query: Pseudogene PTENP1 regulates the expression of PTEN by functioning as an miRNA decoy.

positive: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology The canonical role of messenger RNA (mRNA) is to deliver protein-coding information to sites of protein synthesis. However, given that microRNAs bind to RNAs, we hypothesized that RNAs could possess a regulatory role that relies on their ability to compete for microRNA binding, independently of their protein-coding function. As a model for the protein-coding-independent role of RNAs, we describe the functional relationship between the mRNAs produced by the PTEN tumour suppressor gene and its pseudogene PTENP1 and the critical consequences of this interaction. We find that PTENP1 is biologically active as it can regulate cellular levels of PTEN and exert a growth-suppressive role. We also show that the PTENP1 locus is selectively lost in human cancer. We extended our analysis to other cancer-related genes that possess pseudogenes, such as oncogenic KRAS. We also demonstrate that the transcripts of protein-coding genes such as PTEN are biologically active. These findings attribute a novel biological role to expressed pseudogenes, as they can regulate coding gene expression, and reveal a non-coding function for mRNAs.

negative: Expression of Versican 3′-Untranslated Region Modulates Endogenous MicroRNA Functions BACKGROUND Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. METHODS AND FINDINGS Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector. CONCLUSION Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities.

negative: Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

negative: LncRNA-GAS5 induces PTEN expression through inhibiting miR-103 in endometrial cancer cells BACKGROUND Growth arrest-specific 5 (GAS5) was reported to be implicated and aberrantly express in multiple cancers. However, the expression and mechanism of action of GAS5 were largely poor understood in endometrial carcinoma. RESULTS According to the result of real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and flow cytometry analysis, we identified that GAS5 was down-regulated in endometrial cancer cells and stimulated the apoptosis of endometrial cancer cells. To investigate the expression of GAS5, PTEN and miR-103, RT-PCR was performed. And we found that the expression of PTEN was up-regulated when endometrial cancer cells overexpressed GAS5. The prediction of bioinformatics online revealed that GAS5 could bind to miR-103, which was further found to be regulated by GAS5. Finally, we found that miR-103 mimic could decrease the mRNA and protein levels of PTEN through luciferase reporter assay and western blotting, and GAS5 plasmid may reverse this regulation effect in endometrial cancer cells. CONCLUSION In summary, we demonstrate that GAS5 acts as an tumor suppressor lncRNA in endometrial cancer. Through inhibiting the expression of miR-103, GAS5 significantly enhanced the expression of PTEN to promote cancer cell apoptosis, and, thus, could be an important mediator in the pathogenesis of endometrial cancer.

negative: PTEN Regulates PI(3,4)P2 Signaling Downstream of Class I PI3K The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.

negative: The widespread regulation of microRNA biogenesis, function and decay MicroRNAs (miRNAs) are a large family of post-transcriptional regulators of gene expression that are ∼21 nucleotides in length and control many developmental and cellular processes in eukaryotic organisms. Research during the past decade has identified major factors participating in miRNA biogenesis and has established basic principles of miRNA function. More recently, it has become apparent that miRNA regulators themselves are subject to sophisticated control. Many reports over the past few years have reported the regulation of miRNA metabolism and function by a range of mechanisms involving numerous protein–protein and protein–RNA interactions. Such regulation has an important role in the context-specific functions of miRNAs.

negative: Dual Regulation of miRNA Biogenesis Generates Target Specificity in Neurotrophin-Induced Protein Synthesis Control of translation is a fundamental source of regulation in gene expression. The induction of protein synthesis by brain-derived neurotrophic factor (BDNF) critically contributes to enduring modifications of synaptic function, but how BDNF selectively affects only a minority of expressed mRNAs is poorly understood. We report that BDNF rapidly elevates Dicer, increasing mature miRNA levels and inducing RNA processing bodies in neurons. BDNF also rapidly induces Lin28, causing selective loss of Lin28-regulated miRNAs and a corresponding upregulation in translation of their target mRNAs. Binding sites for Lin28-regulated miRNAs are necessary and sufficient to confer BDNF responsiveness to a transcript. Lin28 deficiency, or expression of a Lin28-resistant Let-7 precursor miRNA, inhibits BDNF translation specificity and BDNF-dependent dendrite arborization. Our data establish that specificity in BDNF-regulated translation depends upon a two-part posttranscriptional control of miRNA biogenesis that generally enhances mRNA repression in association with GW182 while selectively derepressing and increasing translation of specific mRNAs.

negative: miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. MicroRNAs (miRNAs) are ∼22 nt non-coding RNAs that typically bind to the 3' UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.

negative: Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA. T cells transformed by Herpesvirus saimiri express seven viral U-rich noncoding RNAs of unknown function called HSURs. We noted that conserved sequences in HSURs 1 and 2 constitute potential binding sites for three host-cell microRNAs (miRNAs). Coimmunoprecipitation experiments confirmed that HSURs 1 and 2 interact with the predicted miRNAs in virally transformed T cells. The abundance of one of these miRNAs, miR-27, is dramatically lowered in transformed cells, with consequent effects on the expression of miR-27 target genes. Transient knockdown and ectopic expression of HSUR 1 demonstrate that it directs degradation of mature miR-27 in a sequence-specific and binding-dependent manner. This viral strategy illustrates use of a ncRNA to manipulate host-cell gene expression via the miRNA pathway.

negative: LIN28B induces neuroblastoma and enhances MYCN levels via let-7 suppression LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B–let-7–MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.

negative: Epigenetic regulation of NANOG by miR-302 cluster-MBD2 completes induced pluripotent stem cell reprogramming. While most somatic cells undergoing induced pluripotent stem (iPS) cell reprogramming with Yamanaka factors accumulate at stable partially reprogrammed stages, the molecular mechanisms required to achieve full reprogramming are unknown. MicroRNAs (miRNAs) fine-tune mRNA translation and are implicated in reprogramming, but miRNA functional targets critical for complete iPS cell reprogramming remain elusive. We identified methyl-DNA binding domain protein 2 (MBD2) as an epigenetic suppressor, blocking full reprogramming of somatic to iPS cells through direct binding to NANOG promoter elements preventing transcriptional activation. When we overexpressed miR-302 cluster we observed a significant increase in conversion of partial to fully reprogrammed iPS cells by suppressing MBD2 expression, thereby increasing NANOG expression. Thus, expression of exogenous miR-302 cluster (without miR-367) is efficient in attaining a fully reprogrammed iPS state in partially reprogrammed cells by relieving MBD2-mediated inhibition of NANOG expression. Our studies provide a direct molecular mechanism involved in generating complete human iPS cell reprogramming to study disease pathogenesis, drug screening, and for potential cell-based therapies.

negative: MicroRNA-7: A miRNA with expanding roles in development and disease. MicroRNAs (miRNAs) are a family of short, non-coding RNA molecules (∼22nt) involved in post-transcriptional control of gene expression. They act via base-pairing with mRNA transcripts that harbour target sequences, resulting in accelerated mRNA decay and/or translational attenuation. Given miRNAs mediate the expression of molecules involved in many aspects of normal cell development and functioning, it is not surprising that aberrant miRNA expression is closely associated with many human diseases. Their pivotal role in driving a range of normal cellular physiology as well as pathological processes has established miRNAs as potential therapeutics, as well as potential diagnostic and prognostic tools in human health. MicroRNA-7 (miR-7) is a highly conserved miRNA which displays restricted spatiotemporal expression during development and in maturity. In humans and mice, mature miR-7 is generated from three different genes, illustrating unexpected redundancy and also the importance of this miRNA in regulating key cellular processes. In this review we examine the expanding role of miR-7 in the context of health, with emphasis on organ differentiation and development, as well as in various mammalian diseases, particularly of the brain, heart, endocrine pancreas and skin, as well as in cancer. The more we learn about miR-7, the more we realise the complexity of its regulation and potential functional application both from a biomarker and therapeutic perspective.

negative: Competition between target sites of regulators shapes post-transcriptional gene regulation Post-transcriptional gene regulation (PTGR) of mRNA turnover, localization and translation is mediated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). These regulators exert their effects by binding to specific sequences within their target mRNAs. Increasing evidence suggests that competition for binding is a fundamental principle of PTGR. Not only can miRNAs be sequestered and neutralized by the targets with which they interact through a process termed 'sponging', but competition between binding sites on different RNAs may also lead to regulatory crosstalk between transcripts. Here, we quantitatively model competition effects under physiological conditions and review the role of endogenous sponges for PTGR in light of the key features that emerge.

negative: Altered microRNA expression in bovine skeletal muscle with age. Age-dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. We therefore performed a comprehensive investigation for miRNAs from five different periods of bovine skeletal muscle development using next-generation small RNA sequencing. In total, 511 miRNAs, including one putatively novel miRNA, were identified. Thirty-six miRNAs were differentially expressed between prenatal and postnatal stages of muscle development including several myomiRs (miR-1, miR-206 and let-7 families). Compared with miRNA expression between different muscle tissues, 14 miRNAs were up-regulated and 22 miRNAs were down-regulated in the muscle of postnatal stage. In addition, a novel miRNA was predicted and submitted to the miRBase database as bta-mir-10020. A dual luciferase reporter assay was used to demonstrate that bta-mir-10020 directly targeted the 3'-UTR of the bovine ANGPT1 gene. The overexpression of bta-mir-10020 significantly decreased the DsRed fluorescence in the wild-type expression cassette compared to the mutant type. Using three computational approaches - miranda, pita and rnahybrid - these differentially expressed miRNAs were also predicted to target 3609 bovine genes. Disease and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Our miRNA expression analysis findings from different states of muscle development and aging significantly expand the repertoire of bovine miRNAs now shown to be expressed in muscle and could contribute to further studies on growth and developmental disorders in this tissue type.

negative: Virus-encoded microRNAs. MicroRNAs (miRNAs) are the subject of enormous interest. They are small non-coding RNAs that play a regulatory role in numerous and diverse cellular processes such as immune function, apoptosis and tumorigenesis. Several virus families have been shown to encode miRNAs, and an appreciation for their roles in the viral infectious cycle continues to grow. Despite the identification of numerous (>225) viral miRNAs, an in depth functional understanding of most virus-encoded miRNAs is lacking. Here we focus on a few viral miRNAs with well-defined functions. We use these examples to extrapolate general themes of viral miRNA activities including autoregulation of viral gene expression, avoidance of host defenses, and a likely important role in maintaining latent and persistent infections. We hypothesize that although the molecular mechanisms and machinery are similar, the majority of viral miRNAs may utilize a target strategy that differs from host miRNAs. That is, many viral miRNAs may have evolved to regulate viral-encoded transcripts or networks of host genes that are unique to viral miRNAs. Included in this latter category is a likely abundant class of viral miRNAs that may regulate only one or a few principal host genes. Key steps forward for the field are discussed, including the need for additional functional studies that utilize surgical viral miRNA mutants combined with relevant models of infection.

negative: Poor prognosis in carcinoma is associated with a gene expression signature of aberrant PTEN tumor suppressor pathway activity. Pathway-specific therapy is the future of cancer management. The oncogenic phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in solid tumors; however, currently, no reliable test for PI3K pathway activation exists for human tumors. Taking advantage of the observation that loss of PTEN, the negative regulator of PI3K, results in robust activation of this pathway, we developed and validated a microarray gene expression signature for immunohistochemistry (IHC)-detectable PTEN loss in breast cancer (BC). The most significant signature gene was PTEN itself, indicating that PTEN mRNA levels are the primary determinant of PTEN protein levels in BC. Some PTEN IHC-positive BCs exhibited the signature of PTEN loss, which was associated to moderately reduced PTEN mRNA levels cooperating with specific types of PIK3CA mutations and/or amplification of HER2. This demonstrates that the signature is more sensitive than PTEN IHC for identifying tumors with pathway activation. In independent data sets of breast, prostate, and bladder carcinoma, prediction of pathway activity by the signature correlated significantly to poor patient outcome. Stathmin, encoded by the signature gene STMN1, was an accurate IHC marker of the signature and had prognostic significance in BC. Stathmin was also pathway-pharmacodynamic in vitro and in vivo. Thus, the signature or its components such as stathmin may be clinically useful tests for stratification of patients for anti-PI3K pathway therapy and monitoring therapeutic efficacy. This study indicates that aberrant PI3K pathway signaling is strongly associated with metastasis and poor survival across carcinoma types, highlighting the enormous potential impact on patient survival that pathway inhibition could achieve.

negative: Functional proteomics identifies miRNAs to target a p27/Myc/phospho-Rb signature in breast and ovarian cancer The myc oncogene is overexpressed in almost half of all breast and ovarian cancers, but attempts at therapeutic interventions against myc have proven to be challenging. Myc regulates multiple biological processes, including the cell cycle, and as such is associated with cell proliferation and tumor progression. We identified a protein signature of high myc, low p27 and high phospho-Rb significantly correlated with poor patient survival in breast and ovarian cancers. Screening of a miRNA library by functional proteomics in multiple cell lines and integration of data from patient tumors revealed a panel of five microRNAs (miRNAs) (miR-124, miR-365, miR-34b*, miR-18a and miR-506) as potential tumor suppressors capable of reversing the p27/myc/phospho-Rb protein signature. Mechanistic studies revealed an RNA-activation function of miR-124 resulting in direct induction of p27 protein levels by binding to and inducing transcription on the p27 promoter region leading to a subsequent G1 arrest. Additionally, in vivo studies utilizing a xenograft model demonstrated that nanoparticle-mediated delivery of miR-124 could reduce tumor growth and sensitize cells to etoposide, suggesting a clinical application of miRNAs as therapeutics to target the functional effect of myc on tumor growth.

negative: MicroRNAs can generate thresholds in target gene expression MicroRNAs (miRNAs) are short, highly conserved noncoding RNA molecules that repress gene expression in a sequence-dependent manner. We performed single-cell measurements using quantitative fluorescence microscopy and flow cytometry to monitor a target gene's protein expression in the presence and absence of regulation by miRNA. We find that although the average level of repression is modest, in agreement with previous population-based measurements, the repression among individual cells varies dramatically. In particular, we show that regulation by miRNAs establishes a threshold level of target mRNA below which protein production is highly repressed. Near this threshold, protein expression responds sensitively to target mRNA input, consistent with a mathematical model of molecular titration. These results show that miRNAs can act both as a switch and as a fine-tuner of gene expression.

negative: A microRNA feedback loop regulates global microRNA abundance during aging. Expression levels of many microRNAs (miRNAs) change during aging, notably declining globally in a number of organisms and tissues across taxa. However, little is known about the mechanisms or the biological relevance for this change. We investigated the network of genes that controls miRNA transcription and processing during C. elegans aging. We found that miRNA biogenesis genes are highly networked with transcription factors and aging-associated miRNAs. In particular, miR-71, known to influence life span and itself up-regulated during aging, represses alg-1/Argonaute expression post-transcriptionally during aging. Increased ALG-1 abundance in mir-71 loss-of-function mutants led to globally increased miRNA expression. Interestingly, these mutants demonstrated widespread mRNA expression dysregulation and diminished levels of variability both in gene expression and in overall life span. Thus, the progressive molecular decline often thought to be the result of accumulated damage over an organism's life may be partially explained by a miRNA-directed mechanism of age-associated decline.

negative: MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation The Polycomb Group (PcG) of chromatin modifiers regulates pluripotency and differentiation. Mammalian genomes encode multiple homologs of the Polycomb repressive complex 1 (PRC1) components, including five orthologs of the Drosophila Polycomb protein (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8). We have identified Cbx7 as the primary Polycomb ortholog of PRC1 complexes in embryonic stem cells (ESCs). The expression of Cbx7 is downregulated during ESC differentiation, preceding the upregulation of Cbx2, Cbx4, and Cbx8, which are directly repressed by Cbx7. Ectopic expression of Cbx7 inhibits differentiation and X chromosome inactivation and enhances ESC self-renewal. Conversely, Cbx7 knockdown induces differentiation and derepresses lineage-specific markers. In a functional screen, we identified the miR-125 and miR-181 families as regulators of Cbx7 that are induced during ESC differentiation. Ectopic expression of these miRNAs accelerates ESC differentiation via regulation of Cbx7. These observations establish a critical role for Cbx7 and its regulatory miRNAs in determining pluripotency.

negative: Androgen regulation of micro-RNAs in prostate cancer. BACKGROUND Androgens play a critical role in the growth of both androgen dependent and castration-resistant prostate cancer (CRPC). Only a few micro-RNAs (miRNAs) have been suggested to be androgen regulated. We aim to identify androgen regulated miRNAs. METHODS We utilized LNCaP derived model, we have established, and which overexpresses the androgen receptor (AR), the VCaP cell line, and 13 intact-castrated prostate cancer (PC) xenograft pairs, as well as clinical specimens of untreated (PC) and CRPC. The expression of miRNAs was analyzed by microarrays and quantitative RT-PCR (Q-RT-PCR). Transfection of pre-miR-141 and anti-miR-141 was also used. RESULTS Seventeen miRNAs were > 1.5-fold up- or downregulated upon dihydrotestosterone (DHT) treatment in the cell lines, and 42 after castration in the AR-positive xenografts. Only four miRNAs (miR-10a, miR-141, miR-150*, and miR-1225-5p) showed similar androgen regulation in both cell lines and xenografts. Of those, miR-141 was found to be expressed more in PC and CRPC compared to benign prostate hyperplasia. Additionally, the overexpression of miR-141 enhanced growth of parental LNCaP cells while inhibition of miR-141 by anti-miR-141 suppressed the growth of the LNCaP subline overexpressing AR. CONCLUSIONS Only a few miRNAs were found to be androgen-regulated in both cell lines and xenografts models. Of those, the expression of miR-141 was upregulated in cancer. The ectopic overexpression of miR-141 increased growth of LNCaP cell suggesting it may contribute to the progression of PC.

negative: Epigenetic-induced repression of microRNA-205 is associated with MED1 activation and a poorer prognosis in localized prostate cancer Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n=14), when compared with matched normal prostate (n=7). An expanded patient cohort (tumor n=149, matched normal n=30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio=2.005, 95% confidence interval (1.109, 3.625), P=0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.

negative: Cytomegalovirus microRNAs Facilitate Persistent Virus Infection in Salivary Glands Micro (mi)RNAs are small non-coding RNAs that regulate the expression of their targets' messenger RNAs through both translational inhibition and regulation of target RNA stability. Recently, a number of viruses, particularly of the herpesvirus family, have been shown to express their own miRNAs to control both viral and cellular transcripts. Although some targets of viral miRNAs are known, their function in a physiologically relevant infection remains to be elucidated. As such, no in vivo phenotype of a viral miRNA knock-out mutant has been described so far. Here, we report on the first functional phenotype of a miRNA knock-out virus in vivo. During subacute infection of a mutant mouse cytomegalovirus lacking two viral miRNAs, virus production is selectively reduced in salivary glands, an organ essential for virus persistence and horizontal transmission. This phenotype depends on several parameters including viral load and mouse genetic background, and is abolished by combined but not single depletion of natural killer (NK) and CD4+ T cells. Together, our results point towards a miRNA-based immunoevasion mechanism important for long-term virus persistence.

negative: Engineered RNA viral synthesis of microRNAs. MicroRNAs (miRNAs) are short noncoding RNAs that exert posttranscriptional gene silencing and regulate gene expression. In addition to the hundreds of conserved cellular miRNAs that have been identified, miRNAs of viral origin have been isolated and found to modulate both the viral life cycle and the cellular transcriptome. Thus far, detection of virus-derived miRNAs has been largely limited to DNA viruses, suggesting that RNA viruses may be unable to exploit this aspect of transcriptional regulation. Lack of RNA virus-produced miRNAs has been attributed to the replicative constraints that would incur following RNase III processing of a genomic hairpin. To ascertain whether the generation of viral miRNAs is limited to DNA viruses, we investigated whether influenza virus could be designed to deliver functional miRNAs without affecting replication. Here, we describe a modified influenza A virus that expresses cellular microRNA-124 (miR-124). Insertion of the miR-124 hairpin into an intron of the nuclear export protein transcript resulted in endogenous processing and functional miR-124. We demonstrate that a viral RNA genome incorporating a hairpin does not result in segment instability or miRNA-mediated genomic targeting, thereby permitting the virus to produce a miRNA without having a negative impact on viral replication. This work demonstrates that RNA viruses can produce functional miRNAs and suggests that this level of transcriptional regulation may extend beyond DNA viruses.

negative: Hedgehog signaling regulates brain tumor-initiating cell proliferation and portends shorter survival for patients with PTEN-coexpressing glioblastomas. The identification of brain tumor stem-like cells (BTSCs) has implicated a role of biological self-renewal mechanisms in clinical brain tumor initiation and propagation. The molecular mechanisms underlying the tumor-forming capacity of BTSCs, however, remain unknown. Here, we have generated molecular signatures of glioblastoma multiforme (GBM) using gene expression profiles of BTSCs and have identified both Sonic Hedgehog (SHH) signaling-dependent and -independent BTSCs and their respective glioblastoma surgical specimens. BTSC proliferation could be abrogated in a pathway-dependent fashion in vitro and in an intracranial tumor model in athymic mice. Both SHH-dependent and -independent brain tumor growth required phosphoinositide 3-kinase-mammalian target of rapamycin signaling. In human GBMs, the levels of SHH and PTCH1 expression were significantly higher in PTEN-expressing tumors than in PTEN-deficient tumors. In addition, we show that hyperactive SHH-GLI signaling in PTEN-coexpressing human GBM is associated with reduced survival time. Thus, distinct proliferation signaling dependence may underpin glioblastoma propagation by BTSCs. Modeling these BTSC proliferation mechanisms may provide a rationale for individualized glioblastoma treatment.

negative: MicroRNA sponges: progress and possibilities. The microRNA (miRNA) "sponge" method was introduced three years ago as a means to create continuous miRNA loss of function in cell lines and transgenic organisms. Sponge RNAs contain complementary binding sites to a miRNA of interest, and are produced from transgenes within cells. As with most miRNA target genes, a sponge's binding sites are specific to the miRNA seed region, which allows them to block a whole family of related miRNAs. This transgenic approach has proven to be a useful tool to probe miRNA functions in a variety of experimental systems. Here we will discuss the ways sponge and related constructs can be optimized and review recent applications of this method with particular emphasis on stable expression in cancer studies and in transgenic animals.

negative: CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control. MicroRNAs (miRNAs) are a recently identified class of epigenetic elements consisting of small noncoding RNAs that bind to the 3' untranslated region of mRNAs and down-regulate their translation to protein. miRNAs play critical roles in many different cellular processes including metabolism, apoptosis, differentiation, and development. We found 33 miRNAs expressed in CD34+ hematopoietic stem-progenitor cells (HSPCs) from normal human bone marrow and mobilized human peripheral blood stem cell harvests. We then combined these data with human HSPC mRNA expression data and with miRNA-mRNA target predictions, into a previously undescribed miRNA:mRNA interaction database called the Transcriptome Interaction Database. The in silico predictions from the Transcriptome Interaction Database pointed to miRNA control of hematopoietic differentiation through translational control of mRNAs critical to hematopoiesis. From these predictions, we formulated a model for miRNA control of stages of hematopoiesis in which many of the genes specifying hematopoietic differentiation are expressed by HSPCs, but are held in check by miRNAs until differentiation occurs. We validated miRNA control of several of these target mRNAs by demonstrating that their translation in fact is decreased by miRNAs. Finally, we chose miRNA-155 for functional characterization in hematopoiesis, because we predicted that it would control both myelopoiesis and erythropoiesis. As predicted, miRNA-155 transduction greatly reduced both myeloid and erythroid colony formation of normal human HSPCs.

negative: Resistance to gefitinib in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3'-kinase/Akt pathway signaling. PURPOSE Tyrosine kinase (TK) inhibitors are emerging as a promising new approach to the treatment of HER overexpressing tumors, however optimal use of these agents awaits further definition of the downstream signaling pathways that mediate their effects. We reported previously that both EGFR- and Her2-overexpressing tumors are sensitive to the new EGFR-selective TK inhibitor gefitinib (ZD1839, "Iressa"), and sensitivity to this agent correlated with its ability to down-regulate Akt. However, EGFR-overexpressing MDA-468 cells, which lack PTEN function, are resistant to ZD1839, and ZD1839 is unable to down-regulate Akt activity in these cells. EXPERIMENTAL DESIGN To study the role of PTEN function, we generated MDA468 cells with tet-inducible PTEN expression. RESULTS We show here that the resistance of MDA-468 cells to ZD1839 is attributable to EGFR-independent constitutive Akt activation caused by loss of PTEN function in these cells. Reconstitution of PTEN function through tet-inducible expression restores ZD1839 sensitivity to these cells and reestablishes EGFR-stimulated Akt signaling. Although restoration of PTEN function to tumors is difficult to implement clinically, much of the effects of PTEN loss are attributable to overactive PI3K/Akt pathway signaling, and this overactivity can be modulated by pharmacologic approaches. We show here that pharmacologic down-regulation of constitutive PI3K/Akt pathway signaling using the PI3K inhibitor LY294002 similarly restores EGFR-stimulated Akt signaling and sensitizes MDA-468 cells to ZD1839. CONCLUSIONS Sensitivity to ZD1839 requires intact growth factor receptor-stimulated Akt signaling activity. PTEN loss leads to uncoupling of this signaling pathway and results in ZD1839 resistance, which can be reversed with reintroduction of PTEN or pharmacologic down-regulation of constitutive PI3K/Akt pathway activity. These data have important predictive and therapeutic clinical implications.

negative: MicroRNA-Containing T-Regulatory-Cell-Derived Exosomes Suppress Pathogenic T Helper 1 Cells Foxp3(+) T regulatory (Treg) cells prevent inflammatory disease but the mechanistic basis of suppression is not understood completely. Gene silencing by RNA interference can act in a cell-autonomous and non-cell-autonomous manner, providing mechanisms of intercellular regulation. Here, we demonstrate that non-cell-autonomous gene silencing, mediated by miRNA-containing exosomes, is a mechanism employed by Treg cells to suppress T-cell-mediated disease. Treg cells transferred microRNAs (miRNA) to various immune cells, including T helper 1 (Th1) cells, suppressing Th1 cell proliferation and cytokine secretion. Use of Dicer-deficient or Rab27a and Rab27b double-deficient Treg cells to disrupt miRNA biogenesis or the exosomal pathway, respectively, established a requirement for miRNAs and exosomes for Treg-cell-mediated suppression. Transcriptional analysis and miRNA inhibitor studies showed that exosome-mediated transfer of Let-7d from Treg cell to Th1 cells contributed to suppression and prevention of systemic disease. These studies reveal a mechanism of Treg-cell-mediated suppression mediated by miRNA-containing exosomes.

negative: Targeting TLR4 signaling by TLR4 Toll/IL-1 receptor domain-derived decoy peptides: identification of the TLR4 Toll/IL-1 receptor domain dimerization interface. Agonist-induced dimerization of TLR4 Toll/IL-1R (TIR) domains initiates intracellular signaling. Therefore, identification of the TLR4-TIR dimerization interface is one key to the rational design of therapeutics that block TLR4 signaling. A library of cell-permeating decoy peptides, each of which represents a nonfragmented patch of the TLR4 TIR surface, was designed such that the peptides entirely encompass the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating Antennapedia homeodomain sequence and tested for the ability to inhibit early cytokine mRNA expression and MAPK activation in LPS-stimulated primary murine macrophages. Five peptides--4R1, 4R3, 4BB, 4R9, and 4αE--potently inhibited all manifestations of TLR4, but not TLR2 signaling. When tested for their ability to bind directly to TLR4 TIR by Förster resonance energy transfer using time-resolved fluorescence spectroscopy, Bodipy-TMR-X-labeled 4R1, 4BB, and 4αE quenched fluorescence of TLR4-Cerulean expressed in HeLa or HEK293T cells, whereas 4R3 was partially active, and 4R9 was least active. These findings suggest that the area between the BB loop of TLR4 and its fifth helical region mediates TLR4 TIR dimerization. Moreover, our data provide direct evidence for the utility of the decoy peptide approach, in which peptides representing various surface-exposed segments of a protein are initially probed for the ability to inhibit protein function, and then their specific targets are identified by Förster resonance energy transfer to define recognition sites in signaling proteins that may be targeted therapeutically to disrupt functional transient protein interactions.

negative: microRNA-29a suppresses cell proliferation by targeting SPARC in hepatocellular carcinoma. In the present study, we constructed a lentivirus vector encoding the miR-29a             precursor and established two stably infected cell lines, PLC-29a and 97L-29a. The overexpression of miR-29a was confirmed by TaqMan RT-PCR and significantly             suppressed the growth of the hepatocellular carcinoma cell lines MHCC-97L and             PLC. Dual-luciferase reporter assays indicated that the SPARC mRNA 3'UTR was directly             targeted by miR-29a since the mutated 3'UTR was not affected. Silencing SPARC             expression by RNAi knockdown resulted in a similar effect as miR-29a overexpression             on hepatocellular carcinoma (HCC) cell growth regulation. Anti-miR-29a oligonucleotides             (AMOs) upregulated the levels of SPARC in the HCC cells. The phosphorylation of             AKT/mTOR downstream of SPARC was inhibited in miR-29a-overexpressing HCC cells. We further examined and compared the expression levels of miR-29a in HCC tissues             and the corresponding nearby non-cancerous liver tissues of 110 patients with             HCC by qRT-PCR, and significantly lower expression of miR-29a was observed in             the tissues affected by HCC. Our findings demonstrate that the expression of miR-29a             is important in the regulation of the SPARC-AKT pathway and HCC growth.

negative: Exosomal levels of miRNA-21 from cerebrospinal fluids associated with poor prognosis and tumor recurrence of glioma patients Glioma is a most common type of primary brain tumors. Extracellular vesicles, in the form of exosomes, are known to mediate cell-cell communication by transporting cell-derived proteins and nucleic acids, including various microRNAs (miRNAs). Here we examined the cerebrospinal fluid (CSF) from patients with recurrent glioma for the levels of cancer-related miRNAs, and evaluated the values for prognosis by comparing the measures of CSF-, serum-, and exosome-contained miR-21 levels. Samples from seventy glioma patients following surgery were compared with those from brain trauma patients as a non-tumor control group. Exosomal miR-21 levels in the CSF of glioma patients were found significantly higher than in the controls; whereas no difference was detected in serum-derived exosomal miR-21 expression. The CSF-derived exosomal miR-21 levels correlated with tumor spinal/ventricle metastasis and the recurrence with anatomical site preference. From additional 198 glioma tissue samples, we verified that miR-21 levels associated with tumor grade of diagnosis and negatively correlated with the median values of patient overall survival time. We further used a lentiviral inhibitor to suppress miR-21 expression in U251 cells. The results showed that the levels of miR-21 target genes of PTEN, RECK and PDCD4 were up-regulated at protein levels. Therefore, we concluded that the exosomal miR-21 levels could be demonstrated as a promising indicator for glioma diagnosis and prognosis, particularly with values to predict tumor recurrence or metastasis.

negative: Circular RNA and miR-7 in cancer. MicroRNAs (miRNA) play important roles in fine-tuning gene expression and are often deregulated in cancer. The identification of competing endogenous RNA and circular RNA (circRNA) as important regulators of miRNA activity underscores the increasing complexity of ncRNA-mediated regulatory networks. Particularly, the recently identified circular RNA, ciRS-7, which acts as a designated miR-7 inhibitor/sponge, has conceptually changed the mechanistic understanding of miRNA networks. As miR-7 modulates the expression of several oncogenes, disclosing the regulation of miR-7 activity will likely advance the understanding of various cancer etiologies. Here, we review the current knowledge about the ciRS-7/miR-7 axis in cancer-related pathways and discuss possible models explaining the relevance of coexpressing miR-7 along with a circRNA inhibitor.


query: ITAM phosphorylation prevents the transfer of the T cell receptor (TCR) signal from the echo-domain to the cytoplasmic tail of the T cell receptor (TCR).

positive: Full activation of the T cell receptor requires both clustering and conformational changes at CD3. T cell receptor (TCR-CD3) triggering involves both receptor clustering and conformational changes at the cytoplasmic tails of the CD3 subunits. The mechanism by which TCRalphabeta ligand binding confers conformational changes to CD3 is unknown. By using well-defined ligands, we showed that induction of the conformational change requires both multivalent engagement and the mobility restriction of the TCR-CD3 imposed by the plasma membrane. The conformational change is elicited by cooperative rearrangements of two TCR-CD3 complexes and does not require accompanying changes in the structure of the TCRalphabeta ectodomains. This conformational change at CD3 reverts upon ligand dissociation and is required for T cell activation. Thus, our permissive geometry model provides a molecular mechanism that rationalizes how the information of ligand binding to TCRalphabeta is transmitted to the CD3 subunits and to the intracellular signaling machinery.

negative: NSOM/QD-Based Direct Visualization of CD3-Induced and CD28-Enhanced Nanospatial Coclustering of TCR and Coreceptor in Nanodomains in T Cell Activation Direct molecular imaging of nano-spatial relationship between T cell receptor (TCR)/CD3 and CD4 or CD8 co-receptor before and after activation of a primary T cell has not been reported. We have recently innovated application of near-field scanning optical microscopy (NSOM) and immune-labeling quantum dots (QD) to image Ag-specific TCR response during in vivo clonal expansion, and now up-graded the NSOM/QD-based nanotechnology through dipole-polarization and dual-color imaging. Using this imaging system scanning cell-membrane molecules at a best-optical lateral resolution, we demonstrated that CD3, CD4 or CD8 molecules were distinctly distributed as single QD-bound molecules or nano-clusters equivalent to 2-4 QD fluorescence-intensity/size on cell-membrane of un-stimulated primary T cells, and approximately 6-10% of CD3 were co-clustering with CD4 or CD8 as 70-110 nm nano-clusters without forming nano-domains. The ligation of TCR/CD3 on CD4 or CD8 T cells led to CD3 nanoscale co-clustering or interaction with CD4 or CD8 co-receptors forming 200-500 nm nano-domains or >500 nm micro-domains. Such nano-spatial co-clustering of CD3 and CD4 or CD3 and CD8 appeared to be an intrinsic event of TCR/CD3 ligation, not purely limited to MHC engagement, and be driven by Lck phosphorylation. Importantly, CD28 co-stimulation remarkably enhanced TCR/CD3 nanoscale co-clustering or interaction with CD4 co-receptor within nano- or micro-domains on the membrane. In contrast, CD28 co-stimulation did not enhance CD8 clustering or CD3-CD8 co-clustering in nano-domains although it increased molecular number and density of CD3 clustering in the enlarged nano-domains. These nanoscale findings provide new insights into TCR/CD3 interaction with CD4 or CD8 co-receptor in T-cell activation.

negative: Generation of T cell receptor–retrogenic mice: improved retroviral-mediated stem cell gene transfer The use of retrogenic mice offers a rapid and flexible approach to T cell receptor (TCR)-transgenic mice. By transducing bone marrow progenitor cells with a retrovirus that encodes a given TCR-α/β subunit, TCR-retrogenic mice can be generated in as few as 4–6 weeks, whereas conventional TCR transgenics can take 6 months or longer. In this updated protocol, we have increased the efficiency of the bone marrow transduction and bone marrow reconstitution compared with our previously published protocol. The main departure from the previous protocol is the implementation of spin transduction with the viral supernatant instead of coculture with the viral producer cell line. The changes in this protocol improve bone marrow viability, increase consistency of the bone marrow transduction and bone marrow engraftment, and they reduce the ratio of bone marrow donor mice to bone marrow recipients.

negative: Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells. Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.

negative: The pre-B-cell receptor: selector of fitting immunoglobulin heavy chains for the B-cell repertoire In this Opinion article, I address the role of the pre-B-cell receptor (pre-BCR) in the development of antigen-specific B cells in terms of immunoglobulin heavy chain (IgH) variable-region repertoire selection, precursor B-cell differentiation and proliferation, and IgH allelic exclusion. Comparisons with the role of the pre-T-cell receptor (pre-TCR) in T-cell development raise provocative questions. Why do B- and T-cell lineages both use a surrogate chain — the surrogate light chain and the pre-TCR α-chain, respectively — as a step to develop their repertoires of antigen-recognizing cells? What are the functions of the pre-BCR and pre-TCR in lymphocyte differentiation and antigen-receptor allelic exclusion? This article, together with the accompanying article by Harald von Boehmer, hopes to answer some of these questions.

negative: Two Subsets of Naive T Helper Cells with Distinct T Cell Receptor Excision Circle Content in Human Adult Peripheral Blood During ageing thymic function declines and is unable to meet the demand for peripheral T helper (Th) cell replenishment. Therefore, population maintenance of naive Th cells must be at least partly peripherally based. Such peripheral postthymic expansion of recent thymic emigrants (RTEs) during ageing consequently should lead to loss or dilution of T cell receptor excision circles (TRECs) from a subset of naive T cells. We have identified two subsets of naive Th cells in human adult peripheral blood characterized by a striking unequal content of TRECs, indicating different peripheral proliferative histories. TRECs are highly enriched in peripheral naive CD45RA+ Th cells coexpressing CD31 compared with peripheral naive CD45RA+ Th cells lacking CD31 expression, in which TRECs can hardly be detected. Furthermore we show that CD31−CD45RA+ Th cells account for increasing percentages of the naive peripheral Th cell pool during ageing but retain phenotypic and functional features of naive Th cells. As CD31 is lost upon T cell receptor (TCR) engagement in vitro, we hypothesize that TCR triggering is a prerequisite for homeostatically driven peripheral postthymic expansion of human naive RTEs. We describe here the identification of peripherally expanded naive Th cells in human adult blood characterized by the loss of CD31 expression and a highly reduced TREC content.

negative: Quantitative assessment of T-cell repertoire recovery after hematopoietic stem cell transplantation Delayed T cell recovery and restricted T cell receptor (TCR) diversity after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are associated with increased risks of infection and cancer relapse. Technical challenges have limited faithful measurement of TCR diversity after allo-HSCT. Here we combined 5' rapid amplification of complementary DNA ends PCR with deep sequencing to quantify TCR diversity in 28 recipients of allo-HSCT using a single oligonucleotide pair. Analysis of duplicate blood samples confirmed that we accurately determined the frequency of individual TCRs. After 6 months, cord blood-graft recipients approximated the TCR diversity of healthy individuals, whereas recipients of T cell-depleted peripheral-blood stem cell grafts had 28-fold and 14-fold lower CD4(+) and CD8(+) T cell diversities, respectively. After 12 months, these deficiencies had improved for the CD4(+) but not the CD8(+) T cell compartment. Overall, this method provides unprecedented views of T cell repertoire recovery after allo-HSCT and may identify patients at high risk of infection or relapse.

negative: TCR ligand density and affinity determine peripheral induction of Foxp3 in vivo T cell receptor (TCR) ligation is required for the extrathymic differentiation of forkhead box p3(+) (Foxp3(+)) regulatory T cells. Several lines of evidence indicate that weak TCR stimulation favors induction of Foxp3 in the periphery; however, it remains to be determined how TCR ligand potency influences this process. We characterized the density and affinity of TCR ligand favorable for Foxp3 induction and found that a low dose of a strong agonist resulted in maximal induction of Foxp3 in vivo. Initial Foxp3 induction by weak agonist peptide could be enhanced by disruption of TCR-peptide major histocompatibility complex (pMHC) interactions or alteration of peptide dose. However, time course experiments revealed that Foxp3-positive cells induced by weak agonist stimulation are deleted, along with their Foxp3-negative counterparts, whereas Foxp3-positive cells induced by low doses of the strong agonist persist. Our results suggest that, together, pMHC ligand potency, density, and duration of TCR interactions define a cumulative quantity of TCR stimulation that determines initial peripheral Foxp3 induction. However, in the persistence of induced Foxp3(+) T cells, TCR ligand potency and density are noninterchangeable factors that influence the route to peripheral tolerance.

negative: ImmTAC-redirected tumour cell killing induces and potentiates antigen cross-presentation by dendritic cells Antigen cross-presentation by dendritic cells (DCs) is thought to play a critical role in driving a polyclonal and durable T cell response against cancer. It follows, therefore, that the capacity of emerging immunotherapeutic agents to orchestrate tumour eradication may depend on their ability to induce antigen cross-presentation. ImmTACs [immune-mobilising monoclonal TCRs (T cell receptors) against cancer] are a new class of soluble bi-specific anti-cancer agents that combine pico-molar affinity TCR-based antigen recognition with T cell activation via a CD3-specific antibody fragment. ImmTACs specifically recognise human leucocyte antigen (HLA)-restricted tumour-associated antigens, presented by cancer cells, leading to T cell redirection and a potent anti-tumour response. Using an ImmTAC specific for a HLA-A*02-restricted peptide derived from the melanoma antigen gp100 (termed IMCgp100), we here observe that ImmTAC-driven melanoma-cell death leads to cross-presentation of melanoma antigens by DCs. These, in turn, can activate both melanoma-specific T cells and polyclonal T cells redirected by IMCgp100. Moreover, activation of melanoma-specific T cells by cross-presenting DCs is enhanced in the presence of IMCgp100; a feature that serves to increase the prospect of breaking tolerance in the tumour microenvironment. The mechanism of DC cross-presentation occurs via ‘cross-dressing’ which involves the rapid and direct capture by DCs of membrane fragments from dying tumour cells. DC cross-presentation of gp100-peptide-HLA complexes was visualised and quantified using a fluorescently labelled soluble TCR. These data demonstrate how ImmTACs engage with the innate and adaptive components of the immune system enhancing the prospect of mediating an effective and durable anti-tumour response in patients.

negative: Tumor-specific Th17-polarized cells eradicate large established melanoma. CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-gamma (IFN-gamma) production, whereas depletion of interleukin (IL)-17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer-based immunotherapy of human malignancies.

negative: Radiation and Dual Checkpoint Blockade Activates Non-Redundant Immune Mechanisms in Cancer Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

negative: Biochemical differences in the alphabeta T cell receptor.CD3 surface complex between CD8+ and CD4+ human mature T lymphocytes. We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.

negative: CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung Allergic asthma is a T helper type 2 (T(H)2)-dominated disease of the lung. In people with asthma, a fraction of CD4(+) T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4(+) T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT T(H)2 cells. We found that CX3CR1 signaling promoted T(H)2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient T(H)2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for T(H)1 cells upon airway inflammation but not under homeostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma.

negative: Differentiation of effector CD4 T cell populations (*). CD4 T cells play critical roles in mediating adaptive immunity to a variety of pathogens. They are also involved in autoimmunity, asthma, and allergic responses as well as in tumor immunity. During TCR activation in a particular cytokine milieu, naive CD4 T cells may differentiate into one of several lineages of T helper (Th) cells, including Th1, Th2, Th17, and iTreg, as defined by their pattern of cytokine production and function. In this review, we summarize the discovery, functions, and relationships among Th cells; the cytokine and signaling requirements for their development; the networks of transcription factors involved in their differentiation; the epigenetic regulation of their key cytokines and transcription factors; and human diseases involving defective CD4 T cell differentiation.

negative: Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells. Although TNF is a major proinflammatory cytokine, increasing evidence indicates that TNF also has immunosuppressive feedback effects. We have demonstrated in this study that, in both resting and activated states, mouse peripheral CD4(+)CD25(+) T regulatory cells (Tregs) expressed remarkably higher surface levels of TNFR2 than CD4(+)CD25(-) T effector cells (Teffs). In cocultures of Tregs and Teffs, inhibition of proliferation of Teffs by Tregs was initially transiently abrogated by exposure to TNF, but longer exposure to TNF restored suppressive effects. Cytokine production by Teffs remained continually suppressed by Tregs. The profound anergy of Tregs in response to TCR stimulation was overcome by TNF, which expanded the Treg population. Furthermore, in synergy with IL-2, TNF expanded Tregs even more markedly up-regulated expression of CD25 and FoxP3 and phosphorylation of STAT5, and enhanced the suppressive activity of Tregs. Unlike TNF, IL-1beta and IL-6 did not up-regulate FoxP3-expressing Tregs. Furthermore, the number of Tregs increased in wild-type mice, but not in TNFR2(-/-) mice following sublethal cecal ligation and puncture. Depletion of Tregs significantly decreased mortality following cecal ligation and puncture. Thus, the stimulatory effect of TNF on Tregs resembles the reported costimulatory effects of TNF on Teffs, but is even more pronounced because of the higher expression of TNFR2 by Tregs. Moreover, our study suggests that the slower response of Tregs than Teffs to TNF results in delayed immunosuppressive feedback effects.

negative: Critical role of CD28 in protective immunity against Salmonella typhimurium. Efficient T cell activation requires both TCR signals and costimulatory signals. CD28 is one of the molecules that provide costimulatory signals for T cells. We used mice deficient in CD28 expression (CD28-/- mice) to analyze the role of CD28 in the immune response against the intracellular bacterium Salmonella typhimurium, the causative agent of murine typhoid fever. CD28-/- mice were highly susceptible to infection with wild-type S. typhimurium and even failed to control infection with attenuated aroA- S. typhimurium. More detailed analysis revealed that CD28-/- animals did not mount a T-dependent Ab response and were highly impaired in the production of IFN-gamma. Thus, CD28 cosignaling is crucial for immunity against S. typhimurium. To our knowledge, this is the first report describing an essential role for CD28 in protective immunity against an intracellular microbial pathogen.

negative: Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments Receptor endocytosis is a fundamental step in controlling the magnitude, duration, and nature of cell signaling events. Confluent endothelial cells are contact inhibited in their growth and respond poorly to the proliferative signals of vascular endothelial growth factor (VEGF). In a previous study, we found that the association of vascular endothelial cadherin (VEC) with VEGF receptor (VEGFR) type 2 contributes to density-dependent growth inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. 2003. J. Cell Biol. 161:793–804). In the present study, we describe the mechanism through which VEC reduces VEGFR-2 signaling. We found that VEGF induces the clathrin-dependent internalization of VEGFR-2. When VEC is absent or not engaged at junctions, VEGFR-2 is internalized more rapidly and remains in endosomal compartments for a longer time. Internalization does not terminate its signaling; instead, the internalized receptor is phosphorylated, codistributes with active phospholipase C–γ, and activates p44/42 mitogen-activated protein kinase phosphorylation and cell proliferation. Inhibition of VEGFR-2 internalization reestablishes the contact inhibition of cell growth, whereas silencing the junction-associated density-enhanced phosphatase-1/CD148 phosphatase restores VEGFR-2 internalization and signaling. Thus, VEC limits cell proliferation by retaining VEGFR-2 at the membrane and preventing its internalization into signaling compartments.

negative: ImmTACs for targeted cancer therapy: Why, what, how, and which. Overcoming immunosuppression and activating a cytotoxic T cell response has the potential to halt the progression of cancer and, in some circumstances, eradicate it. Designing therapeutic interventions that achieve this goal has proven challenging, but now a greater understanding of the complexities of immune responses is beginning to produce some notable breakthroughs. ImmTACs (immune-mobilising monoclonal TCRs against cancer) are a new class of bispecific reagents, based on soluble monoclonal T cell receptors, which have been engineered to possess extremely high affinity for cognate tumour antigen. In this way, ImmTACs overcome the problem of low affinity tumour-specific T cells imposed by thymic selection and provide access to the large number of antigens presented as peptide-HLA complexes. Once bound to tumour cells the anti-CD3 effector end of the ImmTAC drives recruitment of polyclonal T cells to the tumour site, leading to a potent redirected T cell response and tumour cell destruction. Extensive in vitro testing coupled with promising early clinical data has provided an enhanced appreciation of ImmTAC function in vivo and indicates their potential therapeutic benefit in terms of a durable response and ultimately the breaking of T cell tolerance. This review introduces ImmTACs in the context of immunotherapy, and outlines their design, construction and mechanism of action, as well as examining target selection and aspects of preclinical safety testing.

negative: Safety, pharmacokinetics and efficacy of IMCgp100, a first-in-class soluble TCR-antiCD3 bispecific t cell redirector with solid tumour activity: Results from the FIH study in melanoma. 3016Background: T cell-based bispecific agents have shown activity in hematologic cancers, but solid tumor efficacy remains elusive. IMCgp100 is a bispecific biologic comprising an affinityenhanced TCR specific for gp100 and an anti-CD3 scFV. In vitro, IMCgp100 binds gp100+ melanoma cells causing redirection of cytotoxicity and induction of potent immune effects. Methods: The Phase I was conducted in HLA-A2+ pts with advanced melanoma, using a 3+3 design to define the MTD. Pts were treated with IMCgp100 (iv) weekly (QW, Arm 1) or daily (4QD3W, Arm 2) to evaluate safety, PK and efficacy. The recommended phase 2 regimen (RP2D-QW) was defined. Results: In the Ph I dose escalation,31 pts received doses from 5ng/kg to 900ng/kg. In arm 1 dose-limiting toxicity of gr 3 or 4 hypotension was seen and associated with rapid trafficking of peripheral lymphocytes to skin and tumor. The MTD was determined to be 600ng/kg QW. IMCgp100 has an approximately dose-proportional profile with a plasma T1/2 of 5-6 hrs at the RP2...

negative: GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. GITR (glucocorticoid-induced TNFR family related gene) is a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes. Because of a high homology in its cytoplasmic region with other known costimulatory members of the TNFRSF, we investigated whether GITR played a costimulatory role in T lymphocyte subpopulations. Our results show that the proliferation response of CD8+ and CD4+ peripheral T cell subpopulations was potentiated when a GITR costimulus was added to an anti-CD3 stimulus. Furthermore, expression of the main activation-induced receptor (IL-2Ralpha) and production of IL-2 and IFN-gamma were increased more with a GITR costimulus than with anti-CD3 alone. GITR stimulation also enhanced anti-CD3-induced ERK phosphorylation, suggesting that GITR is involved in MAPK-pathway activation. Interestingly, CD4+CD25+ regulatory T cell (Treg cell) proliferation was triggered by the GITR costimulus; Treg cell proliferation was paralleled by the loss of the anergic phenotype and suppressor activity. Nevertheless, unstimulated GITR(-/-) CD4+CD25+ and GITR(+/+) CD4+CD25+ cells were equally able to exert suppressor activity on CD4+CD25- responder cells. These results indicate a novel function for GITR as costimulatory molecule of T cell subsets.

negative: Stage-Specific and Differential Notch Dependency at the αβ and γδ T Lineage Bifurcation Summary Signals transduced by Notch receptors are indispensable for T cell specification and differentiation of αβ T lineage cells. However, the role of Notch signals during αβ versus γδ T lineage decision remains controversial. Here, we addressed this question by employing a clonal analysis of CD4 − CD8 − (DN) progenitor potential to position the divergence of αβ and γδ T cell lineages to the late DN2 to DN3 developmental stages. Accordingly, αβ and γδ precursor frequencies within these T cell progenitor subsets were determined, both in the presence and absence of Notch signaling through Delta-like 1. Notch signals were found to be critical for the DN to CD4 + CD8 + (DP) transition, irrespective of the identity (pTαβ or γδ) of the inducing T cell receptor complex, whereas γδ T cells developed from γδTCR-expressing T cell progenitors in the absence of further Notch ligand interaction. Collectively, our findings demonstrate a differential, stage-specific requirement for Notch receptor-ligand interactions in the differentiation of αβ and γδ T cells from T cell progenitors.

negative: Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Adoptive T cell transfer for cancer and chronic infection is an emerging field that shows promise in recent trials. Synthetic-biology-based engineering of T lymphocytes to express high-affinity antigen receptors can overcome immune tolerance, which has been a major limitation of immunotherapy-based strategies. Advances in cell engineering and culture approaches to enable efficient gene transfer and ex vivo cell expansion have facilitated broader evaluation of this technology, moving adoptive transfer from a "boutique" application to the cusp of a mainstream technology. The major challenge currently facing the field is to increase the specificity of engineered T cells for tumors, because targeting shared antigens has the potential to lead to on-target off-tumor toxicities, as observed in recent trials. As the field of adoptive transfer technology matures, the major engineering challenge is the development of automated cell culture systems, so that the approach can extend beyond specialized academic centers and become widely available.

negative: CD8+ T cells are activated in an antigen-independent manner in HIV-infected individuals. Hyperactivation of T cells, particularly of CD8(+) T cells, is a hallmark of chronic HIV 1 (HIV-1) infection. Little is known about the antigenic specificities and the mechanisms by which HIV-1 causes activation of CD8(+) T cells during chronic infection. We report that CD8(+) T cells were activated during in vivo HIV-1 replication irrespective of their Ag specificity. Cytokines present during untreated HIV-1 infection, most prominently IL-15, triggered proliferation and expression of activation markers in CD8(+) T cells, but not CD4(+) T cells, in the absence of TCR stimulation. Moreover, LPS or HIV-1-activated dendritic cells (DCs) stimulated CD8(+) T cells in an IL-15-dependent but Ag-independent manner, and IL-15 expression was highly increased in DCs isolated from viremic HIV-1 patients, suggesting that CD8(+) T cells are activated by inflammatory cytokines in untreated HIV-1 patients independent of Ag specificity. This finding contrasts with CD4(+) T cells whose in vivo activation seems biased toward specificities for persistent Ags. These observations explain the higher abundance of activated CD8(+) T cells compared with CD4(+) T cells in untreated HIV-1 infection.

negative: CCR7 provides localized access to IL-2 and defines homeostatically distinct regulatory T cell subsets Immune tolerance and activation depend on precise control over the number and function of immunosuppressive Foxp3(+) regulatory T (T reg) cells, and the importance of IL-2 in maintaining tolerance and preventing autoimmunity is clear. However, the homeostatic requirement for IL-2 among specific populations of peripheral T reg cells remains poorly understood. We show that IL-2 selectively maintains a population of quiescent CD44(lo)CD62L(hi) T reg cells that gain access to paracrine IL-2 produced in the T cell zones of secondary lymphoid tissues due to their expression of the chemokine receptor CCR7. In contrast, CD44(hi)CD62L(lo)CCR7(lo) T reg cells that populate nonlymphoid tissues do not access IL-2-prevalent regions in vivo and are insensitive to IL-2 blockade; instead, their maintenance depends on continued signaling through the co-stimulatory receptor ICOS (inducible co-stimulator). Thus, we define a fundamental homeostatic subdivision in T reg cell populations based on their localization and provide an integrated framework for understanding how T reg cell abundance and function are controlled by unique signals in different tissue environments.

negative: Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

negative: A direct role for IFN-gamma in regulation of Th1 cell development. IL-12 has been identified as a major cytokine influencing the differentiation of CD4 cells to a Th1 phenotype, whereas a role for IFN-gamma is controversial. We investigated the interrelationship between IL-12 and IFN-gamma in promoting Th1 responses using naive CD4 cells reactive with pigeon cytochrome c from TCR transgenics and memory CD4 cells derived by in vivo priming with KLH. Without exogenous rIL-12 or rIFN-gamma, primary and memory effectors induced by Ag or anti-CD3 and anti-CD28 secreted variable levels of IL-2 and IFN-gamma. The level of IFN-gamma secreted by effectors correlated with endogenous IFN-gamma produced in primary cultures, and anti-IFN-gamma largely inhibited the development of effectors producing IFN-gamma. With optimal TCR stimulation and costimulation, endogenous IFN-gamma, without IL-12, was sufficient to elicit Th1 cells via an autocrine mechanism, whereas with suboptimal stimulation, exogenous rIFN-gamma or rIL-12 was required for Th1 development. However, rIL-12 was more effective than rIFN-gamma, partially because rIL-12 greatly enhanced autocrine production of IFN-gamma, and optimal development of the Th1 phenotype was mediated by the synergistic actions of both cytokines. Thus, both IFN-gamma and IL-12 can independently regulate Th1 development, but because of IFN-gamma-mediated feedback, their relative contributions are determined by the conditions of T cell stimulation. The extent of differentiation to a Th1 phenotype may, therefore, depend on the availability of both APC-derived IL-12 and autocrine IFN-gamma consequent to the overall strength of T cell stimulation.

negative: Amelioration of colitis by genetically engineered murine regulatory T cells redirected by antigen-specific chimeric receptor. BACKGROUND & AIMS The therapeutic application of regulatory T cells (Tregs) for the treatment of inflammatory diseases is limited by the scarcity of antigen-specific Tregs. A preferred approach to endow effector T cells (Teff) with a desired specificity uses chimeric immune receptors with antibody-type specificity. Accordingly, employing such chimeric immune receptors to redirect Tregs to sites of inflammation may be a useful therapeutic approach to alleviate a broad scope of diseases in which an uncontrolled inflammatory response plays a major role. METHODS To enable application of the approach in clinical setting, which requires the genetic modification of the patient's own Tregs, we describe here a novel protocol that allows the efficient retroviral transduction and 2,4,6-trinitrophenol-specific expansion of murine naturally occurring regulatory T cells (nTregs), with a 2,4,6-trinitrophenol-specific tripartite chimeric receptor. RESULTS Transduced Tregs maintained their Foxp3 level, could undergo repeated expansion upon ex vivo encounter with their cognate antigen in a major histocompatibility complex-independent, costimulation-independent, and contact-dependent manner and specifically suppressed Teff cells. Adoptive transfer of small numbers of the transduced nTregs was associated with antigen-specific, dose-dependent amelioration of trinitrobenzenesulphonic acid colitis. CONCLUSIONS This study demonstrates that nTregs can be efficiently transduced to express functional, antigen-specific chimeric receptors that enable the specific suppression of effector T cells both in vitro and in vivo. This approach may enable future cell-based therapeutic application in inflammatory bowel disease, as well as other inflammatory disorders.

negative: The Transcription Factors Egr2 and Egr3 Are Essential for the Control of Inflammation and Antigen-Induced Proliferation of B and T Cells Lymphocytes provide optimal responses against pathogens with minimal inflammatory pathology. However, the intrinsic mechanisms regulating these responses are unknown. Here, we report that deletion of both transcription factors Egr2 and Egr3 in lymphocytes resulted in a lethal autoimmune syndrome with excessive serum proinflammatory cytokines but also impaired antigen receptor-induced proliferation of B and T cells. Egr2- and Egr3-defective B and T cells had hyperactive signal transducer and activator of transcription-1 (STAT1) and STAT3 while antigen receptor-induced activation of transcription factor AP-1 was severely impaired. We discovered that Egr2 and/or Egr3 directly induced expression of suppressor of cytokine signaling-1 (SOCS1) and SOCS3, inhibitors of STAT1 and STAT3, and also blocked the function of Batf, an AP-1 inhibitor, in B and T cells. Thus, Egr2 and Egr3 regulate B and T cell function in adaptive immune responses and homeostasis by promoting antigen receptor signaling and controlling inflammation.

negative: Cord factor and peptidoglycan recapitulate the Th17-promoting adjuvant activity of mycobacteria through mincle/CARD9 signaling and the inflammasome. Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1β/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1β processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1β expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.

negative: Integration of Smad and MAPK pathways: a link and a linker revisited. Cells develop by reading mixed signals. Nowhere is this clearer than in the highly dynamic processes that propel embryogenesis, when critical cell-fate decisions are made swiftly in response to well-orchestrated growthfactor combinations. Learning how diverse signaling pathways are integrated is therefore essential for understanding physiology. This requires the identification, in tangible molecular terms, of key nodes for pathway integration that operate in vivo. A report in this issue, on the integration of Smad and Ras/MAPK pathways during neural induction (Pera et al. 2003), provides timely insights into the relevance of one such node. Pera et al. (2003) report that FGF8 and IGF2—two growth factors that activate the Ras/MAPK pathway— favor neural differentiation and mesoderm dorsalization in Xenopus by inhibiting BMP (Bone Morphogenetic Protein) signaling. Mesoderm is formed from ectoderm in response to Nodal-related signals from the endoderm at the blastula stage and beyond (Fig. 1; for review, see De Robertis et al. 2000). BMP induces differentiation of ectoderm into epidermal cell fates at the expense of neural fates, and it ventralizes the mesoderm at the expense of dorsal fates (for review, see Weinstein and HemmatiBrivanlou 1999; De Robertis et al. 2000). Accordingly, neural differentiation and dorsal mesoderm formation are favored when BMP signaling is attenuated. Noggin, Chordin, Cerberus, and Follistatin, secreted by the Spemann organizer on the dorsal side at the gastrula stage, facilitate the formation of neural tissue by sequestering BMP (Weinstein and Hemmati-Brivanlou 1999; De Robertis et al. 2000). Experimentally blocking BMP signaling with a dominant-negative BMP receptor has a similar effect of promoting ectoderm neuralization (Weinstein and Hemmati-Brivanlou 1999). As it turns out, neural induction can also be achieved with FGF (fibroblast growth factor; Kengaku and Okamoto 1993; Lamb and Harland 1995; Hongo et al. 1999; Hardcastle et al. 2000; Streit et al. 2000; Wilson et al. 2000) and IGF (insulin-like growth factor; Pera et al. 2001; Richard-Parpaillon et al. 2002). Injection of transcripts encoding FGF8 or IFG2 into one animal-pole blastomere of a fourto eight-cell embryo results in an expanded neural plate at the injected side (Pera et al. 2003). Surprisingly, expression of a dominant-negative FGF receptor prevents neuralization of ectoderm explants by the BMP blocker Noggin (Launay et al. 1996). Likewise, the potent neuralizing effect of Chordin can be blocked by a dominant-negative FGF receptor or a morpholino oligonucleotide targeting the IGF receptor (Pera et al. 2003). Thus, the neuralizing effect of BMP inhibitors is somehow tied to FGF and IFG signaling. The question is, how? Because FGF8 and IFG2 activate MAPK, Pera et al. (2003) took heed from previous work showing that MAPK inhibits the BMP signal-transduction factor Smad1 (Kretzschmar et al. 1997a). Smad1 is directly phosphorylated by the BMP receptor, resulting in Smad1 activation (Kretzschmar et al. 1997b), and by MAPK in response to EGF, resulting in Smad1 inhibition (Kretzschmar et al. 1997a; Fig. 2). Smad transcription factors mediate gene responses to the entire TGF (Transforming Growth Factor) family, to which the BMPs belong (for review, see Massague 2000; Derynck and Zhang 2003). Smads 1, 5, and 8 act primarily downstream of BMP receptors and Smads 2 and 3 downstream of TGF , Activin and Nodal receptors. Smad proteins have two conserved globular domains—the MH1 and MH2 domains (Fig. 2). The MH1 domain is involved in DNA binding and the MH2 domain in binding to cytoplasmic retention factors, activated receptors, nucleoporins in the nuclear pore, and DNA-binding cofactors, coactivators, and corepressors in the nucleus (for review, see Shi and Massague 2003). Receptor-mediated phosphorylation occurs at the carboxy-terminal sequence SXS. This enables the nuclear accumulation of Smads and their association with the shared partner Smad4 to form transcriptional complexes that are interpreted by the cell as a function of the context (Massague 2000). Between the MH1 and MH2 domains lies a linker region of variable sequence and length. Attention was drawn to this region when it was found that EGF (epidermal growth factor), a classical activator of the Ras/ MAPK pathway, causes phosphorylation of the Smad1 linker at four MAPK sites (PXSP sequences; Kretzschmar et al. 1997a). This prevents the nuclear localization of Smad1 and inhibits BMP signaling. Mutation of these E-MAIL j-massague@ski.mskcc.org; FAX (212) 717-3298. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1167003.

negative: Penaeus monodon Dscam (PmDscam) has a highly diverse cytoplasmic tail and is the first membrane-bound shrimp Dscam to be reported. Down syndrome cell adhesion molecule (Dscam) seems likely to play a key role in the "alternative adaptive immunity" that has been reported in invertebrates. Dscam consists of a cytoplasmic tail that is involved in signal transduction and a hypervariable extracellular region that might use a pathogen recognition mechanism similar to that used by the vertebrate antibodies. In our previous paper, we isolated a unique tail-less form of Dscam from Litopenaeus vannamei. In this study, we report the first membrane-bound form of shrimp Dscam: PmDscam was isolated from Penaeus monodon, and it occurred in both membrane-bound and tail-less forms. Phylogenetic analysis showed that while the crustacean Dscams from shrimp and water flea did not share a single subclade, they were distinct from the invertebrate Dscam-like molecules and from the insecta Dscams. In the extracellular region, the variable regions of PmDscam were located in N-terminal Ig2, N-terminal Ig3 and the entire Ig7 domain. The PmDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events. The cytoplasmic tail variants were produced by exon inclusion/exclusion. Based on the genomic organization of Daphnia Dscam's cytoplasmic tail, we propose a model of how the shrimp Dscam genomic locus might use Type III polyadenylation to generate both the tail-less and membrane-bound forms.

negative: Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150). CD4 T cell help is critical for the generation and maintenance of germinal centers (GCs), and T follicular helper (T(FH)) cells are the CD4 T cell subset required for this process. Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP [SH2D1A]) expression in CD4 T cells is essential for GC development. However, SAP-deficient mice have only a moderate defect in T(FH) differentiation, as defined by common T(FH) surface markers. CXCR5(+) T(FH) cells are found within the GC, as well as along the boundary regions of T/B cell zones. In this study, we show that GC-associated T follicular helper (GC T(FH)) cells can be identified by their coexpression of CXCR5 and the GL7 epitope, allowing for phenotypic and functional analysis of T(FH) and GC T(FH) populations. GC T(FH) cells are a functionally discrete subset of further polarized T(FH) cells, with enhanced B cell help capacity and a specialized ability to produce IL-4 in a T(H)2-independent manner. Strikingly, SAP-deficient mice have an absence of the GC T(FH) cell subset and SAP(-) T(FH) cells are defective in IL-4 and IL-21 production. We further demonstrate that SLAM (Slamf1, CD150), a surface receptor that uses SAP signaling, is specifically required for IL-4 production by GC T(FH) cells. GC T(FH) cells require IL-4 and -21 production for optimal help to B cells. These data illustrate complexities of SAP-dependent SLAM family receptor signaling, revealing a prominent role for SLAM receptor ligation in IL-4 production by GC CD4 T cells but not in T(FH) cell and GC T(FH) cell differentiation.