Adaptive Immune Receptor Repertoire Research Articles

Inferred Allelic Variants of Immunoglobulin Receptor Genes: A System for Their Evaluation, Documentation, and Naming.

Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT®, the international ImMunoGeneTics information system® (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT®. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC—focusing, to begin with, on human IGHV genes—with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species.

Identification of Subject-Specific Immunoglobulin Alleles From Expressed Repertoire Sequencing Data.

The adaptive immune receptor repertoire (AIRR) contains information on an individuals' immune past, present and potential in the form of the evolving sequences that encode the B cell receptor (BCR) repertoire. AIRR sequencing (AIRR-seq) studies rely on databases of known BCR germline variable (V), diversity (D), and joining (J) genes to detect somatic mutations in AIRR-seq data via comparison to the best-aligning database alleles. However, it has been shown that these databases are far from complete, leading to systematic misidentification of mutated positions in subsets of sample sequences. We previously presented TIgGER, a computational method to identify subject-specific V gene genotypes, including the presence of novel V gene alleles, directly from AIRR-seq data. However, the original algorithm was unable to detect alleles that differed by more than 5 single nucleotide polymorphisms (SNPs) from a database allele. Here we present and apply an improved version of the TIgGER algorithm which can detect alleles that differ by any number of SNPs from the nearest database allele, and can construct subject-specific genotypes with minimal prior information. TIgGER predictions are validated both computationally (using a leave-one-out strategy) and experimentally (using genomic sequencing), resulting in the addition of three new immunoglobulin heavy chain V (IGHV) gene alleles to the IMGT repertoire. Finally, we develop a Bayesian strategy to provide a confidence estimate associated with genotype calls. All together, these methods allow for much higher accuracy in germline allele assignment, an essential step in AIRR-seq studies.

Augmenting adaptive immunity: progress and challenges in the quantitative engineering and analysis of adaptive immune receptor repertoires

The adaptive immune system is a natural diagnostic sensor and therapeutic.

Antibody Repertoire Analysis of Hepatitis C Virus Infections Identifies Immune Signatures Associated With Spontaneous Clearance.

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.

AIRR Data Under the E.U. Trade Secrets Directive: Aligning Scientific Practices with Commercial Realities

Whether the E.U. Trade Secrets Directive sufficiently and appropriately covers cutting-edge complex technologies is of critical interest to policy-makers, scientists, and commercial developers alike. One such technology—adaptive immune receptor repertoire sequencing, or AIRR-seq—raises difficult questions concerning what information is and should be protected under the new Directive, and how to best align scientific practices with commercial realities. The ‘raw’ form AIRR-seq data—massive genetic datasets of hundreds of millions of individuals’ immune cells—tends to be freely shared among academic researchers, thus typically destroying the protectability of the underlying information. But follow-on data—essentially, information interpreting that data—is nonetheless protectable under the Directive because it is both economically valuable and not readily available from an examination of the raw data itself. Protecting this follow-on information while encouraging the free sharing of AIRR-seq data best accords the purpose of the Trade Secrets Directive. Lessons from the case of AIRR-seq data also sheds light on other puzzles concerning the tensions between disclosure and intellectual property protections, such as the mutual exclusivity of patents and trade secrets, the sharing of clinical trial data, and protecting genetic diagnostics. (Less)

High-Throughput Sequencing of the T-Cell Receptor Beta Chain Gene Repertoire in Chronic Lymphocytic Leukemia.

High-throughput, next-generation sequencing (NGS) offers a unique opportunity for in-depth characterization of adaptive immune receptor repertoires. Nevertheless, limitations and pitfalls exist in every step of both the experimental and the analytical procedure, leading to discrepancies in the literature and incomprehensive and/or altogether misleading results. Thus, standardization of protocols in NGS immunogenetics is urgently needed.Here, we describe the experimental protocol that we developed for T-cell receptor beta chain (TRB) gene repertoire analysis in chronic lymphocytic leukemia, aiming to provide a reproducible and biologically meaningful output. Although optimized for TRBV-TRBD-TRBJ gene rearrangements, this protocol may be customized for other adaptive immune receptor sequences, as well.

AIRR Community Standardized Representations for Annotated Immune Repertoires.

Increased interest in the immune system's involvement in pathophysiological phenomena coupled with decreased DNA sequencing costs have led to an explosion of antibody and T cell receptor sequencing data collectively termed “adaptive immune receptor repertoire sequencing” (AIRR-seq or Rep-Seq). The AIRR Community has been actively working to standardize protocols, metadata, formats, APIs, and other guidelines to promote open and reproducible studies of the immune repertoire. In this paper, we describe the work of the AIRR Community's Data Representation Working Group to develop standardized data representations for storing and sharing annotated antibody and T cell receptor data. Our file format emphasizes ease-of-use, accessibility, scalability to large data sets, and a commitment to open and transparent science. It is composed of a tab-delimited format with a specific schema. Several popular repertoire analysis tools and data repositories already utilize this AIRR-seq data format. We hope that others will follow suit in the interest of promoting interoperable standards.

ImmuneDB, a Novel Tool for the Analysis, Storage, and Dissemination of Immune Repertoire Sequencing Data.

ImmuneDB is a system for storing and analyzing high-throughput immune receptor sequencing data. Unlike most existing tools, which utilize flat-files, ImmuneDB stores data in a well-structured MySQL database, enabling efficient data queries. It can take raw sequencing data as input and annotate receptor gene usage, infer clonotypes, aggregate results, and run common downstream analyses such as calculating selection pressure and constructing clonal lineages. Alternatively, pre-annotated data can be imported and analyzed data can be exported in a variety of common Adaptive Immune Receptor Repertoire (AIRR) file formats. To validate ImmuneDB, we compare its results to those of another pipeline, MiXCR. We show that the biological conclusions drawn would be similar with either tool, while ImmuneDB provides the additional benefits of integrating other common tools and storing data in a database. ImmuneDB is freely available on GitHub at https://github.com/arosenfeld/immunedb, on PyPi at https://pypi.org/project/ImmuneDB, and a Docker container is provided at https://hub.docker.com/r/arosenfeld/immunedb. Full documentation is available at http://immunedb.com.

The CAIRR Pipeline for Submitting Standards-Compliant B and T Cell Receptor Repertoire Sequencing Studies to the National Center for Biotechnology Information Repositories.

The adaptation of high-throughput sequencing to the B cell receptor and T cell receptor has made it possible to characterize the adaptive immune receptor repertoire (AIRR) at unprecedented depth. These AIRR sequencing (AIRR-seq) studies offer tremendous potential to increase the understanding of adaptive immune responses in vaccinology, infectious disease, autoimmunity, and cancer. The increasingly wide application of AIRR-seq is leading to a critical mass of studies being deposited in the public domain, offering the possibility of novel scientific insights through secondary analyses and meta-analyses. However, effective sharing of these large-scale data remains a challenge. The AIRR community has proposed minimal information about adaptive immune receptor repertoire (MiAIRR), a standard for reporting AIRR-seq studies. The MiAIRR standard has been operationalized using the National Center for Biotechnology Information (NCBI) repositories. Submissions of AIRR-seq data to the NCBI repositories typically use a combination of web-based and flat-file templates and include only a minimal amount of terminology validation. As a result, AIRR-seq studies at the NCBI are often described using inconsistent terminologies, limiting scientists’ ability to access, find, interoperate, and reuse the data sets. In order to improve metadata quality and ease submission of AIRR-seq studies to the NCBI, we have leveraged the software framework developed by the Center for Expanded Data Annotation and Retrieval (CEDAR), which develops technologies involving the use of data standards and ontologies to improve metadata quality. The resulting CEDAR-AIRR (CAIRR) pipeline enables data submitters to: (i) create web-based templates whose entries are controlled by ontology terms, (ii) generate and validate metadata, and (iii) submit the ontology-linked metadata and sequence files (FASTQ) to the NCBI BioProject, BioSample, and Sequence Read Archive databases. Overall, CAIRR provides a web-based metadata submission interface that supports compliance with the MiAIRR standard. This pipeline is available at http://cairr.miairr.org, and will facilitate the NCBI submission process and improve the metadata quality of AIRR-seq studies.

IgE repertoire and immunological memory: compartmental regulation and antibody function.

It is now generally recognized that bone marrow is the survival niche for antigen-specific plasma cells with long-term immunological memory. These cells release antibodies into the circulation, needed to prime effector cells in the secondary immune response. These antibodies participate in the surveillance for antigen and afford immune defence against pathogens and toxins previously encountered in the primary immune response. IgE antibodies function together with their effector cells, mast cells, to exert ‘immediate hypersensitivity’ in mucosal tissues at the front line of immune defence. The constant supply of IgE antibodies from bone marrow plasma cells allows the rapid ‘recall response’ by mast cells upon re-exposure to antigen even after periods of antigen absence. The speed and sensitivity of the IgE recall response and potency of the effector cell functions are advantageous in the early detection and elimination of pathogens and toxins at the sites of attack. Local antigen provocation also stimulates de novo synthesis of IgE or its precursors of other isotypes that undergo IgE switching in the mucosa. This process, however, introduces a delay before mast cells can be sensitized and resume activity; this is terminated shortly after the antigen is eliminated. Recent results from adaptive immune receptor repertoire sequencing of immunoglobulin genes suggest that the mucosal IgE+ plasmablasts, which have undergone affinity maturation in the course of their evolution in vivo, are a source of long-lived IgE+ plasma cells in the bone marrow that are already fully functional.

IReceptor: A platform for querying and analyzing antibody/B-cell and T-cell receptor repertoire data across federated repositories.

Next-generation sequencing allows the characterization of the adaptive immune receptor repertoire (AIRR) in exquisite detail. These large-scale AIRR-seq data sets have rapidly become critical to vaccine development, understanding the immune response in autoimmune and infectious disease, and monitoring novel therapeutics against cancer. However, at present there is no easy way to compare these AIRR-seq data sets across studies and institutions. The ability to combine and compare information for different disease conditions will greatly enhance the value of AIRR-seq data for improving biomedical research and patient care. The iReceptor Data Integration Platform (gateway.ireceptor.org) provides one implementation of the AIRR Data Commons envisioned by the AIRR Community (airr-community.org), an initiative that is developing protocols to facilitate sharing and comparing AIRR-seq data. The iReceptor Scientific Gateway links distributed (federated) AIRR-seq repositories, allowing sequence searches or metadata queries across multiple studies at multiple institutions, returning sets of sequences fulfilling specific criteria. We present a review of the development of iReceptor, and how it fits in with the general trend toward sharing genomic and health data, and the development of standards for describing and reporting AIRR-seq data. Researchers interested in integrating their repositories of AIRR-seq data into the iReceptor Platform are invited to contact support@ireceptor.org.

The Bayesian optimist's guide to adaptive immune receptor repertoire analysis.

Probabilistic modeling is fundamental to the statistical analysis of complex data. In addition to forming a coherent description of the data-generating process, probabilistic models enable parameter inference about given datasets. This procedure is well developed in the Bayesian perspective, in which one infers probability distributions describing to what extent various possible parameters agree with the data. In this paper, we motivate and review probabilistic modeling for adaptive immune receptor repertoire data then describe progress and prospects for future work, from germline haplotyping to adaptive immune system deployment across tissues. The relevant quantities in immune sequence analysis include not only continuous parameters such as gene use frequency but also discrete objects such as B-cell clusters and lineages. Throughout this review, we unravel the many opportunities for probabilistic modeling in adaptive immune receptor analysis, including settings for which the Bayesian approach holds substantial promise (especially if one is optimistic about new computational methods). From our perspective, the greatest prospects for progress in probabilistic modeling for repertoires concern ancestral sequence estimation for B-cell receptor lineages, including uncertainty from germline genotype, rearrangement, and lineage development.

Human adaptive immune receptor repertoire analysis-Past, present, and future.

The genes encoding adaptive immune antigen receptors, namely the immunoglobulins expressed in membrane-bound or secreted forms by B cells, and the cell surface T cell receptors, are unique in human biology because they are generated by combinatorial rearrangement of the genomic DNA. The diversity of receptors so generated in populations of lymphocytes enables the human immune system to recognize antigens expressed by pathogens, but also underlies the pathological specificity of autoimmune diseases and the mistargeted immunity in allergies. Several recent technological developments, foremost among them the invention of high-throughput DNA sequencing instruments, have enabled much deeper and thorough evaluation of clones of human B cells and T cells and the antigen receptors they express during physiological and pathogenic immune responses. The evolutionary struggles between host adaptive immune responses and populations of pathogens are now open to greater scrutiny, elucidation of the underlying reasons for successful or failed immunity, and potential predictive modeling, than ever before. Here we give an overview of the foundations, recent progress, and future prospects in this dynamic area of research.

Computational Strategies for Dissecting the High-Dimensional Complexity of Adaptive Immune Repertoires.

The adaptive immune system recognizes antigens via an immense array of antigen-binding antibodies and T-cell receptors, the immune repertoire. The interrogation of immune repertoires is of high relevance for understanding the adaptive immune response in disease and infection (e.g., autoimmunity, cancer, HIV). Adaptive immune receptor repertoire sequencing (AIRR-seq) has driven the quantitative and molecular-level profiling of immune repertoires, thereby revealing the high-dimensional complexity of the immune receptor sequence landscape. Several methods for the computational and statistical analysis of large-scale AIRR-seq data have been developed to resolve immune repertoire complexity and to understand the dynamics of adaptive immunity. Here, we review the current research on (i) diversity, (ii) clustering and network, (iii) phylogenetic, and (iv) machine learning methods applied to dissect, quantify, and compare the architecture, evolution, and specificity of immune repertoires. We summarize outstanding questions in computational immunology and propose future directions for systems immunology toward coupling AIRR-seq with the computational discovery of immunotherapeutics, vaccines, and immunodiagnostics.

Mechanisms underlying B cell immune dysregulation and autoantibody production in MuSK myasthenia gravis.

Pathogenic autoantibodies to muscle-specific tyrosine kinase (MuSK) can be found in patients with myasthenia gravis (MG) who do not have detectable antibodies to the acetylcholine receptor. Although the autoantibody-mediated pathology is well understood, much remains to be learned about the cellular immunology that contributes to autoantibody production. To that end, our laboratory has investigated particular components associated with the cellular immunopathology of MuSK MG. First, we found that B cell tolerance defects contribute to the abnormal development of the naive repertoire, which indicates that dysregulation occurs before the production of autoantibodies. Second, both the naive and antigen-experienced memory B cell repertoire, which we examined through the application of high-throughput adaptive immune receptor repertoire sequencing, include abnormalities not found in healthy controls. This highlights a broad immune dysregulation. Third, using complementary approaches, including production of human monoclonal antibodies, we determined that circulating plasmablasts directly contribute to the production of MuSK-specific autoantibodies in patients experiencing relapse following B cell depletion therapy. These collective findings contribute to defining a mechanistic model that describes MuSK MG immunopathogenesis.

Adaptive Immune Receptor Repertoire Community recommendations for sharing immune-repertoire sequencing data.

High-throughput sequencing of B and T cell receptors is routinely being applied in studies of adaptive immunity. The Adaptive Immune Receptor Repertoire (AIRR) Community was formed in 2015 to address issues in AIRR sequencing studies, including the development of reporting standards for the sharing of data sets.

Reproducibility and Reuse of Adaptive Immune Receptor Repertoire Data.

High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell receptor repertoires has increased dramatically since the technique was introduced in 2009 (1–3). This experimental approach explores the maturation of the adaptive immune system and its response to antigens, pathogens, and disease conditions in exquisite detail. It holds significant promise for diagnostic and therapy-guiding applications. New technology often spreads rapidly, sometimes more rapidly than the understanding of how to make the products of that technology reliable, reproducible, or usable by others. As complex technologies have developed, scientific communities have come together to adopt common standards, protocols, and policies for generating and sharing data sets, such as the MIAME protocols developed for microarray experiments. The Adaptive Immune Receptor Repertoire (AIRR) Community formed in 2015 to address similar issues for HTS data of immune repertoires. The purpose of this perspective is to provide an overview of the AIRR Community’s founding principles and present the progress that the AIRR Community has made in developing standards of practice and data sharing protocols. Finally, and most important, we invite all interested parties to join this effort to facilitate sharing and use of these powerful data sets (join@airr-community.org).

Novel Method for High-Throughput Full-Length IGHV-D-J Sequencing of the Immune Repertoire from Bulk B-Cells with Single-Cell Resolution.

Efficient and accurate high-throughput DNA sequencing of the adaptive immune receptor repertoire (AIRR) is necessary to study immune diversity in healthy subjects and disease-related conditions. The high complexity and diversity of the AIRR coupled with the limited amount of starting material, which can compromise identification of the full biological diversity makes such sequencing particularly challenging. AIRR sequencing protocols often fail to fully capture the sampled AIRR diversity, especially for samples containing restricted numbers of B lymphocytes. Here, we describe a library preparation method for immunoglobulin sequencing that results in an exhaustive full-length repertoire where virtually every sampled B-cell is sequenced. This maximizes the likelihood of identifying and quantifying the entire IGHV-D-J repertoire of a sample, including the detection of rearrangements present in only one cell in the starting population. The methodology establishes the importance of circumventing genetic material dilution in the preamplification phases and incorporates the use of certain described concepts: (1) balancing the starting material amount and depth of sequencing, (2) avoiding IGHV gene-specific amplification, and (3) using Unique Molecular Identifier. Together, this methodology is highly efficient, in particular for detecting rare rearrangements in the sampled population and when only a limited amount of starting material is available.

Novel Library Method for High-Throughput Full-Length Sequencing of the Immune Repertoire from Unseparated B-Cells with Single-Cell Resolutionution

High-throughput DNA sequencing of the adaptive immune receptor repertoire is a relatively new and fast growing technology used to study the immune response in health and disease. In B and T cell lymphoproliferative disorders, antigen receptor sequencing can be used to study clonal diversity and evolution of the disease in treatment free condition and in response to treatment. Furthermore, it can be used for the detection of minimal residual disease (MRD), providing information on the relationship between the presence and number of pre-treatment clone(s) and their relationship and responsibility for a subsequent relapse. The characteristics and quality of the data generated by high-throughput DNA sequencing of immune receptor signatures are the results of three major components: library preparation, sequencing platform, and software tools.For both the library and software, there are no standard protocols and tools. Indeed, new approaches are continually being developed to accommodate new sequencing platform features and shortcomings, such as errors and read length restrictions. Two major technical challenges are: procuring an unbiased repertoire library that for B lymphocytes obtains and retains the full length IGHV-D-J along with (sub)isotype information, and resolving data to a single cell level, crucial for detection of MRD and rare clonal variants existing in the early phase of the disease, which might emerge and be involved in future relapse or progression.We describe here a library preparation method for use with the Illumina MiSeq platform that results in an exhaustive full-length repertoire where virtually every B cell is sequenced, thereby maximizing the likelihood of identifying and quantifying the “real” IGHV-D-J repertoire of the sample analyzed. The method also allows the detection of very infrequent rearrangements and maintains IG sub-isotype information without compromising data quality. From 0.5 - 1 million human B cells can be sequenced in a single MiSeq 2x300 run with this approach. Key aspects of the technique are: 1) start from a well defined number of B lymphocytes 2) avoid V-gene specific PCR amplification and genetic material dilution in the pre-amplification phases 3) the specific depth of sequencing should depend on the starting B (or T) cell subset (i.e. na•ve, memory or plasma cell), and should be proportional to the number of starting cells. High quality sub-isotype information can be obtained with a second round of sequencing of shorter read length, e.g., with the Illumina 2x150 platform.We used 58 different CLL clones with known IGH sequence mixed all together with polyclonal B cell from a donor PBMC (Figure 1). The mixed lysate is used to test the ability to detect the different clones.The following describes how the absence of genetic material dilution in the pre-amplification phases impact on the ability to obtain a comprehensive repertoire. These are crucial in MRD detection, since diluting the genetic material (RNA and/or cDNA) prior PCR amplification compromises the ability to accurately and consistently detect the clonal variants, reducing the de facto sensitivity and reproducibility of the analysis.As a final example of the method's utility, we also demonstrate how different chronic lymphocytic leukemia clones present considerable variability in IG mRNA expression level that correlate with the number of unique mRNA molecule sequenced (Figure 3), which, if using a method with sub-optimal efficiency, could lead to a reduced clone-specific ability of detection by PCR based techniques. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Assembly-based inference of B-cell receptor repertoires from short read RNA sequencing data with V'DJer.

Motivation: B-cell receptor (BCR) repertoire profiling is an important tool for understanding the biology of diverse immunologic processes. Current methods for analyzing adaptive immune receptor repertoires depend upon PCR amplification of VDJ rearrangements followed by long read amplicon sequencing spanning the VDJ junctions. While this approach has proven to be effective, it is frequently not feasible due to cost or limited sample material. Additionally, there are many existing datasets where short-read RNA sequencing data are available but PCR amplified BCR data are not.Results: We present here V’DJer, an assembly-based method that reconstructs adaptive immune receptor repertoires from short-read RNA sequencing data. This method captures expressed BCR loci from a standard RNA-seq assay. We applied this method to 473 Melanoma samples from The Cancer Genome Atlas and demonstrate V’DJer’s ability to accurately reconstruct BCR repertoires from short read mRNA-seq data.Availability and Implementation: V’DJer is implemented in C/C ++, freely available for academic use and can be downloaded from Github: https://github.com/mozack/vdjerContact: benjamin_vincent@med.unc.edu or parkerjs@email.unc.eduSupplementary information: Supplementary data are available at Bioinformatics online.