ICOS is essential for effective T-helper-cell responses.

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 immunity. ICOS, 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-gamma.

The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo.

Mammalian telomerase is essential for the maintenance of telomere length [1-5]. Its catalytic core comprises a reverse transcriptase component (TERT) and an RNA component. While the biochemical role of mammalian TERT is well established [6-11], it is unknown whether it is sufficient for telomere-length maintenance, chromosome stability or other cellular processes. Cells from mice in which the mTert gene had been disrupted showed progressive loss of telomere DNA, a phenotype similar to cells in which the gene encoding the telomerase RNA component (mTR) has been disrupted [1,12]. On prolonged growth, mTert-deficient embryonic stem (ES) cells exhibited genomic instability, aneuploidy and telomeric fusions. ES cells heterozygous for the mTert disruption also showed telomere attrition, a phenotype that differs from heterozygous mTR cells [12]. Thus, telomere maintenance in mammals is carried out by a single, limiting TERT.

Telomerase-associated protein TEP1 is not essential for telomerase activity or telomere length maintenance in vivo.

TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription.

Induction of NF-kappaB-dependent transcription requires phosphorylation and subsequent degradation of I-kappaB, an inhibitor of NF-kappaB, followed by nuclear translocation and DNA binding of NF-kappaB. Tumor necrosis factor receptor-associated factor 2 (TRAF2) plays a role in NF-kappaB activation in response to cytokines such as tumor necrosis factor alpha (TNFalpha). In this study, we purified and characterized a novel kinase (T2K, also known as TBK1 or NAK), which associates with TRAF2 and exhibits kinase activity towards I-kappaBalpha in vitro. The physiological function of T2K was investigated using T2K-deficient mice. Heterozygotes appear normal, but t2k(-/-) animals die at approximately E14.5 of massive liver degeneration and apoptosis. Never theless, hematopoietic progenitors from T2K-deficient fetal liver support normal lymphocyte development. Furthermore, t2k(-/-) embryonic fibroblasts and thymocytes do not display increased sensitivity to TNFalpha-induced apoptosis. In response to either TNFalpha or IL-1 induction, t2k(-/-) embryonic fibroblasts exhibit normal degradation of I-kappaB and kappaB-binding activity. However, NF-kappaB-directed transcription is dramatically reduced. These results demonstrate that, like I-kappaB kinase beta and the RelA subunit of NF-kappaB, T2K is critical in protecting embryonic liver from apoptosis. However, T2K has a unique role in the activation of NF-kappaB-directed transcription, apparently independent of I-kappaB degradation and NF-kappaB DNA binding.

Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development.

Casper (c-FLIP) associates with FADD and caspase-8 in signaling complexes downstream of death receptors like Fas. We generated Casper-deficient mice and cells and noted a duality in the physiological functions of this molecule. casper-/- embryos do not survive past day 10.5 of embryogenesis and exhibit impaired heart development. This phenotype is reminiscent of that reported for FADD-/- and caspase-8-/- embryos. However, unlike FADD-/- and caspase-8-/- cells, casper-/- embryonic fibroblasts are highly sensitive to FasL- or TNF-induced apoptosis and show rapid induction of caspase activities. NF-kappaB and JNK/SAPK activation is intact in TNF-stimulated casper-/- cells. These results suggest that Casper has two distinct roles: to cooperate with FADD and caspase-8 during embryonic development and to mediate cytoprotection against death factor-induced apoptosis.

Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration.

Phosphoinositide 3-kinases (PI3Ks) regulate fundamental cellular responses such as proliferation, apoptosis, cell motility, and adhesion. Viable gene-targeted mice lacking the p110 catalytic subunit of PI3Kgamma were generated. We show that PI3Kgamma controls thymocyte survival and activation of mature T cells but has no role in the development or function of B cells. PI3Kgamma-deficient neutrophils exhibited severe defects in migration and respiratory burst in response to heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR) agonists and chemotactic agents. PI3Kgamma links GPCR stimulation to the formation of phosphatidylinositol 3,4,5-triphosphate and the activation of protein kinase B, ribosomal protein S6 kinase, and extracellular signal-regulated kinases 1 and 2. Thus, PI3Kgamma regulates thymocyte development, T cell activation, neutrophil migration, and the oxidative burst.

Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b.

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.

Targeted disruption in murine cells reveals variable requirement for Smad4 in transforming growth factor beta-related signaling.

The tumor suppressor gene Smad4 has been proposed to be a common mediator of transforming growth factor beta (TGFbeta)-related signaling pathways. We investigated the role of Smad4 in TGFbeta-related pathways by targeted disruption of its locus in murine cell lines. TGFbeta responses, including growth arrest, induction of the endogenous PAI-1 gene, and other extracellular matrix components, were normal in Smad4-deficient fibroblasts. Assembly of a TGFbeta-induced DNA-binding complex on one of two regulatory regions in the human plasminogen activator inhibitor (PAI)-1 promoter did not require Smad4 but was, instead, dependent on a TFE-3 binding site. In contrast, Smad4 was required for activation of the Xenopus Mix.2 promoter in response to TGFbeta/activin. Smad4 was also involved in the regulation of the Msx homeobox protein family members in response to bone morphogenetic protein (BMP). Interestingly, the expression of the endogenous Msx-2 was reduced, whereas that of Msx-3 was activated in differentiating Smad4(-/-) ES cells relative to wild-type cells. Moreover, reporter assays of the Msx-2 promoter revealed an absolute requirement for Smad4 in fibroblasts and ES cells for activation. Our results indicate that Smad4 is dispensable for critical TGFbeta-induced responses but is required for others in murine fibroblasts. We have identified transcriptional targets for Smad4 in the BMP signaling pathway, which may contribute to the genetic defect observed in the Smad4-deficient embryos.

Interleukin 13 is secreted by and stimulates the growth of Hodgkin and Reed-Sternberg cells.

Gene expression patterns can provide vital clues to the pathogenesis of neoplastic diseases. We investigated the expression of 950 genes in Hodgkin's disease (HD) by analyzing differential mRNA expression using microarrays. In two independent microarray experiments, the HD-derived cell lines L428 and KMH2 were compared with an Epstein-Barr virus (EBV)-immortalized lymphoblastoid B cell line, LCL-GK. Interleukin (IL)-13 and IL-5 were found to be highly expressed in the HD-derived cell lines. Examination of IL-13 and IL-5 expression by Northern blot analysis and enzyme-linked immunosorbent assay confirmed these results and revealed the expression of IL-13 in a third HD-derived cell line, HDLM2. Control LCL and EBV-negative non-Hodgkin lymphoma-derived cell lines did not express IL-13. In situ hybridization of lymph node tissue from HD patients showed that elevated levels of IL-13 were specifically expressed by Hodgkin/Reed-Sternberg (H/RS) tumor cells. Treatment of a HD-derived cell line with a neutralizing antibody to IL-13 resulted in a dose-dependent inhibition of H/RS cell proliferation. These data suggest that H/RS cells produce IL-13 and that IL-13 plays an important role in the stimulation of H/RS cell growth, possibly by an autocrine mechanism. Modulation of the IL-13 signaling pathway may be a logical objective for future therapeutic strategies.

TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling.

Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.

SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival.

SHIP is an inositol 5' phosphatase that hydrolyzes the PI3'K product PI(3,4,5)P3. We show that SHIP-deficient mice exhibit dramatic chronic hyperplasia of myeloid cells resulting in splenomegaly, lymphadenopathy, and myeloid infiltration of vital organs. Neutrophils and bone marrow-derived mast cells from SHIP-/- mice are less susceptible to programmed cell death induced by various apoptotic stimuli or by growth factor withdrawal. Engagement of IL3-R and GM-CSF-R in these cells leads to increased and prolonged PI3'K-dependent PI(3,4,5)P3 accumulation and PKB activation. These data indicate that SHIP is a negative regulator of growth factor-mediated PKB activation and myeloid cell survival.

Neuroendocrine dysplasia in mice lacking protein tyrosine phosphatase sigma.

Protein tyrosine phosphatase sigma (PTP-sigma, encoded by the Ptprs gene) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases that is highly expressed during mammalian embryonic development in the germinal cell layer lining the lateral ventricles of the developing brain, dorsal root ganglia, Rathke's pouch, olfactory epithelium, retina and developing lung and heart. On the basis of its expression and homology with the Drosophila melanogasterorthologues DPTP99 and DPTP100A (refs 5,6), which have roles in the targeting of axonal growth cones, we hypothesized that PTP-sigma may also have a modulating function in cell-cell interactions, as well as in axon guidance during mammalian embryogenesis. To investigate its function in vivo, we generated Ptprs-deficient mice. The resulting Ptprs-/-animals display retarded growth, increased neonatal mortality, hyposmia and hypofecundity. Anatomical and histological analyses showed a decrease in overall brain size with a severe depletion of luteinizing hormone-releasing hormone (LHRH)-immunoreactive cells in Ptprs-/- hypothalamus. Ptprs-/- mice have an enlarged intermediate pituitary lobe, but smaller anterior and posterior lobes. These results suggest that tyrosine phosphorylation-dependent signalling pathways regulated by PTP-sigma influence the proliferation and/or adhesiveness of various cell types in the developing hypothalamo-pituitary axis.

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.

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.

High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice.

BACKGROUND: Germ-line and sporadic mutations in the tumor suppressor gene PTEN (also known as MMAC or TEP1), which encodes a dual-specificity phosphatase, cause a variety of cancers such as Cowden disease, glioblastoma, endometrial carcinoma and prostatic cancer. PTEN is widely expressed, and Cowden disease consistently affects various organ systems, suggesting that the PTEN protein must have an important, although as yet poorly understood, function in cellular physiology. RESULTS: Homozygous mutant mice lacking exons 3-5 of the PTEN gene (mPTEN3-5) had severely expanded and abnormally patterned cephalic and caudal regions at day 8.5 of gestation. Embryonic death occurred by day 9.5 and was associated with defective chorio-allantoic development. Heterozygous mPTEN3-5 mice had an increased incidence of tumors, especially T-cell lymphomas; gamma-irradiation reduced the time lapse of tumor formation. DNA analysis of these tumors revealed the deletion of the mPTEN gene due to loss of heterozygosity of the wild-type allele. Tumors associated with loss of heterozygosity in mPTEN showed elevated phosphorylation of protein kinase B (PKB, also known as Akt kinase), thus providing a functional connection between mPTEN and a murine proto-oncogene (c-Akt) involved in the development of lymphomas. CONCLUSIONS: The mPTEN gene is fundamental for embryonic development in mice, as mPTEN3-5 mutant embryos died by day 9.5 of gestation, with patterning defects in cephalic and caudal regions and defective placentation. Heterozygous mice developed lymphomas associated with loss of heterozygosity of the wild-type mPTEN allele, and tumor appearance was accelerated by gamma-irradiation. These lymphomas had high levels of activated Akt/PKB, the protein product of a murine proto-oncogene with anti-apoptotic function, associated with thymic lymphomas. This suggests that tumors associated with mPTEN loss of heterozygosity may arise as a consequence of an acquired survival advantage. We provide direct evidence of the role of mPTEN as a tumor suppressor gene in mice, and establish the mPTEN mutant mouse as an experimental model for investigating the role of PTEN in cancer progression.

The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo.

Mutations in the SMAD4/DPC4 tumor suppressor gene, a key signal transducer in most TGFbeta-related pathways, are involved in 50% of pancreatic cancers. Homozygous Smad4 mutant mice die before day 7.5 of embryogenesis. Mutant embryos have reduced size, fail to gastrulate or express a mesodermal marker, and show abnormal visceral endoderm development. Growth retardation of the Smad4-deficient embryos results from reduced cell proliferation rather than increased apoptosis. Aggregation of mutant Smad4 ES cells with wild-type tetraploid morulae rescues the gastrulation defect. These results indicate that Smad4 is initially required for the differentiation of the visceral endoderm and that the gastrulation defect in the epiblast is secondary and non-cell autonomous. Rescued embryos show severe anterior truncations, indicating a second important role for Smad4 in anterior patterning during embryogenesis.

Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice.

The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP-deficient mutant mice. The three genotypes (+/+, +/-, -/-) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3-5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.

Brca2 is required for embryonic cellular proliferation in the mouse.

Mutations of the tumor suppressor gene BRCA2 are associated with predisposition to breast and other cancers. Homozygous mutant mice in which exons 10 and 11 of the Brca2 gene were deleted by gene targeting (Brca2(10-11)) die before day 9.5 of embryogenesis. Mutant phenotypes range from severely developmentally retarded embryos that do not gastrulate to embryos with reduced size that make mesoderm and survive until 8.5 days of development. Although apoptosis is normal, cellular proliferation is impaired in Brca2(10-11) mutants, both in vivo and in vitro. In addition, the expression of the cyclin-dependent kinase inhibitor p21 is increased. Thus, Brca2(10-11) mutants are similar in phenotype to Brca1(5-6) mutants but less severely affected. Expression of either of these two genes was unaffected in mutant embryos of the other. This study shows that Brca2, like Brca1, is required for cellular proliferation during embryogenesis. The similarity in phenotype between Brca1 and Brca2 mutants suggests that these genes may have cooperative roles or convergent functions during embryogenesis.

Increased expression of proopiomelanocortin (POMC) mRNA in adrenal glands of mice undergoing graft-versus-host disease (GVHD): association with persistent elevated plasma corticosterone levels.

GVHD in animal models induces severe thymic atrophy as a result of prolonged secretion of high concentrations of adrenal glucocorticoids. In this study we investigated the mechanism responsible for the persistent stimulation of the adrenal glands to secrete glucocorticoids in mice undergoing GVHD. GVHD was induced across the major and multiple minor histocompatibility antigen difference in unirradiated C57Bl/6 x AF1 hybrid mice by the intravenous injection of A strain parental lymphoid cells. Our results showed plasma corticosterone (CS) levels were elevated in association with high concentrations of corticotropin (ACTH) in both the GVHD and control syngeneic (SYN) groups on day 9. By days 16 and 24, plasma CS and ACTH in the SYN mice returned to basal levels. In contrast, plasma CS levels remained elevated in the GVHD animals on days 16 and 24 despite decreasing concentrations of plasma ACTH. Reverse transcription-polymerase chain reaction (RT-PCR) showed several-fold increase in POMC mRNA in the adrenal glands of GVHD mice compared with SYN animals. In addition, high mRNA levels for murine prohormone convertase 1, the enzyme that cleaves POMC into ACTH, were also detected in GVHD adrenals. Histological analysis of GVHD adrenals failed to show any sign of adrenalitis, and RT-PCR of GVHD adrenals also failed to detect mRNA for interferon-gamma (IFN-gamma), a cytokine expressed by activated T and natural killer (NK) cells. However, mRNA for IL-12, a cytokine produced by activated macrophages, was increased in GVHD adrenals, suggesting that resident adrenal macrophages were activated during GVHD. Our findings suggest that persistent elevated levels of plasma glucocorticoids during GVHD could be mediated by intra-adrenal ACTH produced by resident adrenal macrophages activated as a consequence of GVHD.