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 in order 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 towards coupling AIRR-seq with the computational discovery of immunotherapeutics, vaccines, and immunodiagnostics.Comment: 27 pages, 2 figure

Dietary restriction mitigates the age-associated decline in mouse B cell receptor repertoire diversity

Aging impairs the capacity to respond to novel antigens, reducing immune protection against pathogens and vaccine efficacy. Dietary restriction (DR) extends life- and health span in diverse animals. However, little is known about the capacity of DR to combat the decline in immune function. Here, we study the changes in B cell receptor (BCR) repertoire during aging in DR and control mice. By sequencing the variable region of the BCR heavy chain in the spleen, we show that DR preserves diversity and attenuates the increase in clonal expansions throughout aging. Remarkably, mice starting DR in mid-life have repertoire diversity and clonal expansion rates indistinguishable from chronic DR mice. In contrast, in the intestine, these traits are unaffected by either age or DR. Reduced within-individual B cell repertoire diversity and increased clonal expansions are correlated with higher morbidity, suggesting a potential contribution of B cell repertoire dynamics to health during aging

Benchmarking of T cell receptor repertoire profiling methods reveals large systematic biases

Monitoring the T cell receptor (TCR) repertoire in health and disease can provide key insights into adaptive immune responses, but the accuracy of current TCR sequencing (TCRseq) methods is unclear. In this study, we systematically compared the results of nine commercial and academic TCRseq methods, including six rapid amplification of complementary DNA ends (RACE)-polymerase chain reaction (PCR) and three multiplex-PCR approaches, when applied to the same T cell sample. We found marked differences in accuracy and intra- and inter-method reproducibility for T cell receptor α (TRA) and T cell receptor β (TRB) TCR chains. Most methods showed a lower ability to capture TRA than TRB diversity. Low RNA input generated non-representative repertoires. Results from the 5' RACE-PCR methods were consistent among themselves but differed from the RNA-based multiplex-PCR results. Using an in silico meta-repertoire generated from 108 replicates, we found that one genomic DNA-based method and two non-unique molecular identifier (UMI) RNA-based methods were more sensitive than UMI methods in detecting rare clonotypes, despite the better clonotype quantification accuracy of the latter

The Pipeline Repertoire for Ig-Seq Analysis

With the advent of high-throughput sequencing of immunoglobulin genes (Ig-Seq), the understanding of antibody repertoires and their dynamics among individuals and populations has become an exciting area of research. There is an increasing number of computational tools that aid in every step of the immune repertoire characterization. However, since not all tools function identically, every pipeline has its unique rationale and capabilities, creating a rich blend of useful features that may appear intimidating for newcomer laboratories with the desire to plunge into immune repertoire analysis to expand and improve their research; hence, all pipeline strengths and differences may not seem evident. In this review we provide a practical and organized list of the current set of computational tools, focusing on their most attractive features and differences in order to carry out the characterization of antibody repertoires so that the reader better decides a strategic approach for the experimental design, and computational pathways for the analyses of immune repertoires

sumrep: a summary statistic framework for immune receptor repertoire comparison and model validation

In studying the binding of host antibodies to the surface antigens of pathogens, the structural and functional characterization of antibody–antigen complexes by X-ray crystallography and binding assay is important. However, the characterization requires experiments that are typically time consuming and expensive: thus, many antibody–antigen complexes are under-characterized. For vaccine development and disease surveillance, it is often vital to assess the impact of amino acid substitutions on antibody binding. For example, are there antibody substitutions capable of improving binding without a loss of breadth, or antigen substitutions that lead to antigenic escape? The questions cannot be answered reliably from sequence variation alone, exhaustive substitution assays are usually impractical, and alanine scans provide at best an incomplete identification of the critical residue–residue interactions. Here, we show that, given an initial structure of an antibody bound to an antigen, molecular dynamics simulations using the energy method molecular mechanics with Generalized Born surface area (MM/GBSA) can model the impact of single amino acid substitutions on antibody–antigen binding energy. We apply the technique to three broad-spectrum antibodies to influenza A hemagglutinin and examine both previously characterized and novel variant strains observed in the human population that may give rise to antigenic escape. We find that in some cases the impact of a substitution is local, while in others it causes a reorientation of the antibody with wide-ranging impact on residue–residue interactions: this explains, in part, why the change in chemical properties of a residue can be, on its own, a poor predictor of overall change in binding energy. Our estimates are in good agreement with experimental results—indeed, they approximate the degree of agreement between different experimental techniques. Simulations were performed on commodity computer hardware; hence, this approach has the potential to be widely adopted by those undertaking infectious disease research. Novel aspects of this research include the application of MM/GBSA to investigate binding between broadly binding antibodies and a viral glycoprotein; the development of an approach for visualizing substrate–ligand interactions; and the use of experimental assay data to rescale our predictions, allowing us to make inferences about absolute, as well as relative, changes in binding energy

T-Cell Receptor Repertoire Analysis with Computational Tools—An Immunologist’s Perspective

Over the last few years, there has been a rapid expansion in the application of information technology to biological data. Particularly the field of immunology has seen great strides in recent years. The development of next-generation sequencing (NGS) and single-cell technologies also brought forth a revolution in the characterization of immune repertoires. T-cell receptor (TCR) repertoires carry comprehensive information on the history of an individual’s antigen exposure. They serve as correlates of host protection and tolerance, as well as biomarkers of immunological perturbation by natural infections, vaccines or immunotherapies. Their interrogation yields large amounts of data. This requires a suite of highly sophisticated bioinformatics tools to leverage the meaning and complexity of the large datasets. Many different tools and methods, specifically designed for various aspects of immunological research, have recently emerged. Thus, researchers are now confronted with the issue of having to choose the right kind of approach to analyze, visualize and ultimately solve their task at hand. In order to help immunologists to choose from the vastness of available tools for their data analysis, this review addresses and compares commonly used bioinformatics tools for TCR repertoire analysis and illustrates the advantages and limitations of these tools from an immunologist’s perspective

Characterizing the Role of the T Cell Receptor Repertoire in T Cell Development and Function

Expansion and memory of immune cells in response to stimulation of diversified antigen receptors is the hallmark of adaptive immunity. Here, we use antigen receptor sequencing and in vivo analysis of monoclonal cell populations to elucidate the development and function of two T cell populations: Foxp3+RORγt+ CD4+ T cells and γδ T cells. Foxp3+RORγt+ T cells have recently been characterized as an immunoregulatory population highly enriched in the colon lamina propria. However, their developmental origin and relation to RORγt- Treg and RORγt+ TH17 cells remains unclear. Here, we show that despite sharing a subset of TCR specificities with TH17 cells, Foxp3+RORγt+ T cells first acquire a Foxp3+RORγt- phenotype before co-expressing RORγt, suggesting that Foxp3+RORγt+ cell development can occur via an RORγt- Treg intermediate. While γδ T cells are considerably well studied relative to Foxp3+RORγt+ T cells, the importance antigen receptor diversification to γδ T cell function is still poorly understood. In order to comprehensively assess the paired-chain γδ T cell repertoire during inflammation, we developed a fixed-TCRδ system. We show that experimental autoimmune encephalomyelitis (EAE) results in dramatic clonal expansion of γδ T cells and that a single expanded TCR clone is sufficient to exacerbate immune pathology. Together, this suggests that γδ T cells can exhibit the clonal expansion characteristic of an adaptive immune response and that this response is physiologically significant to the outcome of EAE

Characterising antibody immunity and ageing in a short-lived teleost

Ageing individuals exhibit a pervasive decline in adaptive immune function, with important implications for health and lifespan. Systemic changes observed in the structure and diversity of antibody repertoires with age are thought to play an important role in this immunosenescent phenotype; however, the relatively long lifespan of most vertebrate model organisms makes thorough investigation of the ageing repertoire challenging. As a naturally short-lived vertebrate, the turquoise killifish (Nothobranchius furzeri) offers an exciting new opportunity to study the ageing of the adaptive immune system in general and antibody repertoires in particular. In this thesis, I used a combination of existing genomic assemblies and new sequencing data to assemble and characterise the immunoglobulin heavy chain (IGH) locus sequence in the turquoise killifish and compare it to those of closely related species, revealing a history of dynamic locus evolution and repeated duplication and loss of the specialised mucosal isotype IGHZ. The N. furzeri locus itself lacks IGHZ, making it one of the few known teleost species not to possess this isotype. These results support a high rate of evolution in teleost IGH loci and set a strong foundation for the study of comparative evolutionary immunology in cyprionodontiform fishes. Having characterised the IGH locus sequence in N. furzeri, I used it to establish targeted immunoglobulin sequencing in this species, enabling quantitative interrogation of the antibody repertoire. Applying this protocol to whole-body killifish samples revealed complex and individualised antibody repertoires which decline rapidly in within-individual diversity and increase in between-individual variability with age, demonstrating that turquoise killifish exhibit a rapid repertoire-ageing phenotype in line with their short lifespans. This loss of diversity with age was particularly strong in isolated gut samples, a phenomenon that may be related to the constant strong antigenic exposure experienced at mucosal surfaces and has not been previously investigated in a vertebrate model. Taken together, these results establish the turquoise killifish as a novel model for vertebrate immunosenescence and lay the groundwork for future interrogation of -- and intervention in -- adaptive-immune ageing