60 results on '"Francois Vigneault"'
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. Single T Cell Sequencing Demonstrates the Functional Role of αβ TCR Pairing in Cell Lineage and Antigen Specificity
- Author
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Jason A. Carter, Jonathan B. Preall, Kristina Grigaityte, Stephen J. Goldfless, Eric Jeffery, Adrian W. Briggs, Francois Vigneault, and Gurinder S. Atwal
- Subjects
TCR–T cell receptor ,CD4 and CD8 T cell repertoires ,TCR repertoire diversity ,single-cell sequencing ,machine learning ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Although structural studies of individual T cell receptors (TCRs) have revealed important roles for both the α and β chain in directing MHC and antigen recognition, repertoire-level immunogenomic analyses have historically examined the β chain alone. To determine the amount of useful information about TCR repertoire function encoded within αβ pairings, we analyzed paired TCR sequences from nearly 100,000 unique CD4+ and CD8+ T cells captured using two different high-throughput, single-cell sequencing approaches. Our results demonstrate little overlap in the healthy CD4+ and CD8+ repertoires, with shared TCR sequences possessing significantly shorter CDR3 sequences corresponding to higher generation probabilities. We further utilized tools from information theory and machine learning to show that while α and β chains are only weakly associated with lineage, αβ pairings appear to synergistically drive TCR-MHC interactions. Vαβ gene pairings were found to be the TCR feature most informative of T cell lineage, supporting the existence of germline-encoded paired αβ TCR-MHC interaction motifs. Finally, annotating our TCR pairs using a database of sequences with known antigen specificities, we demonstrate that approximately a third of the T cells possess α and β chains that each recognize different known antigens, suggesting that αβ pairing is critical for the accurate inference of repertoire functionality. Together, these findings provide biological insight into the functional implications of αβ pairing and highlight the utility of single-cell sequencing in immunogenomics.
- Published
- 2019
- Full Text
- View/download PDF
4. Antibody Repertoire Analysis of Hepatitis C Virus Infections Identifies Immune Signatures Associated With Spontaneous Clearance
- Author
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Sivan Eliyahu, Oz Sharabi, Shiri Elmedvi, Reut Timor, Ateret Davidovich, Francois Vigneault, Chris Clouser, Ronen Hope, Assy Nimer, Marius Braun, Yaacov Y. Weiss, Pazit Polak, Gur Yaari, and Meital Gal-Tanamy
- Subjects
hepatitis C virus ,antibody repertoire ,neutralizing antibodies ,infectious disease ,immune signature ,Immunologic diseases. Allergy ,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
- Full Text
- View/download PDF
5. 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.
- Published
- 2017
- Full Text
- View/download PDF
6. Correction: The Effects of Somatic Hypermutation on Neutralization and Binding in the PGT121 Family of Broadly Neutralizing HIV Antibodies.
- Author
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Devin Sok, Uri Laserson, Jonathan Laserson, Yi Liu, Francois Vigneault, Jean-Philippe Julien, Bryan Briney, Alejandra Ramos, Karen F. Saye, Khoa Le, Alison Mahan, Shenshen Wang, Mehran Kardar, Gur Yaari, Laura M. Walker, Birgitte B. Simen, Elizabeth P. St. John, Po-Ying Chan-Hui, Kristine Swiderek, Stephen H. Kleinstein, Galit Alter, Michael S. Seaman, Arup K. Chakraborty, Daphne Koller, Ian A. Wilson, George M. Church, Dennis R. Burton, and Pascal Poignard
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Published
- 2013
- Full Text
- View/download PDF
7. The effects of somatic hypermutation on neutralization and binding in the PGT121 family of broadly neutralizing HIV antibodies.
- Author
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Devin Sok, Uri Laserson, Jonathan Laserson, Yi Liu, Francois Vigneault, Jean-Philippe Julien, Bryan Briney, Alejandra Ramos, Karen F Saye, Khoa Le, Alison Mahan, Shenshen Wang, Mehran Kardar, Gur Yaari, Laura M Walker, Birgitte B Simen, Elizabeth P St John, Po-Ying Chan-Hui, Kristine Swiderek, Steven H Kleinstein, Galit Alter, Michael S Seaman, Arup K Chakraborty, Daphne Koller, Ian A Wilson, George M Church, Dennis R Burton, and Pascal Poignard
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121-134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121-134 but were still capable of neutralizing roughly 40-80% of PGT121-134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121-134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121-134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.
- Published
- 2013
- Full Text
- View/download PDF
8. pRESTO: a toolkit for processing high-throughput sequencing raw reads of lymphocyte receptor repertoires.
- Author
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Jason A. Vander Heiden, Gur Yaari, Mohamed Uduman, Joel N. H. Stern, Kevin C. O'Connor, David A. Hafler, Francois Vigneault, and Steven H. Kleinstein
- Published
- 2014
- Full Text
- View/download PDF
9. Mosaic deletion patterns of the human antibody heavy chain gene locus shown by Bayesian haplotyping
- Author
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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
- Subjects
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
10. 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
11. I Feel Awful, Thanks
- Author
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Lara Pickle, François Vigneault, Lara Pickle, and François Vigneault
- Abstract
Joana has dragons inside her. Can she tame them before they burn her life down? Joana is a young witch who secured her dream job with a coven in London, her favorite city, where she can dedicate herself to creating potions, her favorite activity! However, she will soon discover the reality of city life is not so idyllic. Finding a flat is an ordeal, her “dream job” is stressful, and she's totally alone. Little by little, she makes her place, but fatigue, sadness, and doubts threaten to topple her hard-earned success... until she starts talking to a professional who helps her realize in order to take care of herself, she must know herself. Lara Pickle's I Feel Awful, Thanks is a lushly illustrated debut graphic novel that explores the hard-fought journey of self-acceptance in a complex world of swirling emotions and powerful potions!
- Published
- 2024
12. Talli, Daughter of the Moon Vol. 2
- Author
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Sourya, Francois Vigneault, Sourya, and Francois Vigneault
- Abstract
Talli is a Summoner: a nearly extinct people, hunted by those who fear their mysterious powers. As a baby, she was adopted by Lord Borin, and all was well for many years... But Talli has been sent on the run by the forces of Lord Ulric, who wants to exterminate the Summoners. With Ulric's forces led by the ruthless Captain Nina hot on her heels, Talli and her loyal protector Alan find themselves with a motley crew of companions and allies that includes the lethargic-but-incredible swordsman Lélo. A climactic battle has cut them off from their chosen route, and now they find themselves traversing the deadly and mysterious Forbidden Forest. Can Talli and her team make it to the other side without getting eaten by wild animals, and find refuge in the kingdom of Lady Sybbyl? Will she perfect her control over her incredible powers in her moment of need?
- Published
- 2023
13. Pink Lemonade
- Author
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Nick Cagnetti, François Vigneault, Nick Cagnetti, and François Vigneault
- Subjects
- Amnesiacs--Fiction, Amnesiacs--Comic books, strips, etc, Superheroes--Fiction, Superheroes--Comic books, strips, etc
- Abstract
Mysterious past! Colorful costume! Altruistic outlook! Zippy motorcycle! And one very overactive imagination! Meet PINK LEMONADE—the new hero on the scene! She dreams of doing big things, but ultimately, she'll settle for just helping where she can. But when Pink Lemonade accidentally crashes into the set of the next Rex Radical blockbuster, she'll take an unexpected leap into the spotlight...and will soon find herself confronted by a series of increasingly threatening doppelgängers, extraterrestrial fanboys, and nefarious corporate machinations. Collecting PINK LEMONADE #1-6 in one complete volume.
- Published
- 2023
14. Hierarchical Clustering Can Identify B Cell Clones with High Confidence in Ig Repertoire Sequencing Data
- Author
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Francois Vigneault, Sonia Timberlake, Steven H. Kleinstein, Adrian W. Briggs, Namita Gupta, and Kristofor Adams
- Subjects
0301 basic medicine ,Genetics ,Repertoire ,Immunology ,Single-linkage clustering ,Somatic hypermutation ,Hamming distance ,Context (language use) ,Computational biology ,Biology ,Hierarchical clustering ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Immunology and Allergy ,Cluster analysis ,Selection (genetic algorithm) ,030215 immunology - Abstract
Adaptive immunity is driven by the expansion, somatic hypermutation, and selection of B cell clones. Each clone is the progeny of a single B cell responding to Ag, with diversified Ig receptors. These receptors can now be profiled on a large scale by next-generation sequencing. Such data provide a window into the microevolutionary dynamics that drive successful immune responses and the dysregulation that occurs with aging or disease. Clonal relationships are not directly measured, but they must be computationally inferred from these sequencing data. Although several hierarchical clustering-based methods have been proposed, they vary in distance and linkage methods and have not yet been rigorously compared. In this study, we use a combination of human experimental and simulated data to characterize the performance of hierarchical clustering-based methods for partitioning sequences into clones. We find that single linkage clustering has high performance, with specificity, sensitivity, and positive predictive value all >99%, whereas other linkages result in a significant loss of sensitivity. Surprisingly, distance metrics that incorporate the biases of somatic hypermutation do not outperform simple Hamming distance. Although errors were more likely in sequences with short junctions, using the entire dataset to choose a single distance threshold for clustering is near optimal. Our results suggest that hierarchical clustering using single linkage with Hamming distance identifies clones with high confidence and provides a fully automated method for clonal grouping. The performance estimates we develop provide important context to interpret clonal analysis of repertoire sequencing data and allow for rigorous testing of other clonal grouping algorithms.
- Published
- 2017
15. A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Ig Sequencing Data
- Author
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Jason A. Vander Heiden, Adrian W. Briggs, Tamara J. Gilbert, Francois Vigneault, Mark J. Shlomchik, Kevin C. O’Connor, Roberto Di Niro, Kris Adams, Ang Cui, and Steven H. Kleinstein
- Subjects
0301 basic medicine ,DNA Repair ,DNA repair ,Immunology ,Immunoglobulin Variable Region ,Somatic hypermutation ,Mice, Transgenic ,Biology ,medicine.disease_cause ,Article ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Mutation Rate ,medicine ,Animals ,Humans ,Immunology and Allergy ,Mutation frequency ,Clonal Selection, Antigen-Mediated ,Cells, Cultured ,B cell ,Genetics ,B-Lymphocytes ,Mice, Inbred BALB C ,Mutation ,Models, Genetic ,Transition (genetics) ,High-Throughput Nucleotide Sequencing ,Germinal center ,Germinal Center ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Female ,Somatic Hypermutation, Immunoglobulin ,Immunoglobulin Heavy Chains ,DNA ,030215 immunology - Abstract
Analyses of somatic hypermutation (SHM) patterns in B cell Ig sequences have important basic science and clinical applications, but they are often confounded by the intrinsic biases of SHM targeting on specific DNA motifs (i.e., hot and cold spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized B1-8 H chain transgenic mice with nitrophenyl to stimulate nitrophenyl-specific λ+ germinal center B cells and sequenced the unexpressed κ L chains using next-generation methods. Most of these κ sequences had out-of-frame junctions and were presumably uninfluenced by selection. Despite being nonfunctionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We used 39,173 mutations to construct a quantitative SHM targeting model. The model showed targeting biases that were consistent with classic hot and cold spots, yet revealed additional highly mutable motifs. We observed comparable targeting for functional and nonfunctional sequences, suggesting similar biological processes operate at both loci. However, we observed species- and chain-specific targeting patterns, demonstrating the need for multiple SHM targeting models. Interestingly, the targeting of C/G bases and the frequency of transition mutations at C/G bases was higher in mice compared with humans, suggesting lower levels of DNA repair activity in mice. Our models of SHM targeting provide insights into the SHM process and support future analyses of mutation patterns.
- Published
- 2016
16. Single T Cell Sequencing Demonstrates the Functional Role of αβ TCR Pairing in Cell Lineage and Antigen Specificity
- Author
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Jonathan B. Preall, Francois Vigneault, Eric Jeffery, Jason A. Carter, Stephen J. Goldfless, Kristina Grigaityte, Adrian W. Briggs, and Gurinder S. Atwal
- Subjects
0301 basic medicine ,lcsh:Immunologic diseases. Allergy ,Lineage (genetic) ,T cell ,Immunology ,TCR repertoire diversity ,chemical and pharmacologic phenomena ,Computational biology ,single-cell sequencing ,Major histocompatibility complex ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,medicine ,Immunology and Allergy ,Gene ,biology ,T-cell receptor ,TCR–T cell receptor ,CD4 and CD8 T cell repertoires ,030104 developmental biology ,medicine.anatomical_structure ,machine learning ,Single cell sequencing ,biology.protein ,lcsh:RC581-607 ,CD8 ,030215 immunology - Abstract
Although structural studies of individual T cell receptors (TCRs) have revealed important roles for both the α and β chain in directing MHC and antigen recognition, repertoire-level immunogenomic analyses have historically examined the β chain alone. To determine the amount of useful information about TCR repertoire function encoded within αβ pairings, we analyzed paired TCR sequences from nearly 100,000 unique CD4+ and CD8+ T cells captured using two different high-throughput, single-cell sequencing approaches. Our results demonstrate little overlap in the healthy CD4+ and CD8+ repertoires, with shared TCR sequences possessing significantly shorter CDR3 sequences corresponding to higher generation probabilities. We further utilized tools from information theory and machine learning to show that while α and β chains are only weakly associated with lineage, αβ pairings appear to synergistically drive TCR-MHC interactions. Vαβ gene pairings were found to be the TCR feature most informative of T cell lineage, supporting the existence of germline-encoded paired αβ TCR-MHC interaction motifs. Finally, annotating our TCR pairs using a database of sequences with known antigen specificities, we demonstrate that approximately a third of the T cells possess α and β chains that each recognize different known antigens, suggesting that αβ pairing is critical for the accurate inference of repertoire functionality. Together, these findings provide biological insight into the functional implications of αβ pairing and highlight the utility of single-cell sequencing in immunogenomics.
- Published
- 2019
- Full Text
- View/download PDF
17. 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
18. 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
19. Titan
- Author
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François Vigneault and François Vigneault
- Abstract
When MNGR First Class João da Silva arrives on the moon of Titan to take charge of Homestead Station, he finds the massive mining colony plagued by tensions between the giant, genetically-engineered Titan workers and the Terran management. As anger mounts, what began as a routine posting quickly turns into something far more dangerous. Phoebe Mackintosh thought she left her fighting days behind her when she turned her back on the “mixing” circuit. Now, she finds herself caught between a past she'd rather forget and a future she can't predict. Together, they must find a way to pull Homestead back from the brink of disaster... Or Titan might be the spark that sets the entire solar system ablaze.
- Published
- 2020
20. 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
21. 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
22. T-cell receptor αβ chain pairing is associated with CD4+ and CD8+ lineage specification
- Author
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Francois Vigneault, Adrian W. Briggs, Jason A. Carter, Kristina Grigaityte, Stephen J. Goldfless, Gurinder S. Atwal, and Jonathan B. Preall
- Subjects
Genetics ,0303 health sciences ,Lineage (genetic) ,Chemistry ,T-cell receptor ,hemic and immune systems ,chemical and pharmacologic phenomena ,T-Cell Receptor Alpha-Beta ,3. Good health ,03 medical and health sciences ,0302 clinical medicine ,Receptor ,Beta (finance) ,Gene ,030217 neurology & neurosurgery ,Function (biology) ,CD8 ,030304 developmental biology - Abstract
While a highly diverse T-cell receptor (TCR) repertoire is the hallmark of a healthy adaptive immune system, relatively little is understood about how the CD4+ and CD8+ TCR repertoires differ from one another. We here utilize high-throughput single T-cell sequencing to obtain approximately 100,000 TCR αβ chain pairs from human subjects, stratified into CD4+ and CD8+ lineages. We reveal that substantial information about T-cell lineage is encoded by Vαβ gene pairs and, to a lesser extent, by several other TCR features such as CDR3 length and charge. We further find that the strength of association between the β chain and T-cell lineage is surprisingly weak, similar in strength to that of the α chain. Using machine learning classifiers to predict T-cell lineage from TCR features, we demon-strate that αβ chain pairs are significantly more informative than individual chains alone. These findings provide unprecedented insight into the CD4+ and CD8+ TCR repertoires and highlight the importance of αβ chain pairing in TCR function and specificity.
- Published
- 2018
23. Single-cell sequencing reveals αβ chain pairing shapes the T cell repertoire
- Author
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Stephen J. Goldfless, Francois Vigneault, David Koppstein, Ronald J. Hause, Gurinder S. Atwal, Yue Jiang, Kristina Grigaityte, Eric Jeffery, George M. Church, Adrian W. Briggs, and Jason A. Carter
- Subjects
Genetics ,0303 health sciences ,Lymphocyte ,T cell ,T-cell receptor ,Biology ,Acquired immune system ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Single cell sequencing ,Antigen ,030220 oncology & carcinogenesis ,medicine ,Cytotoxic T cell ,CD8 ,030304 developmental biology - Abstract
A diverse T cell repertoire is a critical component of the adaptive immune system, providing protection against invading pathogens and neoplastic changes, relying on the recognition of foreign antigens and neoantigen peptides by T cell receptors (TCRs). However, the statistical properties and function of the T cell pool in an individual, under normal physiological conditions, are poorly understood. In this study, we report a comprehensive, quantitative characterization of the T cell repertoire from over 1.9 million cells, yielding over 200,000 high quality paired αβ sequences in 5 healthy human subjects. The dataset was obtained by leveraging recent biotechnology developments in deep RNA sequencing of lymphocytes via single-cell barcoding in emulsion. We report non-random associations and non-monogamous pairing between the α and β chains, lowering the theoretical diversity of the T cell repertoire, and increasing the frequency of public clones shared among individuals. T cell clone size distributions closely followed a power law, with markedly longer tails for CD8+ cytotoxic T cells than CD4+ helper T cells. Furthermore, clonality estimates based on paired chains from single T cells were lower than that from single chain data. Taken together, these results highlight the importance of sequencing αβ pairs to accurately quantify lymphocyte receptor diversity.
- Published
- 2017
- Full Text
- View/download PDF
24. Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers
- Author
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Sarah P. Preheim, Adrian W. Briggs, Ilana L. Brito, Francois Vigneault, Eric J. Alm, Sarah J. Spencer, Leena K. Pitkänen, Marko Virta, Mira Guo, Manu Tamminen, David A. Weitz, Massachusetts Institute of Technology. Computational and Systems Biology Program, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Civil and Environmental Engineering, Spencer, Sarah J., Tamminen, Manu V., Preheim, Sarah P., and Brito, Ilana Lauren
- Subjects
DNA, Bacterial ,0301 basic medicine ,Molecular Sequence Data ,Computational biology ,Biology ,Microbiology ,03 medical and health sciences ,Bacterial Proteins ,Microbial ecology ,Phylogenetics ,RNA, Ribosomal, 16S ,Genomic library ,Phylogeny ,Ecology, Evolution, Behavior and Systematics ,Genetic diversity ,Massive parallel sequencing ,Bacteria ,Phylogenetic tree ,Ecology ,High-Throughput Nucleotide Sequencing ,Lakes ,030104 developmental biology ,Metagenomics ,Horizontal gene transfer ,Original Article - Abstract
Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer netw, Academy of Finland (Project 257612), National Institute of Environmental Health Sciences (Award P30-ES002109)
- Published
- 2015
25. 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
26. Titan
- Author
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François Vigneault and François Vigneault
- Abstract
Pour l'ADMN João da Silva, il ne s'agit pas d'une simple inspection de routine : la colonie minière de Homestead n'est plus rentable et les ouvriers, des géants que l'on nomme Titans, sont à couteaux tirés avec l'administration. Avec l'aide de Phoebe Mackintosh, représentante syndicale aux allégeances incertaines, João doit découvrir ce qui se trame réellement… avant que la situation dégénère et se transforme en conflit interplanétaire.
- Published
- 2017
27. 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
- View/download PDF
28. Integrating B Cell Lineage Information into Statistical Tests for Detecting Selection in Ig Sequences
- Author
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Mark J. Shlomchik, Francois Vigneault, Mohamed Uduman, George M. Church, and Steven H. Kleinstein
- Subjects
Silent mutation ,Affinity maturation ,Genetics ,Tree structure ,Immunology ,Immunology and Allergy ,Antibody Diversity ,Computational biology ,Biology ,Deep sequencing ,B-Lymphocyte Subsets ,Shape analysis (digital geometry) ,Statistical hypothesis testing - Abstract
Detecting selection in B cell Ig sequences is critical to understanding affinity maturation and can provide insights into Ag-driven selection in normal and pathologic immune responses. The most common sequence-based methods for detecting selection analyze the ratio of replacement and silent mutations using a binomial statistical analysis. However, these approaches have been criticized for low sensitivity. An alternative method is based on the analysis of lineage trees constructed from sets of clonally related Ig sequences. Several tree shape measures have been proposed as indicators of selection that can be statistically compared across cohorts. However, we show that tree shape analysis is confounded by underlying experimental factors that are difficult to control for in practice, including the sequencing depth and number of generations in each clone. Thus, although lineage tree shapes may reflect selection, their analysis alone is an unreliable measure of in vivo selection. To usefully capture the information provided by lineage trees, we propose a new method that applies the binomial statistical framework to mutations identified based on lineage tree structure. This hybrid method is able to detect selection with increased sensitivity in both simulated and experimental data sets. We anticipate that this approach will be especially useful in the analysis of large-scale Ig sequencing data sets generated by high-throughput sequencing technologies.
- Published
- 2014
29. Dysregulation of B Cell Repertoire Formation in Myasthenia Gravis Patients Revealed through Deep Sequencing
- Author
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Francois Vigneault, Tamara J. Gilbert, Kevin C. O’Connor, Teresa J. Broering, Richard Nowak, Steven H. Kleinstein, Mazen M. Dimachkie, Emma Ciafaloni, Panos Stathopoulos, Jason A. Vander Heiden, Luan Chen, Julian Q. Zhou, Richard J. Barohn, and Christopher R. Bolen
- Subjects
0301 basic medicine ,Adult ,Male ,Adolescent ,Somatic cell ,Immunology ,Receptors, Antigen, B-Cell ,Biology ,Deep sequencing ,Article ,Immune tolerance ,03 medical and health sciences ,Young Adult ,0302 clinical medicine ,Myasthenia Gravis ,medicine ,Immune Tolerance ,Immunology and Allergy ,Humans ,Receptors, Cholinergic ,Memory B cell ,B cell ,Autoantibodies ,Genetics ,B-Lymphocytes ,Repertoire ,Autoantibody ,Receptor editing ,High-Throughput Nucleotide Sequencing ,Receptor Protein-Tyrosine Kinases ,Middle Aged ,030104 developmental biology ,medicine.anatomical_structure ,Female ,Immunologic Memory ,Protein Kinases ,030217 neurology & neurosurgery - Abstract
Myasthenia gravis (MG) is a prototypical B cell-mediated autoimmune disease affecting 20–50 people per 100,000. The majority of patients fall into two clinically distinguishable types based on whether they produce autoantibodies targeting the acetylcholine receptor (AChR-MG) or muscle specific kinase (MuSK-MG). The autoantibodies are pathogenic, but whether their generation is associated with broader defects in the B cell repertoire is unknown. To address this question, we performed deep sequencing of the BCR repertoire of AChR-MG, MuSK-MG, and healthy subjects to generate ∼518,000 unique VH and VL sequences from sorted naive and memory B cell populations. AChR-MG and MuSK-MG subjects displayed distinct gene segment usage biases in both VH and VL sequences within the naive and memory compartments. The memory compartment of AChR-MG was further characterized by reduced positive selection of somatic mutations in the VH CDR and altered VH CDR3 physicochemical properties. The VL repertoire of MuSK-MG was specifically characterized by reduced V-J segment distance in recombined sequences, suggesting diminished VL receptor editing during B cell development. Our results identify large-scale abnormalities in both the naive and memory B cell repertoires. Particular abnormalities were unique to either AChR-MG or MuSK-MG, indicating that the repertoires reflect the distinct properties of the subtypes. These repertoire abnormalities are consistent with previously observed defects in B cell tolerance checkpoints in MG, thereby offering additional insight regarding the impact of tolerance defects on peripheral autoimmune repertoires. These collective findings point toward a deformed B cell repertoire as a fundamental component of MG.
- Published
- 2016
30. Systematic identification of edited microRNAs in the human brain
- Author
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Eyal Mor, Francois Vigneault, Shahar Alon, Noam Shomron, Federica Galeano, Eli Eisenberg, George M. Church, Franco Locatelli, Angela Gallo, Ragon Institute of MGH, MIT and Harvard, and Vigneault, Francois
- Subjects
Adenosine ,Adenosine Deaminase ,Method ,RNA-binding protein ,Biology ,Genome ,Databases, Genetic ,microRNA ,Genetics ,Humans ,Genetics (clinical) ,Regulation of gene expression ,Genome, Human ,Sequence Analysis, RNA ,Brain ,Computational Biology ,RNA-Binding Proteins ,Inosine ,MicroRNAs ,RNA silencing ,RNA editing ,ADAR ,Human genome ,RNA Editing ,Algorithms - Abstract
Adenosine-to-inosine (A-to-I) editing modifies RNA transcripts from their genomic blueprint. A prerequisite for this process is a double-stranded RNA (dsRNA) structure. Such dsRNAs are formed as part of the microRNA (miRNA) maturation process, and it is therefore expected that miRNAs are affected by A-to-I editing. Editing of miRNAs has the potential to add another layer of complexity to gene regulation pathways, especially if editing occurs within the miRNA–mRNA recognition site. Thus, it is of interest to study the extent of this phenomenon. Current reports in the literature disagree on its extent; while some reports claim that it may be widespread, others deem the reported events as rare. Utilizing a next-generation sequencing (NGS) approach supplemented by an extensive bioinformatic analysis, we were able to systematically identify A-to-I editing events in mature miRNAs derived from human brain tissues. Our algorithm successfully identified many of the known editing sites in mature miRNAs and revealed 17 novel human sites, 12 of which are in the recognition sites of the miRNAs. We confirmed most of the editing events using in vitro ADAR overexpression assays. The editing efficiency of most sites identified is very low. Similar results are obtained for publicly available data sets of mouse brain-regions tissues. Thus, we find that A-to-I editing does alter several miRNAs, but it is not widespread.
- Published
- 2012
31. Barcoding bias in high-throughput multiplex sequencing of miRNA
- Author
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Francois Vigneault, Jonathan G. Seidman, Shahar Alon, Eli Eisenberg, George M. Church, Seda Eminaga, Danos C. Christodoulou, Ragon Institute of MGH, MIT and Harvard, and Vigneault, Francois
- Subjects
Genetics ,Sequence Analysis, RNA ,Sequence analysis ,Gene Expression Profiling ,Method ,High-Throughput Nucleotide Sequencing ,RNA-Seq ,Biology ,Barcode ,DNA sequencing ,law.invention ,Sequence-tagged site ,Gene expression profiling ,Mice ,MicroRNAs ,Bias ,law ,Animals ,Cluster Analysis ,Humans ,Multiplex ,Genetics (clinical) ,Polymerase chain reaction ,Sequence Tagged Sites - Abstract
Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number., National Human Genome Research Institute (U.S.) (Center for Excellence in Genome Sciences grant), Canadian Institutes of Health Research, National Heart, Lung, and Blood Institute, National Institutes of Health (U.S.) (SysCODE Consortium), United States-Israel Binational Science Foundation (grant no. 2009290), Ragon Institute of MGH, MIT and Harvard (Fellowship), Leducq Foundation
- Published
- 2011
32. Suppression of α5 gene expression is closely related to the tumorigenic properties of uveal melanoma cell lines
- Author
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Steeve Leclerc, Marjorie-Allison Bergeron, Sylvain L. Guérin, Manon Gaudreault, Francois Vigneault, Christian Salesse, Mohib W. Morcos, Solange Landreville, and Frédéric Mouriaux
- Subjects
Nuclear factor I ,biology ,Melanoma ,Repressor ,Dermatology ,medicine.disease ,General Biochemistry, Genetics and Molecular Biology ,Fibronectin ,Oncology ,Cell culture ,Gene expression ,Cancer cell ,biology.protein ,Cancer research ,medicine ,Transcription factor - Abstract
Summary Cancer aggressiveness is related to the ability of cancer cells to escape the anchorage dependency toward the extracellular matrix, a process regulated by the integrin α5β1 and its ligand fibronectin. Here, we characterized the expression of the α5 gene in human uveal melanoma cell lines with distinct tumorigenic properties and investigated some of the mechanisms underlying the variations of their malignancy. Strong and weak expression of α5 was observed in cells with no (T108/T115) and high (T97/T98) tumorigenic properties, respectively. Expression and DNA binding of the transcription factors Sp1, activator protein 1 (AP-1) (both acting as activators), and nuclear factor I (NFI) (a strong repressor) to the α5 promoter were demonstrated in all cell lines. A reduced expression of AP-1 combined with a dramatic increase in NFI correlated with the suppression of α5 expression in T97 and T98 cells. Restoring α5 expression in T97 cells entirely abolished their tumorigenicity in immunodeficient mice. These uveal melanoma cell lines might therefore prove particularly useful as cellular models to investigate α5β1 function in the pathogenesis of invasive uveal melanoma.
- Published
- 2011
33. Transcriptional Profiling of CD4 T Cells Identifies Distinct Subgroups of HIV-1 Elite Controllers
- Author
-
Mathias Lichterfeld, Eric S. Rosenberg, Florencia Pereyra, Jennifer Rychert, Francois Vigneault, Matthew Woods, Seth D. Crosby, Amalio Telenti, Javier Martinez-Picado, Chun Li, Xu G. Yu, George M. Church, and Maria J. Buzon
- Subjects
CD4-Positive T-Lymphocytes ,T cell ,Immunology ,HIV Infections ,Human leukocyte antigen ,CD8-Positive T-Lymphocytes ,Microbiology ,HIV Long-Term Survivors ,03 medical and health sciences ,0302 clinical medicine ,Virology ,Gene expression ,medicine ,Humans ,Allele ,030304 developmental biology ,Genetics ,0303 health sciences ,biology ,Gene Expression Profiling ,Viral Load ,biology.organism_classification ,CD4 Lymphocyte Count ,3. Good health ,Gene expression profiling ,medicine.anatomical_structure ,Insect Science ,Lentivirus ,HIV-1 ,Pathogenesis and Immunity ,Viral load ,CD8 ,030215 immunology - Abstract
Human immunodeficiency virus type 1 (HIV-1) elite controllers maintain undetectable levels of viral replication in the absence of antiretroviral therapy (ART), but their underlying immunological and virological characteristics may vary. Here, we used a whole-genome transcriptional profiling approach to characterize gene expression signatures of CD4 T cells from an unselected cohort of elite controllers. The transcriptional profiles for the majority of elite controllers were similar to those of ART-treated patients but different from those of HIV-1-negative persons. Yet, a smaller proportion of elite controllers showed an alternative gene expression pattern that was indistinguishable from that of HIV-1-negative persons but different from that of highly active antiretroviral therapy (HAART)-treated individuals. Elite controllers with the latter gene expression signature had significantly higher CD4 T cell counts and lower levels of HIV-1-specific CD8 + T cell responses but did not significantly differ from other elite controllers in terms of HLA class I alleles, HIV-1 viral loads determined by ultrasensitive single-copy PCR assays, or chemokine receptor polymorphisms. Thus, these data identify a specific subgroup of elite controllers whose immunological and gene expression characteristics approximate those of HIV-1-negative persons.
- Published
- 2011
34. Abstract 1541: Directed, unbiased mapping of lentiviral integrations with next generation sequencing in CAR-T cells
- Author
-
David Koppstein, Raj Chari, Adrian W. Briggs, Francois Vigneault, Timothy G. Johnstone, Rafael Ponce, and Ronald J. Hause
- Subjects
Cancer Research ,biology ,Computational biology ,biology.organism_classification ,Chimeric antigen receptor ,DNA sequencing ,Chromatin ,law.invention ,Viral vector ,Oncology ,law ,Lentivirus ,Vector (molecular biology) ,GC-content ,Polymerase chain reaction - Abstract
During manufacturing of an exemplary CD19-directed CAR-T cell product, patient T cells were transduced with a viral vector encoding the CD19-directed, 41BBζ endodomain-containing chimeric antigen receptor (CAR) construct, enabling permanent genetic modification of the target T cells. Lentiviral transduction with a self-inactivating (SIN) vector can offer certain advantages in safety and consistency over other DNA delivery methods. Although the integration patterns of wildtype HIV-1 have been well-characterized and shown to have a low risk of insertional transformation, the addition of an artificial construct may have the potential to affect integration patterns. Existing techniques for viral integration mapping, such as ligation-mediated polymerase chain reaction (LM-PCR), have limitations precluding comprehensive, unbiased, high-throughput insertion site mapping. To characterize insertional events in engineered CAR-T cells, we developed an unbiased NGS assay and computational pipeline capable of characterizing viral integration events genome-wide. The method leverages Tn5 transposase, coupled with a long terminal repeat-specific PCR, to map integration events down to nulceotide resolution. This assay identified over 150,000 high-confidence integration events across 23 patient-derived CAR-T cell products. Analysis of these sites revealed a similar integration preference to lentiviruses described in the literature: driven by GC content, local nucleotide content, chromatin status, and transcriptional activity, with no indication of preferential integration at oncogenic loci. In conclusion, in this assay a CD19-directed, CAR-containing lentivirus exhibited a low risk integration profile with no significant evidence of construct-specific risk factors. Furthermore, the findings suggest that the NGS assay may be useful for more efficient, cost-effective, and sensitive measurement of CAR-T integration events for a range of constructs. Citation Format: Timothy G. Johnstone, Rajagopal Chari, David Koppstein, Ronald J. Hause, Rafael Ponce, Adrian W. Briggs, Francois Vigneault. Directed, unbiased mapping of lentiviral integrations with next generation sequencing in CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1541.
- Published
- 2018
35. Reprogramming of T Cells from Human Peripheral Blood
- Author
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In-Hyun Park, Yuin-Han Loh, Chunguang Guo, Suneet Agarwal, Francois Vigneault, James J. Collins, George Q. Daley, Stefan Irion, George M. Church, Hu Li, Garrett C. Heffner, Odelya Hartung, Philip D. Manos, Achia Urbach, Marica Grskovic, M. William Lensch, and Julie M. Sahalie
- Subjects
medicine.diagnostic_test ,T lymphocyte ,Cell Biology ,Biology ,STEMCELL ,Molecular biology ,Article ,Flow cytometry ,Antigen ,Cell culture ,Immunology ,medicine ,Genetics ,Immunohistochemistry ,Molecular Medicine ,Stem cell ,Induced pluripotent stem cell ,Reprogramming - Abstract
A manuscript has appeared online demonstrating isolation of iPSCs from peripheral blood, including a single line that showed evidence for both TCR-β and TCR-γ rearrangement by PCR (Kunisato, A., Wakatsuki, M., Shinba, H., Ota, T., Ishida, I., and Nagao, K. [2010]. Direct generation of induced pluripotent stem cells from human non-mobilized blood. Stem Cells Dev., in press. Published online May 24, 2010. 10.1089/scd.2010.0063).
- Published
- 2010
- Full Text
- View/download PDF
36. In Cellulo DNA Analysis: LMPCR Footprinting
- Author
-
Jean-François Millau, Francois Vigneault, Nathalie Bastien, Isabelle Paradis, and Régen Drouin
- Subjects
chemistry.chemical_compound ,biology ,chemistry ,DNA polymerase ,biology.protein ,DNA footprinting ,Protein–DNA interaction ,Computational biology ,DNase footprinting assay ,DNA ,DNA sequencing ,Footprinting ,Chromatin - Abstract
The in cellulo analysis of protein-DNA interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene expression. The nuclease-hypersensitive sites and sequences bound by transcription factors often correspond to genetic regulatory elements. Using the ligation-mediated polymerase chain reaction (LMPCR) technology, it is possible to precisely analyze these DNA sequences to demonstrate the existence of DNA-protein interactions or unusual DNA structures directly in living cells. Indeed, the ideal chromatin substrate is, of course, found inside intact cells. LMPCR, a genomic sequencing technique that map DNA single-strand breaks at the sequence level of resolution, is the method of choice for in cellulo footprinting and DNA structure studies because it can be used to investigate complex animal genomes, including human. The detailed conventional and automated LMPCR protocols are presented in this chapter.
- Published
- 2015
37. Regulation of gene expression: probing DNA–protein interactionsin vivoandin vitro
- Author
-
Sylvain L. Guérin and Francois Vigneault
- Subjects
Regulation of gene expression ,Genetics ,Therapeutic gene modulation ,DNA ,Computational biology ,Regulatory Sequences, Nucleic Acid ,Biology ,Biochemistry ,ChIP-sequencing ,DNA-Binding Proteins ,SOX4 ,DDB1 ,Gene Expression Regulation ,Genes, Reporter ,GATAD2B ,Trans-Activators ,Transcriptional regulation ,Humans ,E2F1 ,Molecular Biology - Abstract
Tremendous efforts have been put together over the last several years to complete the entire sequencing of the human genome. As we enter the proteomic era, when the major aim is understanding which gene encodes which protein, the time has also come to identify their precise function inside the astonishing signaling network required to accomplish all cellular functions. Understanding when, why and how a gene is expressed has now become a necessity toward identifying all the regulatory pathways that mediate cellular processes such as differentiation, migration, replication, DNA repair and apoptosis. Regulation of gene transcription is a process that is primarily under the influence of nuclear-located transcription factors. Consequently, identifying which protein activates or represses a specific gene is a prerequisite for understanding cell fate and function. The current state of, and recent advances in, transcriptional regulation approaches are reviewed here, with special emphasis on new technologies required when probing for DNA-protein interactions. This review explores different strategies aimed at identifying both the regulatory sequences of any given gene and the trans-acting regulatory factors that recognize these elements as their target sites in the nucleus. Ongoing developments in the fields of nanotechnology, RNA silencing and protein modeling toward the investigation of DNA-protein interactions and their relevance in the battle against cancer are discussed.
- Published
- 2005
38. 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
39. High-resolution antibody dynamics of vaccine-induced immune responses
- Author
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Daphne Koller, Erez Lieberman-Aiden, George Georgiou, George M. Church, Jason A. Vander Heiden, Birgitte B. Simen, Steven H. Kleinstein, Mohamed Uduman, Brendan Hickey, Uri Laserson, Michael Egholm, Jonathan Laserson, Francois Vigneault, Bozena Hanczaruk, Raj Chari, Ido Bachelet, Yi Liu, Je-Hyuk Lee, Sang Taek Jung, Gur Yaari, Daniel Gadala-Maria, and William Kelton
- Subjects
Male ,Phage display ,Time Factors ,Genetic Vectors ,Immunoglobulin Variable Region ,Biology ,Antibodies ,Immune system ,Antibody Repertoire ,Humans ,Genetics ,Multidisciplinary ,Repertoire ,Viral Vaccine ,V(D)J recombination ,Vaccination ,Immunity ,Reproducibility of Results ,Viral Vaccines ,Biological Sciences ,Acquired immune system ,Healthy Volunteers ,V(D)J Recombination ,Clone Cells ,Mutation ,biology.protein ,Antibody - Abstract
The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from 38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge.
- Published
- 2014
40. B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes
- Author
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David A. Hafler, Alyssa Nylander, Steven H. Kleinstein, Francois Vigneault, David Pitt, Jon D. Laman, Bilal A. Siddiqui, Rogier Q. Hintzen, Kevin C. O’Connor, Gur Yaari, Anita Huttner, William Donahue, Joel N. H. Stern, Rashed M. Nagra, Jason A. Vander Heiden, George M. Church, Sriram Ramanan, Neurology, and Immunology
- Subjects
Pathology ,medicine.medical_specialty ,Multiple sclerosis ,Cellular differentiation ,Lymphocyte ,General Medicine ,Biology ,medicine.disease ,Affinity maturation ,Lymphatic system ,medicine.anatomical_structure ,Antigen ,Immunology ,medicine ,biology.protein ,Antibody ,B cell - Abstract
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune-mediated demyelination and neurodegeneration. The CNS of patients with MS harbors expanded clones of antigen-experienced B cells that reside in distinct compartments including the meninges, cerebrospinal fluid (CSF), and parenchyma. It is not understood whether this immune infiltrate initiates its development in the CNS or in peripheral tissues. B cells in the CSF can exchange with those in peripheral blood, implying that CNS B cells may have access to lymphoid tissue that may be the specific compartment(s) in which CNS-resident B cells encounter antigen and experience affinity maturation. Paired tissues were used to determine whether the B cells that populate the CNS mature in the draining cervical lymph nodes (CLNs). High-throughput sequencing of the antibody repertoire demonstrated that clonally expanded B cells were present in both compartments. Founding members of clones were more often found in the draining CLNs. More mature clonal members derived from these founders were observed in the draining CLNs and also in the CNS, including lesions. These data provide new evidence that B cells traffic freely across the tissue barrier, with the majority of B cell maturation occurring outside of the CNS in the secondary lymphoid tissue. Our study may aid in further defining the mechanisms of immunomodulatory therapies that either deplete circulating B cells or affect the intrathecal B cell compartment by inhibiting lymphocyte transmigration into the CNS.
- Published
- 2014
41. Correction: The Effects of Somatic Hypermutation on Neutralization and Binding in the PGT121 Family of Broadly Neutralizing HIV Antibodies
- Author
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George M. Church, Laura M. Walker, Elizabeth P. St. John, Dennis R. Burton, Kristine M. Swiderek, Bryan Briney, Gur Yaari, Devin Sok, Uri Laserson, Jean-Philippe Julien, Arup K. Chakraborty, Alejandra Ramos, Pascal Poignard, Michael S. Seaman, Khoa Le, Alison E. Mahan, Jonathan Laserson, Mehran Kardar, Ian A. Wilson, Karen F. Saye, Shenshen Wang, Stephen H. Kleinstein, Daphne Koller, Yi Liu, Birgitte B. Simen, Po-Ying Chan-Hui, Galit Alter, Francois Vigneault, Institute for Medical Engineering and Science, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Chemical Engineering, Massachusetts Institute of Technology. Department of Mathematics, Massachusetts Institute of Technology. Department of Physics, Laserson, Uri, Wang, Shenshen, Kardar, Mehran, Chakraborty, Arup K., and Church, George M.
- Subjects
Male ,lcsh:Immunologic diseases. Allergy ,Glycan ,Immunogen ,QH301-705.5 ,Immunology ,Somatic hypermutation ,HIV Antibodies ,HIV Envelope Protein gp120 ,medicine.disease_cause ,Microbiology ,Epitope ,Neutralization ,Affinity maturation ,03 medical and health sciences ,0302 clinical medicine ,Polysaccharides ,Virology ,medicine ,Genetics ,Humans ,Biology (General) ,lcsh:QH301-705.5 ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Mutation ,biology ,Correction ,RC581-607 ,Antibodies, Neutralizing ,3. Good health ,lcsh:Biology (General) ,HIV-1 ,biology.protein ,Female ,Parasitology ,Immunologic diseases. Allergy ,Antibody ,lcsh:RC581-607 ,Research Article ,030215 immunology - Abstract
Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121–134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121–134 but were still capable of neutralizing roughly 40–80% of PGT121–134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121–134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121–134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design., National Institutes of Health (U.S.) (Bioinformatics and Integrative Genomics Fellowship), National Institutes of Health (U.S.) (Center for Excellence in Genomic Science)
- Published
- 2013
42. Models of Somatic Hypermutation Targeting and Substitution Based on Synonymous Mutations from High-Throughput Immunoglobulin Sequencing Data
- Author
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Francois Vigneault, Joel N. H. Stern, Namita Gupta, David A. Hafler, Mohamed Uduman, Steven H. Kleinstein, Jason A. Vander Heiden, Gur Yaari, Daniel Gadala-Maria, Kevin C. O’Connor, and Uri Laserson
- Subjects
lcsh:Immunologic diseases. Allergy ,Sequencing data ,Immunology ,Somatic hypermutation ,Affinity maturation ,03 medical and health sciences ,0302 clinical medicine ,substitution ,medicine ,AID ,Immunology and Allergy ,Throughput (business) ,B cell ,targeting ,030304 developmental biology ,Original Research ,Genetics ,affinity maturation ,0303 health sciences ,biology ,business.industry ,Substitution (logic) ,mutability ,somatic hypermutation ,medicine.anatomical_structure ,biology.protein ,ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,Antibody ,business ,Synonymous substitution ,lcsh:RC581-607 ,immunoglobulin ,030215 immunology - Abstract
Analyses of somatic hypermutation (SHM) patterns in B cell immunoglobulin (Ig) sequences contribute to our basic understanding of adaptive immunity, and have broad applications not only for understanding the immune response to pathogens, but also to determining the role of SHM in autoimmunity and B cell cancers. Although stochastic, SHM displays intrinsic biases that can confound statistical analysis, especially when combined with the particular codon usage and base composition in Ig sequences. Analysis of B cell clonal expansion, diversification, and selection processes thus critically depends on an accurate background model for SHM micro-sequence targeting (i.e., hot/cold-spots) and nucleotide substitution. Existing models are based on small numbers of sequences/mutations, in part because they depend on data from non-coding regions or non-functional sequences to remove the confounding influences of selection. Here, we combine high-throughput Ig sequencing with new computational analysis methods to produce improved models of SHM targeting and substitution that are based only on synonymous mutations, and are thus independent of selection. The resulting "S5F" models are based on 806,860 Synonymous mutations in 5-mer motifs from 1,145,182 Functional sequences and account for dependencies on the adjacent four nucleotides (two bases upstream and downstream of the mutation). The estimated profiles can explain almost half of the variance in observed mutation patterns, and clearly show that both mutation targeting and substitution are significantly influenced by neighboring bases. While mutability and substitution profiles were highly conserved across individuals, the variability across motifs was found to be much larger than previously estimated. The model and method source code are made available at http://clip.med.yale.edu/SHM.
- Published
- 2013
43. 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
44. 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
45. Altered Expression of the Poly(ADP-Ribosyl)ation Enzymes in Uveal Melanoma and Regulation of PARG Gene Expression by the Transcription Factor ERM
- Author
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Natasha Dargis, Yvonne Molgat, Mohib W. Morcos, Dan Bergeron, Jean-François St-Laurent, Vanessa Molloy-Simard, Francois Vigneault, Serge Desnoyers, Sylvain L. Guérin, Steeve Leclerc, Daniel Ovid Black, Marie-Christine Guérin, François Boudreau, Yvan de Launoit, Manon Gaudreault, Mécanismes de tumorigenèse et thérapies ciblées, Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Hôpital du Saint-Sacrement [CHU Québec] (HSS), CHU de Québec–Université Laval, Université Laval [Québec] (ULaval)-Université Laval [Québec] (ULaval), Harvard Medical School [Boston] (HMS), Université Laval [Québec] (ULaval), Réseau International des Instituts Pasteur (RIIP), Université de Sherbrooke (UdeS), and Supported by a grant from the Canadian Institutes of Health Research (CIHR) to SD and SG and a studentship from the Réseau de Recherche en Santé de la Vision from the Fonds de la Recherche en Santé du Québec (FRSQ) to VM-S.
- Subjects
Uveal Neoplasms ,Blotting, Western ,Poly (ADP-Ribose) Polymerase-1 ,Mice, Nude ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,Biology ,Polymerase Chain Reaction ,Mice ,03 medical and health sciences ,0302 clinical medicine ,RNA interference ,Cell Line, Tumor ,Gene expression ,Animals ,Melanoma ,Transcription factor ,ComputingMilieux_MISCELLANEOUS ,Oligonucleotide Array Sequence Analysis ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,PARG ,Gene Expression Profiling ,GTPase-Activating Proteins ,Promoter ,Transfection ,Microarray Analysis ,Molecular biology ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Gene expression profiling ,030220 oncology & carcinogenesis ,Cancer research ,Poly(ADP-ribose) Polymerases ,Transcription Factors - Abstract
PURPOSE. Poly(ADP-ribosyl)ation is a reversible post-translational modification that requires the contribution of the enzymes poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Our study explores expression and activity of PARP-1 and PARG in uveal melanoma cell lines with varying tumorigenic properties. METHODS. Gene profiling on microarrays was conducted using RNA prepared from the uveal melanoma cell lines T97, T98, T108, and T115. The activity of PARP-1 and PARG was monitored by enzymatic assays, whereas their expression was measured by Western blot and PCR. The PARG promoter was analyzed using promoter deletions and site-specific mutagenesis in transfection analyses. The transcription factors binding the PARG promoter were studied by electrophoretic mobility shift assay (EMSA) analyses. Suppression of PARP-1 and PARG expression was performed in T97 and T115 cells by RNAi, and their tumorigenic properties monitored by injections into athymic mice. RESULTS. Expression of PARP-1 was found to vary considerably between uveal melanoma cell lines with distinctive tumorigenic properties in vivo. Sp1 and the ETS protein ERM were shown to bind to the PARG gene promoter to ensure basal transcription in uveal melanoma. Importantly, suppression of PARG gene expression in T97 and T115 cells increased their capacity to form tumors in athymic mice, whereas suppression of PARP-1 significantly reduced or almost entirely abolished tumor formation. CONCLUSIONS. Our results suggest that while overexpression of PARP-1 may confer a proliferative advantage to aggressive uveal melanoma tumors, PARG may, on the other hand, support a tumor suppressor function in vivo. (Invest Ophthalmol Vis Sci. 2012;53:6219‐6231) DOI:10.1167/iovs.11-8853
- Published
- 2012
46. High‐Throughput Multiplex Sequencing of miRNA
- Author
-
Shahar Alon, Francois Vigneault, George M. Church, Jonathan G. Seidman, Seda Eminaga, Danos C. Christodoulou, Dmitry Ter-Ovanesyan, and Eli Eisenberg
- Subjects
Cancer genome sequencing ,Genetics ,Massive parallel sequencing ,Sequence Analysis, RNA ,Shotgun sequencing ,High-Throughput Nucleotide Sequencing ,Computational biology ,Biology ,Article ,Deep sequencing ,Massively parallel signature sequencing ,MicroRNAs ,Single cell sequencing ,Humans ,Genetics (clinical) ,Illumina dye sequencing ,Exome sequencing ,Gene Library - Abstract
Next-generation sequencing offers many advantages over other methods of microRNA (miRNA) expression profiling, such as sample throughput and the capability to discover novel miRNAs. As the sequencing depth of current sequencing platforms exceeds what is necessary to quantify miRNAs, multiplexing several samples in one sequencing run offers a significant cost advantage. Although previous studies have achieved this goal by adding bar codes to miRNA libraries at the ligation step, this was recently shown to introduce significant bias into the miRNA expression data. This bias can be avoided, however, by bar coding the miRNA libraries at the PCR step instead. Here, we describe a user-friendly PCR bar-coding method of preparing multiplexed microRNA libraries for Illumina-based sequencing. The method also prevents the production of adapter dimers and can be completed in one day.
- Published
- 2012
47. Rescue of the transcription factors Sp1 and NFI in human skin keratinocytes through a feeder-layer-dependent suppression of the proteasome activity
- Author
-
Francois Vigneault, Sylvain L. Guérin, Manon Gaudreault, Bénédicte Masson-Gadais, Lydia Touzel-Deschênes, Lucie Germain, Patrick J. Rochette, and Céline Duval
- Subjects
Adult ,Keratinocytes ,Proteasome Endopeptidase Complex ,Sp1 Transcription Factor ,Human skin ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Feeder Layer ,Structural Biology ,microRNA ,Animals ,Humans ,Molecular Biology ,Transcription factor ,030304 developmental biology ,0303 health sciences ,Neurofibromin 1 ,integumentary system ,Nuclear factor I ,Cell growth ,Infant, Newborn ,Feeder Cells ,3T3 Cells ,3. Good health ,Cell biology ,Proteasome ,chemistry ,030220 oncology & carcinogenesis ,Immunology ,DNA - Abstract
Co-culturing human skin keratinocytes along with a feeder layer has proven to considerably improve their proliferative properties by delaying massive induction of terminal differentiation. Through a yet unclear mechanism, we recently reported that irradiated 3T3 (i3T3) fibroblasts used as a feeder layer increase the nuclear content of Sp1, a positive transcription factor (TF) that plays a critical role in many cellular functions including cell proliferation, into both adult skin keratinocytes and newborn skin keratinocytes. In this study, we examined the influence of i3T3 on the expression and DNA binding of NFI, another TF important for cell proliferation and cell cycle progression, and attempted to decipher the mechanism by which the feeder layer contributes at maintaining higher levels of these TFs in skin keratinocytes. Our results indicate that co-culturing both adult skin keratinocytes and newborn skin keratinocytes along with a feeder layer dramatically increases glycosylation of NFI and may prevent it from being degraded by the proteasome.
- Published
- 2011
48. Efficient microRNA capture and bar-coding via enzymatic oligonucleotide adenylation
- Author
-
A. Michael Sismour, George M. Church, and Francois Vigneault
- Subjects
Genetics ,chemistry.chemical_classification ,Electronic Data Processing ,DNA Ligases ,Oligonucleotide ,Extramural ,Adenine ,Oligonucleotides ,RNA Ligase (ATP) ,Cell Biology ,Computational biology ,Biology ,Biochemistry ,MicroRNAs ,Enzyme ,chemistry ,microRNA ,Humans ,Multiplex ,Molecular Biology ,Adenylylation ,Biotechnology - Abstract
Here we report a highly efficient and simplified strategy to preadenylate bar-coded oligonucleotides designed for microRNA (miRNA) capture and multiplex analysis. Using this approach, we enzymatically preadenylated bar-coded oligonucleotides with high efficiency when compared to the chemical method currently used by miRNA investigators. As a case study, we used these oligonucleotides in an ATP-independent ligation to miRNAs, suggesting the utility of our method in end-capture protocols and high-throughput sequencing applications.
- Published
- 2009
49. Abstract 643: Identification of novel pancreatic cancer-specific antibodies and their target antigens through a next generation immune sequencing platform
- Author
-
Adrian W. Briggs, Brian J. Belmont, Sonia Timberlake, Francois Vigneault, Teresa J. Broering, David Fabrizio, and Stephen J. Goldfless
- Subjects
Cancer Research ,Tumor-infiltrating lymphocytes ,Cancer ,Biology ,medicine.disease ,Molecular biology ,Cell killing ,Oncology ,Antibody Repertoire ,Antigen ,Pancreatic cancer ,Cancer cell ,biology.protein ,medicine ,Antibody - Abstract
One of the major challenges in developing new therapies for pancreatic cancer is the lack of known specific and sensitive targets to the cancer cells. In this study, we present a powerful way to identify novel specific targets to pancreatic cancer by leveraging the antibody responses of tumor infiltrating lymphocytes (TILs) using a novel next-generation immune sequencing approach. We first deeply characterize the antibody repertoire produced by TILs, followed by antibody expression, screening and functional characterization to identify their biological targets. We aim to manipulate the antibody cancer targeting specificity into potential therapeutics by utilizing antibody-drug conjugation (ADC) technology. We obtained tumor resections and matched normal adjacent tissue from patients diagnosed with pancreatic ductal adenocarcinoma or acinar cell carcinoma, and characterized each sample's TIL immune repertoire using AbVitro's proprietary Next-Generation Immune Sequencing platform. Interestingly, results indicated the presence of millions of B and T cells in both tumor and normal tissues. Strikingly, tumor tissues across all patients were characterized by an abundance of expanded B-cell lineages expressing IgG, whereas normal adjacent tissue and healthy control samples contained almost exclusively IgA expressing cells. Antibody candidates were selected from the sequencing data and heavy and light chains were synthesized and expressed in a mammalian cell-based expression system. The antibodies were then screened for cell surface binding, tissue specificity and cell killing potential against pancreatic cancer cell lines and FFPE human cancer tissues. Selected antibodies from these screens were then chosen as candidates for antigen identification. Our study uncovered multiple antibodies that specifically bind to pancreatic cancer tissues but not to healthy tissues. Interestingly, some of the antibodies also showed strong binding to other types of cancer such as lung squamous cell carcinoma. Initial target identification efforts for these antibodies yielded a short list of antigens which are known to be expressed at high levels in the pancreas or pancreatic cancer cell lines. We are currently performing studies to further understand the antigen specificity and sensitivity of these antibodies and evaluate their cell killing potential as ADCs, as well as investigating the diagnostic value of the novel antigen targets. In summary, our study demonstrates the potential of next-generation sequencing in TIL analysis for the discovery of novel cancer-specific targets of potential therapeutic value. Citation Format: David A. Fabrizio, Sonia Timberlake, Brian Belmont, Stephen J. Goldfless, Adrian W. Briggs, Teresa J. Broering, Francois Vigneault. Identification of novel pancreatic cancer-specific antibodies and their target antigens through a next generation immune sequencing platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 643. doi:10.1158/1538-7445.AM2015-643
- Published
- 2015
50. A model of somatic hypermutation targeting in mice based on high-throughput immunoglobulin sequencing data (TECH2P.910)
- Author
-
Ang Cui, Roberto Diniro, Adrian Briggs, Kris Adams, Jason Vander Heiden, Kevin O'Connor, Francois Vigneault, Mark Shlomchik, and Steven Kleinstein
- Subjects
Immunology ,Immunology and Allergy - Abstract
Analyses of somatic hypermutation (SHM) patterns in B-cell immunoglobulin (Ig) genes have important basic science and clinical applications, but are often confounded by the intrinsic biases of SHM targeting on DNA motifs (i.e., hot/cold spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized heavy-chain transgenic mice with nitrophenyl (NP) to generate NP-specific λ+ B cells, and used next-generation sequencing to examine the unproductive κ light chains. Most of these sequences had out-of-frame junctions and were presumed uninfluenced by selection. Despite being non-functionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We identified 29,005 mutations and used them to construct a quantitative SHM targeting model, which was consistent with classic hot/cold-spots, but revealed additional highly mutable motifs. We observed comparable targeting for functional and non-functional sequences, suggesting similar biological processes operate at both loci. In contrast, the level of C to T transitions in mice was higher than in human κ chains, suggesting lower levels of DNA repair activities. In summary, our model of SHM targeting for mice provides insights into the SHM process and supports future analyses of mutation patterns.
- Published
- 2015
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