severe acute lung injury induced by acid aspiration or sepsis. However, other components of the renin–angiotensin system, including ACE, angiotensin II and the angiotensin II type 1a receptor (AT1a), promote disease pathogenesis, induce lung oedemas and impair lung function. We show that mice deficient for Ace show markedly improved disease, and also that recombinant ACE2 can protect mice from severe acute lung injury. Our data identify a critical function for ACE2 in acute lung injury, pointing to a possible therapy for a syndrome affecting millions of people worldwide every year. Supplementary information The online version of this article (doi:10.1038/nature03712) contains supplementary material, which is available to authorized users.

Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.

The regulation of tyrosine phosphorylation and associated signalling through antigen, growth-factor and cytokine receptors is mediated by the reciprocal activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPases)1. The transmembrane PTPase CD45 is a key regulator of antigen receptor signalling in T and B cells2,3. Src-family kinases have been identified as primary molecular targets for CD45 (ref. 4). However, CD45 is highly expressed in all haematopoietic lineages at all stages of development5, indicating that CD45 could regulate other cell types and might act on additional substrates. Here we show that CD45 suppresses JAK (Janus kinase) kinases and negatively regulates cytokine receptor signalling. Targeted disruption of the cd45 gene leads to enhanced cytokine and interferon-receptor-mediated activation of JAKs and STAT (signal transducer and activators of transcription) proteins. In vitro , CD45 directly dephosphorylates and binds to JAKs. Functionally, CD45 negatively regulates interleukin-3-mediated cellular proliferation, erythropoietin-dependent haematopoieisis and antiviral responses in vitro and in vivo. Our data identify an unexpected and novel function for CD45 as a haematopoietic JAK phosphatase that negatively regulates cytokine receptor signalling.

The signalling thresholds of antigen receptors and co-stimulatory receptors determine immunity or tolerance to self molecules. Changes in co-stimulatory pathways can lead to enhanced activation of lymphocytes and autoimmunity, or the induction of clonal anergy. The molecular mechanisms that maintain immunotolerance in vivo and integrate co-stimulatory signals with antigen receptor signals in T and B lymphocytes are poorly understood. Members of the Cbl/Sli family of molecular adaptors function downstream from growth factor and antigen receptors. Here we show that gene-targeted mice lacking the adaptor Cbl-b develop spontaneous autoimmunity characterized by auto-antibody production, infiltration of activated T and B lymphocytes into multiple organs, and parenchymal damage. Resting cbl-b(-/-) lymphocytes hyperproliferate upon antigen receptor stimulation, and cbl-b(-/-) T cells display specific hyperproduction of the T-cell growth factor interleukin-2, but not interferon-gamma or tumour necrosis factor-alpha. Mutation of Cbl-b uncouples T-cell proliferation, interleukin-2 production and phosphorylation of the GDP/GTP exchange factor Vav1 from the requirement for CD28 co-stimulation. Cbl-b is thus a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity.

Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

Martin and Mundy 1 emphasize the crucial role played by osteoclasts in the metastasis of many tumour types to bone. Our intention was not to question the importance of osteoclasts in bone metastases, but rather to show that RANK ligand (RANKL) exerts additional action on cancer cells beyond its effects on osteoclasts2. We reported that epithelial tumour cells can express RANK, a finding that is supported by earlier work3; that RANKL has chemotactic activity4; and that in vivo inhibition of RANKL by osteoprotegerin reduces tumour burden in bone when assessed in a previously published experimental system5.

Nature 406, 897–902 (2000). In this Letter, we reported that mice lacking the catalytic subunit of PI(3)Kγ on 129J backgrounds develop invasive colorectal tumours. We have observed this cancer phenotype in mouse lines derived from two different targeted embryonic stem-cell clones for at least two years.