22 results on '"Christopher Clouser"'
Search Results
2. Breast cancer is marked by specific, Public T-cell receptor CDR3 regions shared by mice and humans.
- Author
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Miri Gordin, Hagit Philip, Alona Zilberberg, Moriah Gidoni, Raanan Margalit, Christopher Clouser, Kristofor Adams, Francois Vigneault, Irun R Cohen, Gur Yaari, and Sol Efroni
- Subjects
Biology (General) ,QH301-705.5 - Abstract
The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires.
- Published
- 2021
- Full Text
- View/download PDF
3. Exploration of immunoglobulin transcriptomes from mice immunized with three-finger toxins and phospholipases A2 from the Central American coral snake, Micrurus nigrocinctus
- Author
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Andreas H. Laustsen, Mikael Engmark, Christopher Clouser, Sonia Timberlake, Francois Vigneault, José María Gutiérrez, and Bruno Lomonte
- Subjects
Murine immune response ,Venom ,Micrurus nigrocinctus ,High-throughput sequencing ,Antivenom ,Antibody isotyping ,Medicine ,Biology (General) ,QH301-705.5 - Abstract
Snakebite envenomings represent a neglected public health issue in many parts of the rural tropical world. Animal-derived antivenoms have existed for more than a hundred years and are effective in neutralizing snake venom toxins when timely administered. However, the low immunogenicity of many small but potent snake venom toxins represents a challenge for obtaining a balanced immune response against the medically relevant components of the venom. Here, we employ high-throughput sequencing of the immunoglobulin (Ig) transcriptome of mice immunized with a three-finger toxin and a phospholipase A2 from the venom of the Central American coral snake, Micrurus nigrocinctus. Although exploratory in nature, our indicate results showed that only low frequencies of mRNA encoding IgG isotypes, the most relevant isotype for therapeutic purposes, were present in splenocytes of five mice immunized with 6 doses of the two types of toxins over 90 days. Furthermore, analysis of Ig heavy chain transcripts showed that no particular combination of variable (V) and joining (J) gene segments had been selected in the immunization process, as would be expected after a strong humoral immune response to a single antigen. Combined with the titration of toxin-specific antibodies in the sera of immunized mice, these data support the low immunogenicity of three-finger toxins and phospholipases A2found in M. nigrocinctusvenoms, and highlight the need for future studies analyzing the complexity of antibody responses to toxins at the molecular level.
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- 2017
- Full Text
- View/download PDF
4. Mosaic deletion patterns of the human antibody heavy chain gene locus shown by Bayesian haplotyping
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Andrew M. Collins, Ludvig M. Sollid, Ayelet Peres, Francois Vigneault, Gur Yaari, Christopher Clouser, Pazit Polak, Ivana Mikocziova, Moriah Gidoni, Vikas Kumar Sarna, Ida Lindeman, Knut E.A. Lundin, and Omri Snir
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0301 basic medicine ,DNA Copy Number Variations ,Genotype ,Science ,Immunoglobulin Variable Region ,General Physics and Astronomy ,Inference ,Sequence assembly ,Locus (genetics) ,02 engineering and technology ,Computational biology ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Antibody Repertoire ,Humans ,Copy-number variation ,lcsh:Science ,Alleles ,Multidisciplinary ,Haplotype ,Bayes Theorem ,General Chemistry ,021001 nanoscience & nanotechnology ,030104 developmental biology ,ComputingMethodologies_PATTERNRECOGNITION ,Haplotypes ,IGHD ,lcsh:Q ,Immunoglobulin Heavy Chains ,0210 nano-technology ,IGHV@ - Abstract
Analysis of antibody repertoires by high-throughput sequencing is of major importance in understanding adaptive immune responses. Our knowledge of variations in the genomic loci encoding immunoglobulin genes is incomplete, resulting in conflicting VDJ gene assignments and biased genotype and haplotype inference. Haplotypes can be inferred using IGHJ6 heterozygosity, observed in one third of the people. Here, we propose a robust novel method for determining VDJ haplotypes by adapting a Bayesian framework. Our method extends haplotype inference to IGHD- and IGHV-based analysis, enabling inference of deletions and copy number variations in the entire population. To test this method, we generated a multi-individual data set of naive B-cell repertoires, and found allele usage bias, as well as a mosaic, tiled pattern of deleted IGHD and IGHV genes. The inferred haplotypes may have clinical implications for genetic disease predispositions. Our findings expand the knowledge that can be extracted from antibody repertoire sequencing data., High-throughput sequencing and analyzes of antibody repertoire provide important information on immune responses, but current methodologies are limited in sequence assembly precision and haplotype inference validity. Here the authors propose a new Bayesian haplotyping method, and attest its broad application with a large, multi-individual dataset.
- Published
- 2019
5. Breast cancer is marked by specific, Public T-cell receptor CDR3 regions shared by mice and humans
- Author
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Hagit Philip, Gur Yaari, Kristofor Adams, Irun R. Cohen, Raanan Margalit, Moriah Gidoni, Francois Vigneault, Miri Gordin, Christopher Clouser, Sol Efroni, and Alona Zilberberg
- Subjects
0301 basic medicine ,medicine.medical_treatment ,T-Lymphocytes ,Cell ,Immune Receptors ,Biochemistry ,Lung and Intrathoracic Tumors ,White Blood Cells ,Mice ,0302 clinical medicine ,Animal Cells ,Breast Tumors ,Databases, Genetic ,Medicine and Health Sciences ,Biology (General) ,Receptor ,Cells, Cultured ,Immune System Proteins ,Ecology ,T Cells ,Genetically Modified Organisms ,High-Throughput Nucleotide Sequencing ,Animal Models ,medicine.anatomical_structure ,Computational Theory and Mathematics ,Oncology ,Experimental Organism Systems ,030220 oncology & carcinogenesis ,Modeling and Simulation ,Biomarker (medicine) ,Engineering and Technology ,Female ,Cellular Types ,Genetic Engineering ,Research Article ,Biotechnology ,Signal Transduction ,QH301-705.5 ,Immune Cells ,Immunology ,Receptors, Antigen, T-Cell ,Mouse Models ,Bioengineering ,Breast Neoplasms ,Mice, Transgenic ,chemical and pharmacologic phenomena ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Immune system ,Breast cancer ,Model Organisms ,Antigen ,Breast Cancer ,Genetics ,medicine ,Animals ,Humans ,Molecular Biology Techniques ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Blood Cells ,Genetically Modified Animals ,T-cell receptor ,Cancers and Neoplasms ,Biology and Life Sciences ,Proteins ,Immunotherapy ,Cell Biology ,medicine.disease ,Complementarity Determining Regions ,T Cell Receptors ,030104 developmental biology ,Cancer research ,Animal Studies ,Cloning - Abstract
The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires., Author summary More than 1,100 cancer treatments are currently in clinical testing. Of these, 295 are immune-oncology medicines and vaccines. For Checkpoint Immunotherapy alone, 52,539 patients were enrolled in 2017. Of these treatments, 108 are for breast cancer, the leading cancer diagnosed in women. Although checkpoint immunotherapy has yet to be proven beneficial in most types of breast cancer, current observations of which specific T cell go into the tumor, and how these cells are connected to the response to treatment, have lead to a new understating of the role of the immune system. Yet, and in spite of this intense research into the role of T cells in tumors, very little is known about how clones of T cells behave during tumor formation. The paper we present here studies this clonal behavior over time. We study it during mammary tumor development in mice. We see that a set of T cell clones, which comes from a diversity of different nucleotide sequences, stands out in its behavior compared with other T cell clones. Importantly, we show that these mouse T cell clones have a strong connection to human breast cancer. The specific identification of a subset of T cell clones, together with the behavior of these clones over time, is critical to our understanding of the effect of modifications to the immune system, such as the modifications made by immunotherapy treatments, and to our understanding of how to better control the behavior of the immune system.
- Published
- 2021
6. Liver Is a Generative Site for the B Cell Response to Ehrlichia Muris
- Author
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Aditya Radhakrishnan, Francois Vigneault, Nikita Trivedi, Mark J. Shlomchik, Nahed Ismail, Aaron M. Rosenfeld, Christopher Clouser, Muhamuda Kader, and Uri Hershberg
- Subjects
Ehrlichia muris ,medicine.anatomical_structure ,biology ,Antigen ,Ehrlichia ,Somatic cell ,medicine ,Germinal center ,Spleen ,biology.organism_classification ,Molecular biology ,B cell ,Laser capture microdissection - Abstract
Murine infection with Ehrlichia muris models human Ehrlichiosis, with prominent liver involvement. Although Ehrlichia is an obligate intracellular pathogen, B cells are important for protection. The B cell response is dominated by plasmablasts (PBs), with few if any germinal centers (GC), and generates protective IgM memory B cells (MBC) that express T-bet and include some B cells express V region mutation. Here we address the origins, specificity, and location of these mutated MBC, revealing several novel features of the E. muris response and B cell memory in general. We first identified B cells within infected livers and demonstrated that they were proliferating and — via laser capture microdissection — undergoing somatic hyper mutation (SHM) in local liver foci. Similarly, splenic extrafollicular (EF) foci were undergoing active proliferation and mutation. In addition to PBs, we identified proliferating B cell blasts but no GC B cells. Vh region high throughput sequencing (HTS) revealed substantial trafficking of B cell clones between the spleen and liver that was often followed by localized clonal expansion. A subset of responding B cells persisted as memory cells in the spleen and liver of infected mice, even after antigen clearance, including a novel tissue-resident liver MBC. These data demonstrate that liver is a generative site of B cell responses that include V region mutation, MBC generation, and MBC residency.
- Published
- 2019
7. Antibody Repertoire Analysis of Hepatitis C Virus Infections Identifies Immune Signatures Associated With Spontaneous Clearance
- Author
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Christopher Clouser, Yaacov Y. Weiss, Oz Sharabi, Ateret Davidovich, Reut Timor, Pazit Polak, Meital Gal-Tanamy, Sivan Eliyahu, Gur Yaari, Francois Vigneault, Ronen Hope, Marius Braun, Assy Nimer, and Shiri Elmedvi
- Subjects
0301 basic medicine ,hepatitis C virus ,lcsh:Immunologic diseases. Allergy ,Hepatitis C virus ,infectious disease ,Remission, Spontaneous ,Immunology ,immune signature ,Datasets as Topic ,Immune receptor ,Hepacivirus ,medicine.disease_cause ,Virus ,Machine Learning ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Antibody Repertoire ,Viral Envelope Proteins ,Immunity ,Peptide Library ,Cell Line, Tumor ,antibody repertoire ,Immunology and Allergy ,Medicine ,Humans ,neutralizing antibodies ,Original Research ,biology ,business.industry ,Computational Biology ,High-Throughput Nucleotide Sequencing ,Hepatitis C Antibodies ,Hepatitis C, Chronic ,Prognosis ,Antibodies, Neutralizing ,030104 developmental biology ,Infectious disease (medical specialty) ,030220 oncology & carcinogenesis ,biology.protein ,Antibody ,business ,lcsh:RC581-607 - Abstract
Hepatitis C virus (HCV) is a major public health concern, with over 70 million people infected worldwide, who are at risk for developing life-threatening liver disease. No vaccine is available, and immunity against the virus is not well-understood. Following the acute stage, HCV usually causes chronic infections. However, ~30% of infected individuals spontaneously clear the virus. Therefore, using HCV as a model for comparing immune responses between spontaneous clearer (SC) and chronically infected (CI) individuals may empower the identification of mechanisms governing viral infection outcomes. Here, we provide the first in-depth analysis of adaptive immune receptor repertoires in individuals with current or past HCV infection. We demonstrate that SC individuals, in contrast to CI patients, develop clusters of antibodies with distinct properties. These antibodies' characteristics were used in a machine learning framework to accurately predict infection outcome. Using combinatorial antibody phage display library technology, we identified HCV-specific antibody sequences. By integrating these data with the repertoire analysis, we constructed two antibodies characterized by high neutralization breadth, which are associated with clearance. This study provides insight into the nature of effective immune response against HCV and demonstrates an innovative approach for constructing antibodies correlating with successful infection clearance. It may have clinical implications for prognosis of the future status of infection, and the design of effective immunotherapies and a vaccine for HCV.
- Published
- 2018
8. Mice developing mammary tumors evolve T cell sequences shared with human breast cancer patients
- Author
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Hagit Philip, Gur Yaari, Raanan Margalit, Christopher Clouser, Sol Efroni, Moriah Gidoni, Alona Zilberberg, Francois Vigneault, Irun R. Cohen, Kristofor Adams, and Miri Gordin
- Subjects
biology ,Transgene ,T cell ,medicine.medical_treatment ,CD44 ,T-cell receptor ,Immunotherapy ,medicine.anatomical_structure ,Immune system ,Cancer immunotherapy ,biology.protein ,medicine ,Cancer research ,Receptor - Abstract
Cancer immunotherapy by checkpoint blockade proves that an effective immune response to a tumor can be induced clinically. However, little is known about the evolution of tumor-associated T-cell receptor (TCR) repertoires without intervention. Here we studied TCR repertoire evolution in mice spontaneously developing mammary tumors; we sequenced peripheral blood alpha and beta TCRs of CD4+CD62L+CD44− T cells monthly for 8 months in 10 FVB/NJ mice transgenic at the Erbb2 locus, all developing tumors; 5 FVB/NJ mice without the transgene were age-matched controls. Sequences were either private (restricted to one mouse) or public (shared among mice); public sequences were either exclusive to the tumor group or inclusive among different groups. We now report that 1), public AA sequences were each encoded by many different nucleotide sequences (NT) recombinations (convergent recombination; CR); 2) mice developing tumors evolved tumor-exclusive public sequences, derived initially from private or from inclusive public sequences; and 3) tumor-exclusive public sequences in mice were also present among published public TCR sequences from human breast cancer patients. These cross-species tumor-exclusive TCR sequences manifested high CR; but the AA sequences shared by mice and humans did not share NT sequences. Thus, tumor-exclusive TCR AA sequences across species are selected from different NT recombination events. The roles of tumor-exclusive TCR repertoires in advancing or inhibiting tumor development and the effects of tumor immunotherapy on these T cells remain to be seen.
- Published
- 2018
- Full Text
- View/download PDF
9. Mosaic deletion patterns of the human antibody heavy chain gene locus as revealed by Bayesian haplotyping
- Author
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Ida Lindeman, Francois Vigneault, Ayelet Peres, Moriah Gidoni, Ludvig M. Sollid, Knut E.A. Lundin, Ivana Mikocziova, Christopher Clouser, Andrew M. Collins, Omri Snir, Gur Yaari, Pazit Polak, and Vikas Kumar Sarna
- Subjects
education.field_of_study ,Haplotype ,Population ,IGHD ,Locus (genetics) ,Genomics ,Copy-number variation ,Computational biology ,Allele ,Biology ,IGHV@ ,education - Abstract
Analysis of antibody repertoires by high-throughput sequencing is of major importance in understanding adaptive immune responses. Our knowledge of variations in the genomic loci encoding antibody genes is incomplete, mostly due to technical difficulties in aligning short reads to these highly repetitive loci. The partial knowledge results in conflicting V-D-J gene assignments between different algorithms, and biased genotype and haplotype inference. Previous studies have shown that haplotypes can be inferred by taking advantage of IGHJ6 heterozygosity, observed in approximately one third of the population. Here, we propose a robust novel method for determining V-D-J haplotypes by adapting a Bayesian framework. Our method extends haplotype inference to IGHD- and IGHV-based analysis, thereby enabling inference of complex genetic events like deletions and copy number variations in the entire population. We generated the largest multi individual data set, to date, of naïve B-cell repertoires, and tested our method on it. We present evidence for allele usage bias, as well as a mosaic, tiled pattern of deleted and present IGHD and IGHV nearby genes, across the population. The inferred haplotypes and deletion patterns may have clinical implications for genetic predispositions to diseases. Our findings greatly expand the knowledge that can be extracted from antibody repertoire sequencing data.
- Published
- 2018
10. Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing
- Author
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Steven H. Kleinstein, Francois Vigneault, Xiaomei Wang, J. Christopher Love, Kristy O. Murray, Konstantinos Tsioris, Feng Qian, George M. Church, Melissa N. Garcia, Yi Yao, Namita Gupta, Christopher Clouser, Ruth R. Montgomery, Raj Chari, Adrian W. Briggs, Joel N. H. Stern, Adebola O. Ogunniyi, Ross M. Zimnisky, Massachusetts Institute of Technology. Department of Chemical Engineering, Koch Institute for Integrative Cancer Research at MIT, Tsioris, Konstantinos, Ogunniyi, Adebola Oluwakayode, Zimnisky, Ross Martin, and Love, J. Christopher
- Subjects
Adult ,Male ,viruses ,Biophysics ,Disease ,Antibodies, Viral ,Biochemistry ,Asymptomatic ,Article ,DNA sequencing ,Virus ,Cohort Studies ,Young Adult ,Single-cell analysis ,Antibody Specificity ,Immunity ,medicine ,Humans ,Aged ,B-Lymphocytes ,biology ,Repertoire ,High-Throughput Nucleotide Sequencing ,virus diseases ,Middle Aged ,Antibodies, Neutralizing ,Virology ,Immunity, Humoral ,Immunology ,biology.protein ,Female ,Single-Cell Analysis ,medicine.symptom ,Antibody ,West Nile virus ,West Nile Fever - Abstract
West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV – recently infected and post-convalescent subjects – and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases., National Institutes of Health (U.S.) (U19AI089992), National Institutes of Health (U.S.) (AI091816), National Institutes of Health (U.S.) (R01AI104739), National Institutes of Health (U.S.) (1F32AI112359-01), National Institutes of Health (U.S.) (T15LM07056), Human Immunology Project Consortium, Gillson Longenbaugh Foundation
- Published
- 2015
11. Tumor-infiltrating immune repertoires captured by single-cell barcoding in emulsion
- Author
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Steven H. Kleinstein, Dennis R. Burton, Francois Vigneault, Christopher Clouser, David Koppstein, Brian J. Belmont, Adrian W. Briggs, Manu Tamminen, Jason Vander Heiden, Devin Sok, Stephen J. Goldfless, George M. Church, and Sonia Timberlake
- Subjects
biology ,T-cell receptor ,Cell ,breakpoint cluster region ,RNA ,chemical and pharmacologic phenomena ,Computational biology ,Phenotype ,medicine.anatomical_structure ,Immune system ,Immunology ,biology.protein ,medicine ,Antibody ,Receptor - Abstract
Tumor-infiltrating lymphocytes (TILs) are critical to anti-cancer immune responses, but their diverse phenotypes and functions remain poorly understood and challenging to study. We therefore developed a single-cell barcoding technology for deep characterization of TILs without the need for cell-sorting or culture. Our emulsion-based method captures full-length, natively paired B-cell and T-cell receptor (BCR and TCR) sequences from lymphocytes among millions of input cells. We validated the method with 3 million B-cells from healthy human blood and 350,000 B-cells from an HIV elite controller, before processing 400,000 cells from an unsorted dissociated ovarian adenocarcinoma and recovering paired BCRs and TCRs from over 11,000 TILs. We then extended the barcoding method to detect DNA-labeled antibodies, allowing ultra-high throughput, simultaneous protein detection and RNA sequencing from single cells.
- Published
- 2017
- Full Text
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12. Maternal Plasma DNA Analysis with Massively Parallel Sequencing by Ligation for Noninvasive Prenatal Diagnosis of Trisomy 21
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Y.M. Dennis Lo, Christopher Clouser, Kevin McKernan, Hao Sun, Clarence Lee, Ranjit Akolekar, Daixing Zhou, Rossa W.K. Chiu, and Kypros H. Nicolaides
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Genetics ,Massive parallel sequencing ,Chromosomes, Human, Pair 21 ,Biochemistry (medical) ,Clinical Biochemistry ,Aneuploidy ,Chromosome ,Prenatal diagnosis ,DNA ,Sequence Analysis, DNA ,Biology ,medicine.disease ,Pregnancy ,Prenatal Diagnosis ,medicine ,Humans ,Female ,Genetic Testing ,Down Syndrome ,Ploidy ,Trisomy ,Chromosome 21 ,GC-content - Abstract
Background: Noninvasive prenatal diagnosis of trisomy 21 (T21) has recently been shown to be achievable by massively parallel sequencing of maternal plasma on a sequencing-by-synthesis platform. The quantification of several other human chromosomes, including chromosomes 18 and 13, has been shown to be less precise, however, with quantitative biases related to the chromosomal GC content. Methods: Maternal plasma DNA from 10 euploid and 5 T21 pregnancies was sequenced with a sequencing-by-ligation approach. We calculated the genomic representations (GRs) of sequenced reads from each chromosome and their associated measurement CVs and compared the GRs of chromosome 21 (chr21) for the euploid and T21 pregnancies. Results: We obtained a median of 12 × 106 unique reads (21% of the total reads) per sample. The GRs deviated from those expected for some chromosomes but in a manner different from that previously reported for the sequencing-by-synthesis approach. Measurements of the GRs for chromosomes 18 and 13 were less precise than for chr21. z Scores of the GR of chr21 were increased in the T21 pregnancies, compared with the euploid pregnancies. Conclusions: Massively parallel sequencing-by-ligation of maternal plasma DNA was effective in identifying T21 fetuses noninvasively. The quantitative biases observed among the GRs of certain chromosomes were more likely based on analytical factors than biological factors. Further research is needed to enhance the precision for measuring for the representations of chromosomes 18 and 13.
- Published
- 2010
13. Direct control of neurogenesis by selector factors in the fly eye:regulation ofatonalby Ey and So
- Author
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Christopher Clouser, Tianyi Zhang, Francesca Pignoni, Chuan Qi Cai, and Swati Ranade
- Subjects
Electrophoretic Mobility Shift Assay ,Nerve Tissue Proteins ,Biology ,Eye ,Basic Helix-Loop-Helix Transcription Factors ,medicine ,Animals ,Drosophila Proteins ,3' Flanking Region ,Eye Proteins ,Molecular Biology ,Transcription factor ,Progenitor ,Homeodomain Proteins ,Eye morphogenesis ,Mechanism (biology) ,Neurogenesis ,Gene Expression Regulation, Developmental ,Anatomy ,Biological Evolution ,DNA-Binding Proteins ,Enhancer Elements, Genetic ,medicine.anatomical_structure ,Eye development ,Drosophila ,Photoreceptor Cells, Invertebrate ,sense organs ,Neuron ,PAX6 ,Neuroscience ,Protein Binding ,Signal Transduction ,Developmental Biology - Abstract
During eye development, the selector factors of the Eyeless/Pax6 or Retinal Determination (RD) network control specification of organ-type whereas the bHLH-type proneural factor Atonal drives neurogenesis. Although significant progress has been made in dissecting the acquisition of `eye identity' at the transcriptional level, the molecular mechanisms underlying the progression from neuronal progenitor to differentiating neuron remain unclear. A recently proposed model for the integration of organ specification and neurogenesis hypothesizes that atonal expression in the eye is RD-network-independent and that Eyeless works in parallel or downstream of atonal to modify the neurogenetic program. We show here that distinct cis-regulatory elements control atonal expression specifically in the eye and that the RD factors Eyeless and Sine oculis function as direct regulators. We find that these transcription factors interact in vitro and provide indirect evidence that this interaction may be required in vivo. The subordination of neurogenesis to the RD pathway in the eye provides a direct mechanism for the coordination of neurogenesis and tissue specification during sensory organ formation.
- Published
- 2006
14. Partner specificity is essential for proper function of the SIX-type homeodomain proteins Sine oculis and Optix during fly eye development
- Author
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Susan L. Tran, Christopher Clouser, Swati Ranade, Donghui Yang-Zhou, Chuan Qi Cai, Gina Decene, Kristy L. Kenyon, and Francesca Pignoni
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Six2 ,Six1 ,Six3 ,Six6 ,Molecular Sequence Data ,Genes, Insect ,Biology ,medicine.disease_cause ,Eye ,Animals, Genetically Modified ,03 medical and health sciences ,0302 clinical medicine ,Six-co-factors ,In vivo ,medicine ,Animals ,Drosophila Proteins ,Point Mutation ,Sine ,Amino Acid Sequence ,Eye Proteins ,Transcription factor ,Molecular Biology ,Binding selectivity ,Conserved Sequence ,030304 developmental biology ,Genes, Dominant ,Genetics ,Homeodomain Proteins ,0303 health sciences ,Mutation ,Dominant negative ,Mechanism (biology) ,Genes, Homeobox ,Gene Expression Regulation, Developmental ,SoDrl ,DNA ,Cell Biology ,Drl ,Phenotype ,Amino Acid Substitution ,Eye development ,Homeobox ,Drosophila ,SoD ,030217 neurology & neurosurgery ,Protein Binding ,Transcription Factors ,Developmental Biology - Abstract
The development of the Drosophila visual system utilizes two members of the highly conserved Six-Homeobox family of transcription factor, Sine oculis and Optix. Although in vitro studies have detected differences in DNA-binding and interactions with some co-factors, questions remain as to what extent the activity for these two transcriptional regulators is redundant or specific in vivo. In this work, we show that the SoD mutation within the Six domain does not abolish DNA–protein interactions, but alters co-factor binding specificity to resemble that of Optix. A mutation in the same region of Optix alters its activity in vivo. We propose that the dominant mutant phenotype is primarily due to an alteration in binding properties of the Sine oculis protein and that distinct partner interactions is one important mechanism in determining significant functional differences between these highly conserved factors during eye development.
- Published
- 2005
- Full Text
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15. Exploration of immunoglobulin transcriptomes from mice immunized with three-finger toxins and phospholipases A2from the Central American coral snake,Micrurus nigrocinctus
- Author
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Bruno Lomonte, Andreas Hougaard Laustsen, Francois Vigneault, Christopher Clouser, Sonia Timberlake, José María Gutiérrez, and Mikael Engmark
- Subjects
0301 basic medicine ,Snake venom ,food.ingredient ,Bioinformatics ,Immunology ,Antivenom ,lcsh:Medicine ,Toxicology ,complex mixtures ,Antibodies ,General Biochemistry, Genetics and Molecular Biology ,transcriptomics ,Antibody isotyping ,03 medical and health sciences ,food ,SDG 3 - Good Health and Well-being ,antibody ,Micrurus nigrocinctus ,Murine immune response ,Toxins ,Micrurus ,Transcriptomics ,Coral snake ,antivenom ,High-throughput sequencing ,030102 biochemistry & molecular biology ,biology ,General Neuroscience ,Immunogenicity ,lcsh:R ,General Medicine ,biology.organism_classification ,Venom ,Isotype ,030104 developmental biology ,biology.protein ,Synthetic Biology ,Antibody ,General Agricultural and Biological Sciences ,Biotechnology - Abstract
Snakebite envenomings represent a neglected public health issue in many parts of the rural tropical world. Animal-derived antivenoms have existed for more than a hundred years and are effective in neutralizing snake venom toxins when timely administered. However, the low immunogenicity of many small but potent snake venom toxins represents a challenge for obtaining a balanced immune response against the medically relevant components of the venom. Here, we employ high-throughput sequencing of the immunoglobulin (Ig) transcriptome of mice immunized with a three-finger toxin and a phospholipase A2from the venom of the Central American coral snake,Micrurus nigrocinctus.Although exploratory in nature, our indicate results showed that only low frequencies of mRNA encoding IgG isotypes, the most relevant isotype for therapeutic purposes, were present in splenocytes of five mice immunized with 6 doses of the two types of toxins over 90 days. Furthermore, analysis of Ig heavy chain transcripts showed that no particular combination of variable (V) and joining (J) gene segments had been selected in the immunization process, as would be expected after a strong humoral immune response to a single antigen. Combined with the titration of toxin-specific antibodies in the sera of immunized mice, these data support the low immunogenicity of three-finger toxins and phospholipases A2found inM. nigrocinctusvenoms, and highlight the need for future studies analyzing the complexity of antibody responses to toxins at the molecular level.
- Published
- 2017
16. Abstract 1442: Comprehensive TIL profiling by simultaneous DNA barcoding of proteins, RNA and natively paired immune receptors from millions of single cells
- Author
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Adrian W. Briggs, Christopher Clouser, Francois Vigneault, Brian J. Belmont, Katherine L. Connor, Sonia Timberlake, Stephen J. Goldfless, and David Koppstein
- Subjects
Cancer Research ,Immune system ,Oncology ,Profiling (information science) ,RNA ,Computational biology ,Biology ,Bioinformatics ,Receptor ,DNA barcoding - Abstract
We present high resolution single cell profiling of tumor infiltrating lymphocytes (TILs) for immune research and cancer target discovery. TILs hold the key to understanding anti-cancer immune responses but remain challenging to study due to their vast range of phenotypes, functions and abundance. Current approaches to TIL analysis such as immune receptor sequencing, flow cytometry, and RNA expression profiling are limited, since each approach gives only partial insights into a TIL repertoire and their results cannot be connected at the single cell level. We therefore built a technology for simultaneous determination of immune receptor sequences, surface protein phenotypes and RNA expression profiles from single TILs at unprecedented scale. Able to process millions of cells per experiment, the throughput of our emulsion-based DNA barcoding method allows deep and unbiased TIL profiling directly from tumor tissue without the need for cell sorting, stimulation or culture. Briefly, single-cell dissociated biopsy tissue samples are stained with DNA-labeled surface marker antibodies and isolated into individual ∼65-pL microdroplets by a microfluidic emulsion chip. Within the droplets, cellular RNAs are reverse transcribed into cDNA, and a barcoding step attaches droplet-specific DNA barcodes to all of the cDNAs as well as to the DNA labels carried by the marker antibodies. At the end of the reaction, all products associated with a single input cell carry the same DNA barcode sequence, allowing subsequent high throughput sequencing to assign the products to their cell of origin. During analysis the full-length immune receptor pairs for each B- and T-cell are reconstructed and the marker antibody tags are identified and quantified along with dozens of additional mRNA markers, resulting in a high-dimensional dataset containing integrated immune receptor clone, protein marker and RNA expression profiles of thousands to hundreds of thousands of individual lymphocytes in the input sample. We have successfully applied our method to a variety of healthy blood and tissue samples including human cancers such as pancreatic, ovarian and lung cancer, profiling millions of B-cells and T-cells present in the samples. We identify likely tumor-reactive TILs by finding BCR or TCR clonal lineages associated with particular protein and RNA signatures, including expression of immunosuppression markers such as PD-1 and CTLA-4. Following candidate selection, full-length BCR and TCR pairs are synthesized and expressed for functional screening and target antigen identification. By providing an unprecedented view into TIL diversity across a wide range of disease and sample types, our platform also has widespread potential for basic and applied research into the patient immune response to cancer and the dynamics of immunotherapy. Citation Format: Katherine L. Connor, Adrian W. Briggs, Stephen J. Goldfless, Sonia Timberlake, Brian J. Belmont, Christopher R. Clouser, David Koppstein, Francois Vigneault. Comprehensive TIL profiling by simultaneous DNA barcoding of proteins, RNA and natively paired immune receptors from millions of single cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1442.
- Published
- 2016
17. Abstract 3998: Sequencing the B-cell and T-cell repertoire
- Author
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Lynne Apone, Francois Vigneault, Christopher Clouser, Eileen T. Dimalanta, Mehmet Karaca, William Donahue, Bonny Patel, Theodore B. Davis, Fiona J. Stewart, Sonia Timberlake, Salvatore Russello, and Kris Adams
- Subjects
clone (Java method) ,Cancer Research ,Oncology ,Heavy-chain antibody ,biology ,Repertoire ,Consensus sequence ,biology.protein ,Computational biology ,Antibody ,Immunoglobulin D ,Gene ,Isotype - Abstract
Immune sequencing allows for the study of complex immunological diseases by sequencing millions of V(D)J combinations from B-cell antibody and T-cell receptors. The popularity of this technique has increased due to recent throughput and read length improvements in next-generation sequencing technologies. However, structural and sequence complexities of antibody genes have made reliable targeting approaches challenging. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. The method uses a unique barcoding scheme specifically designed to tag every mRNA molecule with a unique identifier (UID) so that all PCR copies of each mRNA fragment can be collapsed into a single consensus sequence. This makes the assay extremely accurate, by resolving PCR bias and sequencing errors as well as allowing quantitative digital molecule counting. Immune sequencing libraries were generated from total RNA extracted from Peripheral Blood Mononuclear Cells in duplicate from a single patient. The use of UIDs enabled absolute quantification of starting RNA molecules present in the original sample and therefore accurate ranking of the antibody clone abundance, by avoiding the bias incorporated by PCR or sequencing when total reads only were measured. Using the same sequencing method, tumor samples were analyzed for abundance of expanded clones via grouping clones by V gene, J gene and CDR3 similarity and ranking by mRNA abundance. Additionally, the use of isotype-specific primers (IgM, IgD, IgG, IgA and IgE) enabled measurement of the heavy chain isotype proportions within the samples. Further, alignment of full-length heavy chain antibody sequences generated using this method to germline genes from reference databases enabled quantitation of the mutation level of each antibody sequence, thereby providing information on the overall maturity and mutational profile of the sample repertoire. Citation Format: Fiona J. Stewart, Mehmet Karaca, Kris Adams, Chris Clouser, Bonny Patel, Sonia Timberlake, William Donahue, Lynne Apone, Salvatore Russello, Eileen T. Dimalanta, Theodore B. Davis, Francois Vigneault. Sequencing the B-cell and T-cell repertoire. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3998.
- Published
- 2016
18. Development of personalized tumor biomarkers using massively parallel sequencing
- Author
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Kerstin Schmidt, Clarence Lee, Francisco M. De La Vega, Rebecca J. Leary, Alena A. Antipova, Luis A. Diaz, Kevin McKernan, Kenneth W. Kinzler, Victor E. Velculescu, Cisilya Duncan, Isaac Kinde, Frank Diehl, Bert Vogelstein, and Christopher Clouser
- Subjects
Sequence analysis ,Molecular Sequence Data ,Computational biology ,Biology ,Bioinformatics ,Polymerase Chain Reaction ,Article ,Translocation, Genetic ,law.invention ,chemistry.chemical_compound ,law ,Neoplasms ,medicine ,Biomarkers, Tumor ,Humans ,Precision Medicine ,Polymerase chain reaction ,Gene Rearrangement ,Massive parallel sequencing ,Genome ,Base Sequence ,business.industry ,Breakpoint ,Cancer ,General Medicine ,Gene rearrangement ,Genomics ,DNA ,Sequence Analysis, DNA ,medicine.disease ,chemistry ,Personalized medicine ,business - Abstract
Clinical management of human cancer is dependent on the accurate monitoring of residual and recurrent tumors. The evaluation of patient-specific translocations in leukemias and lymphomas has revolutionized diagnostics for these diseases. We have developed a method, called personalized analysis of rearranged ends (PARE), which can identify translocations in solid tumors. Analysis of four colorectal and two breast cancers with massively parallel sequencing revealed an average of nine rearranged sequences (range, 4 to 15) per tumor. Polymerase chain reaction with primers spanning the breakpoints was able to detect mutant DNA molecules present at levels lower than 0.001% and readily identified mutated circulating DNA in patient plasma samples. This approach provides an exquisitely sensitive and broadly applicable approach for the development of personalized biomarkers to enhance the clinical management of cancer patients.
- Published
- 2010
19. Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding
- Author
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Stephen F. McLaughlin, Martin G. Reese, Gina Costa, Francisco M. De La Vega, Cynthia L. Hendrickson, Can Alkan, Joel A. Malek, Tanya Sokolsky, Andrew MacBride, Alena A. Antipova, Kathleen C. Hayashibara, Zheng Zhang, Jonathan M. Manning, Alan Blanchard, Swati Ranade, Haoning Fu, Kevin McKernan, Adam Sannicandro, Damon S. Perez, Vineet Bafna, Jeremy R. Stuart, Michael P. Moore, Robert E. Beaudoin, Rajesh Gottimukkala, Yutao Fu, Lei Zhang, Eric F. Tsung, Andrew Sheridan, Cisyla Duncan, Ali Bashir, Fiona Hyland, Jeffrey M. Kidd, Jeffrey K. Ichikawa, Michael W. Laptewicz, Michael D. Rhodes, Brittany E. Coleman, Michael R. Lyons, Eileen T. Dimalanta, Clarence Lee, Shan Yang, Bin Li, Evan E. Eichler, Heather E. Peckham, Christopher Clouser, and Lev Kotler
- Subjects
Nonsynonymous substitution ,Heterozygote ,Genotype ,Genomics ,Biology ,Genome ,Polymorphism, Single Nucleotide ,DNA sequencing ,Deep sequencing ,Structural variation ,Ligases ,Genetics ,Methods ,Humans ,Base Pairing ,Genetics (clinical) ,Base Sequence ,Genome, Human ,Homozygote ,Computational Biology ,Genetic Variation ,Sequence Analysis, DNA ,Reference Standards ,Africa ,Human genome ,Reference genome - Abstract
We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding ∼18× haploid coverage of aligned sequence and close to 300× clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.
- Published
- 2009
20. Nicastrin controls aspects of photoreceptor neuron specification and differentiation in the Drosophila eye
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Tianyi Zhang, Susan L. Tran, Francesca Pignoni, and Christopher Clouser
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Regulation of gene expression ,Genetics ,Cell Nucleus ,Membrane Glycoproteins ,biology ,Receptors, Notch ,Cellular differentiation ,Neurogenesis ,Notch signaling pathway ,Nicastrin ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Eye ,Cell biology ,Drosophila melanogaster ,Phenotype ,Notch proteins ,Lateral inhibition ,Mutation ,Eye development ,biology.protein ,Animals ,Photoreceptor Cells, Invertebrate ,Amyloid Precursor Protein Secretases ,Developmental Biology - Abstract
Nicastrin is a component of the Notch signaling pathway involved in proteolytic release of the Notch receptor intracellular domain. It has been postulated that intracellular Notch is required within the nucleus of fly eye progenitor cells to enhance (pro-neural enhancement) and then repress (lateral inhibition) transcription of pro-neural genes. We present here an analysis of Nicastrin function during eye development and find that Nicastrin is essential to early photoreceptor neuron development. Nicastrin mutant tissue displays neuronal loss or hyperplasia; these phenotypes can be rescued by targeted expression of an intracellular form of Notch. Thus, nuclear translocation of Notch and its direct regulation of gene expression appear to be critical to pro-neural enhancement as well as lateral inhibition. In addition, we show that Nicastrin as well as Notch are required to maintain normal R-cell morphology, because the nuclei of mutant photoreceptor neurons cannot maintain their proper position. Thus, Notch signaling plays a role, not only in cell fate specification, but also in differentiation of photoreceptor neurons.
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- 2005
21. Fly SIX-type homeodomain proteins Sine oculis and Optix partner with different cofactors during eye development
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Dan Jun Li, Francesca Pignoni, Susan L. Tran, Kristy L. Kenyon, and Christopher Clouser
- Subjects
genetic structures ,Molecular Sequence Data ,Eye ,Animals ,Drosophila Proteins ,Eye Proteins ,Sine ,Amino Acid Sequence ,Transcription factor ,Genetics ,Homeodomain Proteins ,biology ,Sequence Homology, Amino Acid ,Gene Expression Regulation, Developmental ,biology.organism_classification ,Phenotype ,eye diseases ,TAF1 ,Drosophila melanogaster ,Larva ,Eye development ,Homeobox ,sense organs ,Sequence Alignment ,Developmental Biology ,Protein Binding ,Transcription Factors - Abstract
Two members from the Six class of homeobox transcription factors, Sine oculis (SO) and Optix, function during development of the fly visual system. Differences in gain-of-function phenotypes and gene expression suggest that these related factors play distinct roles in the formation of the fly eye. However, the molecular nature of their functional differences remains unclear. In this study, we report the identification of two novel factors that participate in specific partnerships with Sine oculis and Optix during photoreceptor neurons formation and in eye progenitor cells. This work shows that different cofactors likely mediate unique functions of Sine oculis and Optix during the development of the fly eye and that the repeated requirement for SO function at multiple stages of eye development reflects the activity of different SO-cofactor complexes.
- Published
- 2005
22. Fly Six‐type homeodomain proteins Sine oculis and Optix partner with different cofactors during eye development.
- Author
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Kristy L. Kenyon, Dan Jun Li, Christopher Clouser, Susan Tran, and Francesca Pignoni
- Published
- 2005
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