scholarly journals Synthetic standards combined with error and bias correction improves the accuracy and quantitative resolution of antibody repertoire sequencing in human naïve and memory B cells

2018 ◽  
Author(s):  
Simon Friedensohn ◽  
John M. Lindner ◽  
Vanessa Cornacchione ◽  
Mariavittoria Iazeolla ◽  
Enkelejda Miho ◽  
...  
Keyword(s):  
B Cell ◽  
Bias Correction ◽  
High Throughput Sequencing ◽  
Somatic Hypermutation ◽  
Sampling Strategy ◽  
Antibody Repertoire ◽  
Human Antibody ◽  
Library Preparation ◽  
Synthetic Antibody

ABSTRACTHigh-throughput sequencing of immunoglobulin repertoires (Ig-seq) is a powerful method for quantitatively interrogating B cell receptor sequence diversity. When applied to human repertoires, Ig-seq provides insight into fundamental immunological questions, and can be implemented in diagnostic and drug discovery projects. However, a major challenge in Ig-seq is ensuring accuracy, as library preparation protocols and sequencing platforms can introduce substantial errors and bias that compromise immunological interpretation. Here, we have established an approach for performing highly accurate human Ig-seq by combining synthetic standards with a comprehensive error and bias correction pipeline. First, we designed a set of 85 synthetic antibody heavy chain standards (in vitro transcribed RNA) to assess correction workflow fidelity. Next, we adapted a library preparation protocol that incorporates unique molecular identifiers (UIDs) for error and bias correction which, when applied to the synthetic standards, resulted in highly accurate data. Finally, we performed Ig-seq on purified human circulating B cell subsets (naïve and memory), combined with a cellular replicate sampling strategy. This strategy enabled robust and reliable estimation of key repertoire features such as clonotype diversity, germline segment and isotype subclass usage, and somatic hypermutation (SHM). We anticipate that our standards and error and bias correction pipeline will become a valuable tool for researchers to validate and improve accuracy in human Ig-seq studies, thus leading to potentially new insights and applications in human antibody repertoire profiling.

scholarly journals Antibody Repertoire Analysis of Tumor-Infiltrating B Cells Reveals Distinct Signatures and Distributions Across Tissues

2021 ◽  
Vol 12 ◽  
Author(s):  
Ligal Aizik ◽  
Yael Dror ◽  
David Taussig ◽  
Adi Barzel ◽  
Yaron Carmi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
High Throughput Sequencing ◽  
Triple Negative ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
Antibody Repertoire ◽  
Class Switch ◽  
Repertoire Analysis

The role of B cells in the tumor microenvironment (TME) has largely been under investigated, and data regarding the antibody repertoire encoded by B cells in the TME and the adjacent lymphoid organs are scarce. Here, we utilized B cell receptor high-throughput sequencing (BCR-Seq) to profile the antibody repertoire signature of tumor-infiltrating lymphocyte B cells (TIL−Bs) in comparison to B cells from three anatomic compartments in a mouse model of triple-negative breast cancer. We found that TIL-Bs exhibit distinct antibody repertoire measures, including high clonal polarization and elevated somatic hypermutation rates, suggesting a local antigen-driven B-cell response. Importantly, TIL-Bs were highly mutated but non-class switched, suggesting that class-switch recombination may be inhibited in the TME. Tracing the distribution of TIL-B clones across various compartments indicated that they migrate to and from the TME. The data thus suggests that antibody repertoire signatures can serve as indicators for identifying tumor-reactive B cells.

scholarly journals Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting

2016 ◽  
Vol 2 (3) ◽  
pp. e1501371 ◽  
Author(s):  
Tarik A. Khan ◽  
Simon Friedensohn ◽  
Arthur R. Gorter de Vries ◽  
Jakub Straszewski ◽  
Hans-Joachim Ruscheweyh ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Diversity Index ◽  
Pcr Amplification ◽  
Antibody Repertoire ◽  
Library Preparation ◽  
Antibody Diversity ◽  
Synthetic Antibody ◽  
Sequencing Errors ◽  
Repertoire Sequencing ◽  
Improved Accuracy

High-throughput antibody repertoire sequencing (Ig-seq) provides quantitative molecular information on humoral immunity. However, Ig-seq is compromised by biases and errors introduced during library preparation and sequencing. By using synthetic antibody spike-in genes, we determined that primer bias from multiplex polymerase chain reaction (PCR) library preparation resulted in antibody frequencies with only 42 to 62% accuracy. Additionally, Ig-seq errors resulted in antibody diversity measurements being overestimated by up to 5000-fold. To rectify this, we developed molecular amplification fingerprinting (MAF), which uses unique molecular identifier (UID) tagging before and during multiplex PCR amplification, which enabled tagging of transcripts while accounting for PCR efficiency. Combined with a bioinformatic pipeline, MAF bias correction led to measurements of antibody frequencies with up to 99% accuracy. We also used MAF to correct PCR and sequencing errors, resulting in enhanced accuracy of full-length antibody diversity measurements, achieving 98 to 100% error correction. Using murine MAF-corrected data, we established a quantitative metric of recent clonal expansion—the intraclonal diversity index—which measures the number of unique transcripts associated with an antibody clone. We used this intraclonal diversity index along with antibody frequencies and somatic hypermutation to build a logistic regression model for prediction of the immunological status of clones. The model was able to predict clonal status with high confidence but only when using MAF error and bias corrected Ig-seq data. Improved accuracy by MAF provides the potential to greatly advance Ig-seq and its utility in immunology and biotechnology.

scholarly journals Antibody repertoire analysis of tumor-infiltrating B cells reveals distinct signatures and distributions across tissues

2021 ◽  
Author(s):  
Ligal Aizik ◽  
Yael Dror ◽  
David Taussig ◽  
Adi Barzel ◽  
Yaron Carmi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
High Throughput Sequencing ◽  
Triple Negative ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
Antibody Repertoire ◽  
Class Switch ◽  
Repertoire Analysis

The role of B cells in the tumor microenvironment (TME) has largely been under-investigated, and data regarding the antibody repertoire encoded by B cells in the TME and the adjacent lymphoid organs are scarce. Here, we utilized B cell receptor high-throughput sequencing (BCR-Seq) to profile the antibody repertoire signature of tumor-infiltrating lymphocyte B cells (TIL Bs) in comparison to B cells from three anatomic compartments in a mouse model of triple-negative breast cancer. We found that TIL-Bs exhibit distinct antibody repertoire measures, including high clonal polarization and elevated somatic hypermutation rates, suggesting a local antigen-driven B-cell response. Importantly, TIL-Bs were highly mutated but non-class switched, suggesting that class-switch recombination may be inhibited in the TME. Tracing the distribution of TIL-B clones across various compartments indicated that they migrate to and from the TME. The data thus suggests that antibody repertoire signatures can serve as indicators for identifying tumor-reactive B cells.

scholarly journals Signatures of selection in the human antibody repertoire: Selective sweeps, competing subclones, and neutral drift

2019 ◽  
Vol 116 (4) ◽  
pp. 1261-1266 ◽  
Author(s):  
Felix Horns ◽  
Christopher Vollmers ◽  
Cornelia L. Dekker ◽  
Stephen R. Quake
Keyword(s):  
B Cell ◽  
Binding Affinity ◽  
Antibody Repertoire ◽  
Human Antibody ◽  
Selective Sweeps ◽  
Human Immune System ◽  
Evolutionary Processes ◽  
Neutral Drift ◽  
Cell Lineages ◽  
Antibody Repertoires

Antibodies are created and refined by somatic evolution in B cell populations, which endows the human immune system with the ability to recognize and eliminate diverse pathogens. However, the evolutionary processes that sculpt antibody repertoires remain poorly understood. Here, using an unbiased repertoire-scale approach, we show that the population genetic signatures of evolution are evident in human B cell lineages and reveal how antibodies evolve somatically. We measured the dynamics and genetic diversity of B cell responses in five adults longitudinally before and after influenza vaccination using high-throughput antibody repertoire sequencing. We identified vaccine-responsive B cell lineages that carry signatures of selective sweeps driven by positive selection, and discovered that they often display evidence for selective sweeps favoring multiple subclones. We also found persistent B cell lineages that exhibit stable population dynamics and carry signatures of neutral drift. By exploiting the relationship between B cell fitness and antibody binding affinity, we demonstrate the potential for using phylogenetic approaches to identify antibodies with high binding affinity. This quantitative characterization reveals that antibody repertoires are shaped by an unexpectedly broad spectrum of evolutionary processes and shows how signatures of evolutionary history can be harnessed for antibody discovery and engineering.

EBV Transformation of Tonsil Germinal Centre B Cells Carrying Functionally Inactivated IgV Gene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3248-3248
Author(s):  
Sridhar Chaganti ◽  
Noelia Begue Pastor ◽  
Mark T. Drayson ◽  
Andy I. Bell ◽  
Alan B. Rickinson
Keyword(s):  
B Cells ◽  
B Cell ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Affinity Maturation ◽  
Germinal Centre ◽  
Chain Gene ◽  
Lymphoid Tissues ◽  
Memory B Cell

Abstract Somatic hypermutation of immunoglobulin (Ig) gene sequences in the germinal centres of lymphoid tissues is necessary for affinity maturation of B cell responses to antigen challenge. This process generates a few clones with improved affinity that are selected into B cell memory and many clones with other non favourable Ig mutations, including some cells with functionally inactivated Ig gene that normally die by apoptosis. It is postulated that infection with Epstein-Barr virus (EBV), a B lymphotropic agent linked to several types of B cell lymphoma, can rescue germinal centre cells with unfavourable mutations. This creates a pool of infected cells at greater risk of developing into lymphomas. In the present work, CD38+ germinal centre B cells were separated from tonsil by negative selection for IgD and CD39. Peripheral blood naïve and memory B cell subpopulations were FACS sorted as IgD+, CD27− and IgD−, CD27+ fractions respectively. These cells were infected with EBV (B95.8 strain) in vitro and seeded at limiting dilutions onto fibroblast feeders. EBV transformed lymphoblastoid cell lines (LCLs) from such cultures were analysed for surface Ig phenotype. Naïve B cell transformants were consistently IgM+, IgD+. Memory B cell transformants were IgM+ in some cases but more frequently IgG+ or IgA+. Germinal centre transformants showed the same spectrum of surface Ig phenotypes as memory cell transformants but in addition we identified six germinal centre derived LCLs which were consistently surface Ig negative. Sequencing from these lines confirmed that in at least three cases EBV had rescued cells with functionally inactivated Ig heavy chain gene.

Pathologic Co-Expression and Physical Interaction of c-MYC and BCL6 in B-Cell Lymphomas.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2-2 ◽  
Author(s):  
Masumichi Saito ◽  
Ryan T. Phan ◽  
Herbert C. Morse ◽  
Laura Pasqualucci ◽  
Riccardo Dalla-Favera
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Transgenic Animals ◽  
Primary Lymphoma ◽  
Physical Interaction

Abstract Deregulated expression of the proto-oncogenes BCL6 and c-MYC caused by chromosomal translocation or somatic hypermutation is common in non-Hodgkin B cell lymphoma derived from germinal center (GC) B cells, including diffuse large cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Normal GC B cells express BCL6, whereas, surprisingly, they do not express c-MYC, suggesting that the expression of this oncogene in BL and DLBCL (20% of cases) is ectopic (Klein, U. et al. Proc Natl Acad Sci U S A100, 2639–2644, 2003). Here we report that c-MYC is absent in proliferating GC B cells because it is transcriptionally suppressed by BCL6, as demonstrated by the presence of specific BCL6 binding sites in the c-MYC promoter region and by chromatin immunoprecipitation experiments showing that BCL6 is bound to these sites in vivo. Thus, c-MYC escapes BCL6-mediated suppression in lymphoma leading to the co-expression of the two transcription factors, an event never observed in immunohistochemical and gene expression profile analysis of normal GC B cells. Surprisingly, co-immunoprecipitation experiments and in vitro binding experiments indicate that, when co-expressed, BCL6 and c-MYC are physically bound in a novel complex detectable in DLBCL and BL cell lines as well as in primary lymphoma cases. The formation of the BCL6/c-MYC complex has several significant functional consequences on the function of both c-MYC and BCL6: 1) a two fold, BCL6-binding dependent increase in c-MYC half-life, an event that has been shown to contribute to its oncogenic activation; 2) a synergistic increase in the ability of both BCL6 and c-MYC to suppress MIZ1-activated transcription of the p21CIP cell cycle arrest gene; 3) MYC-dependent inhibition of BCL6 acetylation by p300, an event that physiologically inactivates BCL6 via c-MYC-mediated recruitment of HDAC. Notably, the pathologic co-expression of c-MYC and BCL6 was shown to have pathologic consequences in vivo, since double transgenic BCL6/c-MYC mice display accelerated lymphoma development and the appearance of a novel GC-derived tumor phenotype not recognizable in single transgenic animals and containing the pathologic c-MYC/BCL6 complex. Thus, the pathologic co-expression and illegitimate physical interaction of BCL6 and c-MYC leads to an increase in the constitutive activity of both oncogenes. These results identify a novel mechanism of oncogenic function for BCL6 and c-MYC and a novel tumor-specific protein complex of potential therapeutic interest.

Oncogenic Pim Kinase Activity Provides Resistance to Mtor Inhibition in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3974-3974
Author(s):  
Jonathan H Schatz ◽  
Hans-Guido Wendel
Keyword(s):  
B Cell ◽  
Conflicts Of Interest ◽  
Somatic Hypermutation ◽  
Enzyme Activation ◽  
Genomic Amplification ◽  
Mtor Inhibition ◽  
Clinically Significant ◽  
The Eu

Abstract Abstract 3974 Poster Board III-910 The PI3K/Akt/mTor pathway is among the most frequently deregulated in human cancers, including many leukemias and lymphomas, and inhibitors targeting it at multiple levels are either clinically available or under development. The Pim family proteins are oncogenic serine/threonine kinases expressed in many malignancies and that have numerous overlapping downstream targets and functional consequences with PI3K/Akt/mTor. We here investigate the role of Pim proteins in oncogenesis and their ability in particular to mediate resistance to mTor inhibition. We find over-expression and genomic amplification of Pim1 and Pim2 in tumor samples and cell lines derived from patients with multiple B lymphomas. We show that both Pim1 and Pim2 powerfully mediate resistance to apoptosis in the murine pro-B cell line FL5-12 when withdrawn from IL-3. In murine tumor cells with constitutive activation of mTor, Pim1 and Pim2 provide resistance to treatment with the mTor inhibitor rapamycin. In vivo, we find that murine Pim2 (mPim2) accelerates tumorigenesis in the Eu-Myc transgenic model of Burkitt's lymphoma in a manner highly similar to Akt. However, unlike Akt, mPim2 leads to tumors that resist sensitization to chemotherapy by co-administration of rapamycin. We also investigate Pim1's role as a target of aberrant somatic hypermutation (ASHM), the process by which the enzyme activation induced deamidase (AID) introduces mutations into the coding and non-coding regions of the Pim1 locus and other proto-oncogenes in many B-cell malignancies. We find that structural Pim1 mutants found in patient tumors samples retain their ability to accelerate tumorigenesis in the Eu-Myc model in vivo and to mediate resistance to apoptosis and rapamycin in vitro. In conclusion, Pim activity is clinically significant, highly oncogenic, and provides resistance to mTor inhibition. Pim inhibition is therefore an attractive therapeutic approach, especially in combination with PI3K/Akt/mTor inhibition, even as ASHM provides a potential mechanism for B-cell tumors to escape it. Disclosures: No relevant conflicts of interest to declare.

scholarly journals B Lymphocyte Chemotaxis Regulated in Association with Microanatomic Localization, Differentiation State, and B Cell Receptor Engagement

1998 ◽  
Vol 187 (5) ◽  
pp. 753-762 ◽  
Author(s):  
Conrad C. Bleul ◽  
Joachim L. Schultze ◽  
Timothy A. Springer
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Messenger Rna ◽  
B Lymphocyte ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Cxc Chemokine

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein–coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell– derived factor (SDF)-1α is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1α also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1α by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1α is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.

scholarly journals A multi-objective based clustering for identifying clonally-related sequences from high-throughput B cell repertoire data

2021 ◽  
Author(s):  
Nika Abdollahi ◽  
Anne Langlois De Septenville ◽  
Hugues Ripoche ◽  
Frederic Davi ◽  
Juliana Silva Bernardes
Keyword(s):  
B Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Somatic Hypermutation ◽  
Clustering Algorithms ◽  
Objective Functions ◽  
Cell Repertoire ◽  
Multi Objective ◽  
B Cell Repertoire ◽  
Attractive Option

The adaptive B cell response is driven by the expansion, somatic hypermutation, and selection of B cell clones. A high number of clones in a B cell population indicates a highly diverse repertoire, while clonal size distribution and sequence diversity within clones can be related to antigen's selective pressure. Identifying clones is fundamental to many repertoire studies, including repertoire comparisons, clonal tracking and statistical analysis. Several methods have been developed to group sequences from high-throughput B cell repertoire data. Current methods use clustering algorithms to group clonally-related sequences based on their similarities or distances. Such approaches create groups by optimizing a single objective that typically minimizes intra-clonal distances. However, optimizing several objective functions can be advantageous and boost the algorithm convergence rate. Here we propose a new method based on multi-objective clustering. Our approach requires V(D)J annotations to obtain the initial clones and iteratively applies two objective functions that optimize cohesion and separation within clones simultaneously. We show that under simulations with varied mutation rates, our method greatly improves clonal grouping as compared to other tools. When applied to experimental repertoires generated from high-throughput sequencing, its clustering results are comparable to the most performing tools. The method based on multi-objective clustering can accurately identify clone members, has fewer parameter settings and presents the lowest running time among existing tools. All these features constitute an attractive option for repertoire analysis, particularly in the clinical context to unravel the mechanisms involved in the development and evolution of B cell malignancies.

scholarly journals AID–RNA polymerase II transcription-dependent deamination of IgV DNA

2019 ◽  
Vol 47 (20) ◽  
pp. 10815-10829 ◽  
Author(s):  
Phuong Pham ◽  
Sohail Malik ◽  
Chiho Mak ◽  
Peter C Calabrese ◽  
Robert G Roeder ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
B Cell ◽  
Rna Polymerase Ii ◽  
Somatic Hypermutation ◽  
Human Memory ◽  
Pol Ii ◽  
Transcription System ◽  
Complementarity Determining Regions ◽  
Factor C

Abstract Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.

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