Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4.

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.

Mechanical stress during confined migration causes aberrant mitoses and c-MYC amplification.

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.

Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Eomesodermin-expressing type 1 regulatory (EOMES+ Tr1)-like T cells: Basic biology and role in immune-mediated diseases.

Type 1 regulatory (Tr1) T cells are currently defined all T cells with regulatory functions that lack FOXP3 expression and produce IL-10. Tr1 cells are heterogeneous, and the different reported properties of Tr1-cell populations have caused some confusion in the field. Moreover, understanding the role of Tr1 cells in immune-mediated diseases has been hampered by the lack of a lineage-defining transcription factor. Several independent studies indicated recently that the transcription factor Eomesodermin (EOMES) could act as a lineage-defining transcription factor in a population of IL-10 and IFN-γ co-producing Tr1-like cells, since EOMES directly induces IFN-γ and cytotoxicity, enhances IL-10, and antagonizes alternative T-cell fates. Here, we review the known properties of EOMES+ Tr1-like cells. They share several key characteristics with other Tr1 cells (i.e., "Tr1-like"), namely high IL-10 production, cytotoxicity, and suppressive capabilities. Notably, they also share some features with FOXP3+ Tregs, like downregulation of IL-7R and CD40L. In addition, they possess several unique, EOMES-dependent features, that is, expression of GzmK and IFN-γ, and downregulation of type-17 cytokines. Published evidence indicates that EOMES+ Tr1-like cells play key roles in graft-versus-host disease, colitis, systemic autoimmunity and in tumors. Thus, EOMES+ Tr1-like cells are key players of the adaptive immune system that are involved in several different immune-mediated diseases.

Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b.

MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA.

OligoMinerApp: a web-server application for the design of genome-scale oligonucleotide in situ hybridization probes through the flexible OligoMiner environment.

Fluorescence in situ hybridization (FISH) is a powerful single-cell technique that harnesses nucleic acid base pairing to detect the abundance and positioning of cellular RNA and DNA molecules in fixed samples. Recent technology development has paved the way to the construction of FISH probes entirely from synthetic oligonucleotides (oligos), allowing the optimization of thermodynamic properties together with the opportunity to design probes against any sequenced genome. However, comparatively little progress has been made in the development of computational tools to facilitate the oligos design, and even less has been done to extend their accessibility. OligoMiner is an open-source and modular pipeline written in Python that introduces a novel method of assessing probe specificity that employs supervised machine learning to predict probe binding specificity from genome-scale sequence alignment information. However, its use is restricted to only those people who are confident with command line interfaces because it lacks a Graphical User Interface (GUI), potentially cutting out many researchers from this technology. Here, we present OligoMinerApp (http://oligominerapp.org), a web-based application that aims to extend the OligoMiner framework through the implementation of a smart and easy-to-use GUI and the introduction of new functionalities specially designed to make effective probe mining available to everyone.

miRiadne: a web tool for consistent integration of miRNA nomenclature.

The miRBase is the official miRNA repository which keeps the annotation updated on newly discovered miRNAs: it is also used as a reference for the design of miRNA profiling platforms. Nomenclature ambiguities generated by loosely updated platforms and design errors lead to incompatibilities among platforms, even from the same vendor. Published miRNA lists are thus generated with different profiling platforms that refer to diverse and not updated annotations. This greatly compromises searches, comparisons and analyses that rely on miRNA names only without taking into account the mature sequences, which is particularly critic when such analyses are carried over automatically. In this paper we introduce miRiadne, a web tool to harmonize miRNA nomenclature, which takes into account the original miRBase versions from 10 up to 21, and annotations of 40 common profiling platforms from nine brands that we manually curated. miRiadne uses the miRNA mature sequence to link miRBase versions and/or platforms to prevent nomenclature ambiguities. miRiadne was designed to simplify and support biologists and bioinformaticians in re-annotating their own miRNA lists and/or data sets. As Ariadne helped Theseus in escaping the mythological maze, miRiadne will help the miRNA researcher in escaping the nomenclature maze. miRiadne is freely accessible from the URL http://www.miriadne.org.

Role of microRNAs and long-non-coding RNAs in CD4(+) T-cell differentiation.

CD4(+) T lymphocytes orchestrate adaptive immune responses by differentiating into various subsets of effector T cells such as T-helper 1 (Th1), Th2, Th17, and regulatory T cells. These subsets have been generally described by master transcription factors that dictate the expression of cytokines and receptors, which ultimately define lymphocyte effector functions. However, the view of T-lymphocyte subsets as stable and terminally differentiated lineages has been challenged by increasing evidence of functional plasticity within CD4(+) T-cell subsets, which implies flexible programming of effector functions depending on time and space of T-cell activation. An outstanding question with broad basic and traslational implications relates to the mechanisms, besides transcriptional regulation, which define the plasticity of effector functions. In this study, we discuss the emerging role of regulatory non-coding RNAs in T-cell differentiation and plasticity. Not only microRNAs have been proven to be important for CD4(+) T-cell differentiation, but it is also likely that the overall T-cell functioning is the result of a multilayered network composed by coding RNAs as well as by short and long non-coding RNAs. The integrated study of all the nodes of this network will provide a comprehensive view of the molecular mechanisms underlying T-cell functions in health and disease.

Community-driven development for computational biology at Sprints, Hackathons and Codefests.

BACKGROUND: Computational biology comprises a wide range of technologies and approaches. Multiple technologies can be combined to create more powerful workflows if the individuals contributing the data or providing tools for its interpretation can find mutual understanding and consensus. Much conversation and joint investigation are required in order to identify and implement the best approaches. Traditionally, scientific conferences feature talks presenting novel technologies or insights, followed up by informal discussions during coffee breaks. In multi-institution collaborations, in order to reach agreement on implementation details or to transfer deeper insights in a technology and practical skills, a representative of one group typically visits the other. However, this does not scale well when the number of technologies or research groups is large. Conferences have responded to this issue by introducing Birds-of-a-Feather (BoF) sessions, which offer an opportunity for individuals with common interests to intensify their interaction. However, parallel BoF sessions often make it hard for participants to join multiple BoFs and find common ground between the different technologies, and BoFs are generally too short to allow time for participants to program together. RESULTS: This report summarises our experience with computational biology Codefests, Hackathons and Sprints, which are interactive developer meetings. They are structured to reduce the limitations of traditional scientific meetings described above by strengthening the interaction among peers and letting the participants determine the schedule and topics. These meetings are commonly run as loosely scheduled "unconferences" (self-organized identification of participants and topics for meetings) over at least two days, with early introductory talks to welcome and organize contributors, followed by intensive collaborative coding sessions. We summarise some prominent achievements of those meetings and describe differences in how these are organised, how their audience is addressed, and their outreach to their respective communities. CONCLUSIONS: Hackathons, Codefests and Sprints share a stimulating atmosphere that encourages participants to jointly brainstorm and tackle problems of shared interest in a self-driven proactive environment, as well as providing an opportunity for new participants to get involved in collaborative projects.

Identification of new autoantigens by protein array indicates a role for IL4 neutralization in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an unresolving inflammation of the liver of unknown cause. Diagnosis requires the exclusion of other conditions and the presence of characteristic features such as specific autoantibodies. Presently, these autoantibodies have relatively low sensitivity and specificity and are identified via immunostaining of cells or tissues; therefore, there is a diagnostic need for better and easy-to-assess markers. To identify new AIH-specific autoantigens, we developed a protein microarray comprising 1626 human recombinant proteins, selected in silico for being secreted or membrane associated. We screened sera from AIH patients on this microarray and compared the reactivity with that of sera from healthy donors and patients with chronic viral hepatitis C. We identified six human proteins that are specifically recognized by AIH sera. Serum reactivity to a combination of four of these autoantigens allows identification of AIH patients with high sensitivity (82%) and specificity (92%). Of the six autoantigens, the interleukin-4 (IL4) receptor fibronectin type III domain of the IL4 receptor (CD124), which is expressed on the surface of both lymphocytes and hepatocytes, showed the highest individual sensitivity and specificity for AIH. Remarkably, patients' sera inhibited STAT6 phosphorylation induced by IL4 binding to CD124, demonstrating that these autoantibodies are functional and suggesting that IL4 neutralization has a pathogenetic role in AIH.

An atlas of transcribed enhancers across helper T cell diversity for decoding human diseases.

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5' single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4+ T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type-specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type-resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.

Biogem: an effective tool-based approach for scaling up open source software development in bioinformatics.

SUMMARY: Biogem provides a software development environment for the Ruby programming language, which encourages community-based software development for bioinformatics while lowering the barrier to entry and encouraging best practices. Biogem, with its targeted modular and decentralized approach, software generator, tools and tight web integration, is an improved general model for scaling up collaborative open source software development in bioinformatics. AVAILABILITY: Biogem and modules are free and are OSS. Biogem runs on all systems that support recent versions of Ruby, including Linux, Mac OS X and Windows. Further information at http://www.biogems.info. A tutorial is available at http://www.biogems.info/howto.html CONTACT: bonnal@ingm.org.

The Ruby UCSC API: accessing the UCSC genome database using Ruby.

BACKGROUND: The University of California, Santa Cruz (UCSC) genome database is among the most used sources of genomic annotation in human and other organisms. The database offers an excellent web-based graphical user interface (the UCSC genome browser) and several means for programmatic queries. A simple application programming interface (API) in a scripting language aimed at the biologist was however not yet available. Here, we present the Ruby UCSC API, a library to access the UCSC genome database using Ruby. RESULTS: The API is designed as a BioRuby plug-in and built on the ActiveRecord 3 framework for the object-relational mapping, making writing SQL statements unnecessary. The current version of the API supports databases of all organisms in the UCSC genome database including human, mammals, vertebrates, deuterostomes, insects, nematodes, and yeast.The API uses the bin index-if available-when querying for genomic intervals. The API also supports genomic sequence queries using locally downloaded *.2bit files that are not stored in the official MySQL database. The API is implemented in pure Ruby and is therefore available in different environments and with different Ruby interpreters (including JRuby). CONCLUSIONS: Assisted by the straightforward object-oriented design of Ruby and ActiveRecord, the Ruby UCSC API will facilitate biologists to query the UCSC genome database programmatically. The API is available through the RubyGem system. Source code and documentation are available at https://github.com/misshie/bioruby-ucsc-api/ under the Ruby license. Feedback and help is provided via the website at http://rubyucscapi.userecho.com/.

RSSsite: a reference database and prediction tool for the identification of cryptic Recombination Signal Sequences in human and murine genomes.

Recombination signal sequences (RSSs) flanking V, D and J gene segments are recognized and cut by the VDJ recombinase during development of B and T lymphocytes. All RSSs are composed of seven conserved nucleotides, followed by a spacer (containing either 12 +/- 1 or 23 +/- 1 poorly conserved nucleotides) and a conserved nonamer. Errors in V(D)J recombination, including cleavage of cryptic RSS outside the immunoglobulin and T cell receptor loci, are associated with oncogenic translocations observed in some lymphoid malignancies. We present in this paper the RSSsite web server, which is available from the address http://www.itb.cnr.it/rss. RSSsite consists of a web-accessible database, RSSdb, for the identification of pre-computed potential RSSs, and of the related search tool, DnaGrab, which allows the scoring of potential RSSs in user-supplied sequences. This latter algorithm makes use of probability models, which can be recasted to Bayesian network, taking into account correlations between groups of positions of a sequence, developed starting from specific reference sets of RSSs. In validation laboratory experiments, we selected 33 predicted cryptic RSSs (cRSSs) from 11 chromosomal regions outside the immunoglobulin and TCR loci for functional testing.

Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer.

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients' metastatic ascites, establishing the conditions for propagating them as 3D cultures that we refer to as single cell-derived metastatic ovarian cancer spheroids (sMOCS). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that sMOCS retain and amplify key subpopulations from the original patients' samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients' specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method provides a powerful instrument for precision oncology in ovarian cancer.

Complete mitochondrial genome sequence of the Tyrolean Iceman.

The Tyrolean Iceman was a witness to the Neolithic-Copper Age transition in Central Europe 5350-5100 years ago, and his mummified corpse was recovered from an Alpine glacier on the Austro-Italian border in 1991 [1]. Using a mixed sequencing procedure based on PCR amplification and 454 sequencing of pooled amplification products, we have retrieved the first complete mitochondrial-genome sequence of a prehistoric European. We have then compared it with 115 related extant lineages from mitochondrial haplogroup K. We found that the Iceman belonged to a branch of mitochondrial haplogroup K1 that has not yet been identified in modern European populations. This is the oldest complete Homo sapiens mtDNA genome generated to date. The results point to the potential significance of complete-ancient-mtDNA studies in addressing questions concerning the genetic history of human populations that the phylogeography of modern lineages is unable to tackle.

BioRuby: bioinformatics software for the Ruby programming language.

SUMMARY: The BioRuby software toolkit contains a comprehensive set of free development tools and libraries for bioinformatics and molecular biology, written in the Ruby programming language. BioRuby has components for sequence analysis, pathway analysis, protein modelling and phylogenetic analysis; it supports many widely used data formats and provides easy access to databases, external programs and public web services, including BLAST, KEGG, GenBank, MEDLINE and GO. BioRuby comes with a tutorial, documentation and an interactive environment, which can be used in the shell, and in the web browser. AVAILABILITY: BioRuby is free and open source software, made available under the Ruby license. BioRuby runs on all platforms that support Ruby, including Linux, Mac OS X and Windows. And, with JRuby, BioRuby runs on the Java Virtual Machine. The source code is available from http://www.bioruby.org/. CONTACT: katayama@bioruby.org

Complete genome sequence of a serotype 11A, ST62 Streptococcus pneumoniae invasive isolate.

BACKGROUND: Streptococcus pneumoniae is an important human pathogen representing a major cause of morbidity and mortality worldwide. We sequenced the genome of a serotype 11A, ST62 S. pneumoniae invasive isolate (AP200), that was erythromycin-resistant due to the presence of the erm(TR) determinant, and carried out analysis of the genome organization and comparison with other pneumococcal genomes. RESULTS: The genome sequence of S. pneumoniae AP200 is 2,130,580 base pair in length. The genome carries 2216 coding sequences (CDS), 56 tRNA, and 12 rRNA genes. Of the CDSs, 72.9% have a predicted biological known function. AP200 contains the pilus islet 2 and, although its phenotype corresponds to serotype 11A, it contains an 11D capsular locus. Chromosomal rearrangements resulting from a large inversion across the replication axis, and horizontal gene transfer events were observed. The chromosomal inversion is likely implicated in the rebalance of the chromosomal architecture affected by the insertions of two large exogenous elements, the erm(TR)-carrying Tn1806 and a functional prophage designated φSpn_200. Tn1806 is 52,457 bp in size and comprises 49 ORFs. Comparative analysis of Tn1806 revealed the presence of a similar genetic element or part of it in related species such as Streptococcus pyogenes and also in the anaerobic species Finegoldia magna, Anaerococcus prevotii and Clostridium difficile. The genome of φSpn_200 is 35,989 bp in size and is organized in 47 ORFs grouped into five functional modules. Prophages similar to φSpn_200 were found in pneumococci and in other streptococcal species, showing a high degree of exchange of functional modules. φSpn_200 viral particles have morphologic characteristics typical of the Siphoviridae family and are capable of infecting a pneumococcal recipient strain. CONCLUSIONS: The sequence of S. pneumoniae AP200 chromosome revealed a dynamic genome, characterized by chromosomal rearrangements and horizontal gene transfers. The overall diversity of AP200 is driven mainly by the presence of the exogenous elements Tn1806 and φSpn_200 that show large gene exchanges with other genetic elements of different bacterial species. These genetic elements likely provide AP200 with additional genes, such as those conferring antibiotic-resistance, promoting its adaptation to the environment.

A Simplified Amino Acidic Alphabet to Unveil the T-Cells Receptors Antigens: A Computational Perspective.

The exposure to pathogens triggers the activation of adaptive immune responses through antigens bound to surface receptors of antigen presenting cells (APCs). T cell receptors (TCR) are responsible for initiating the immune response through their physical direct interaction with antigen-bound receptors on the APCs surface. The study of T cell interactions with antigens is considered of crucial importance for the comprehension of the role of immune responses in cancer growth and for the subsequent design of immunomodulating anticancer drugs. RNA sequencing experiments performed on T cells represented a major breakthrough for this branch of experimental molecular biology. Apart from the gene expression levels, the hypervariable CDR3α/ß sequences of the TCR loops can now be easily determined and modelled in the three dimensions, being the portions of TCR mainly responsible for the interaction with APC receptors. The most direct experimental method for the investigation of antigens would be based on peptide libraries, but their huge combinatorial nature, size, cost, and the difficulty of experimental fine tuning makes this approach complicated time consuming, and costly. We have implemented in silico methodology with the aim of moving from CDR3α/ß sequences to a library of potentially antigenic peptides that can be used in immunologically oriented experiments to study T cells' reactivity. To reduce the size of the library, we have verified the reproducibility of experimental benchmarks using the permutation of only six residues that can be considered representative of all ensembles of 20 natural amino acids. Such a simplified alphabet is able to correctly find the poses and chemical nature of original antigens within a small subset of ligands of potential interest. The newly generated library would have the advantage of leading to potentially antigenic ligands that would contribute to a better understanding of the chemical nature of TCR-antigen interactions. This step is crucial in the design of immunomodulators targeted towards T-cells response as well as in understanding the first principles of an immune response in several diseases, from cancer to autoimmune disorders.