Evaluation of the antigen-experienced B-cell receptor repertoire in healthy children and adults

Upon antigen recognition via their B cell receptor (BR), B cells migrate to the germinal center where they undergo somatic hypermutation (SHM) to increase their affinity for the antigen, and class switch recombination (CSR) to change the effector function of the secreted antibodies. These steps are essential to create an antigen-experienced BR repertoire that efficiently protects the body against pathogens. At the same time, the BR repertoire should be selected to protect against responses to self-antigen or harmless antigens. Insights into the processes of SHM, selection, and CSR can be obtained by studying the antigen-experienced BR repertoire. Currently, a large reference data set of healthy children and adults, which ranges from neonates to the elderly, is not available. In this study, we analyzed the antigen-experienced repertoire of 38 healthy donors (HD), ranging from cord blood to 74 years old, by sequencing IGA and IGG transcripts using next generation sequencing. This resulted in a large, freely available reference data set containing 412,890 IGA and IGG transcripts. We used this data set to study mutation levels, SHM patterns, antigenic selection, and CSR from birth to elderly HD. Only small differences were observed in SHM patterns, while the mutation levels increase in early childhood and stabilize at 6 years of age at around 7%. Furthermore, comparison of the antigen-experienced repertoire with sequences from the naive immune repertoire showed that features associated with autoimmunity such as long CDR3 length and IGHV4-34 usage are reduced in the antigen-experienced repertoire. Moreover, IGA2 and IGG2 usage was increased in HD in higher age categories, while IGG1 usage was decreased. In addition, we studied clonal relationship in the different samples. Clonally related sequences were found with different subclasses. Interestingly, we found transcripts with the same CDR1-CDR3 sequence, but different subclasses. Together, these data suggest that a single antigen can provoke a B-cell response with BR of different subclasses and that, during the course of an immune response, some B cells change their isotype without acquiring additional SHM or can directly switch to different isotypes

ImmunoGlobulin galaxy (IGGalaxy) for simple determination and quantitation of immunoglobulin heavy chain rearrangements from NGS

Background: Sequence analysis of immunoglobulin heavy chain (IGH) gene rearrangements and frequency analysis is a powerful tool for studying the immune repertoire, immune responses and immune dysregulation in health and disease. The challenge is to provide user friendly, secure and reproducible analytical services that are available for both small and large laboratories which are determining VDJ repertoire using NGS technology. Results: In this study we describe ImmunoGlobulin Galaxy (IGGalaxy)- a convenient web based application for analyzing next-generation sequencing results and reporting IGH gene rearrangements for both repertoire and clonality studies. IGGalaxy has two analysis options one using the built in igBLAST algorithm and the second using output from IMGT; in either case repertoire summaries for the B-cell populations tested are available. IGGalaxy supports multi-sample and multi-replicate input analysis for both igBLAST and IMGT/HIGHV-QUEST. We demonstrate the technical validity of this platform using a standard dataset, S22, used for benchmarking the performance of antibody alignment utilities with a 99.9 % concordance with previous results. Re-analysis of NGS data from our samples of RAG-deficient patients demonstrated the validity and user friendliness of this tool. Conclusions: IGGalaxy provides clinical researchers with detailed insight into the repertoire of the B-cell population per individual sequenced and between control and pathogenic genomes. IGGalaxy was developed for 454 NGS results but is capable of analyzing alternative NGS data (e.g. Illumina, Ion Torrent). We demonstrate the use of a Galaxy virtual machine to determine the VDJ repertoire for reference data and from B-cells taken from immune deficient patients. IGGalaxy is available as a VM for download and use on a desktop PC or on a server

Automated Selection of Hotspots (ASH): enhanced automated segmentation and adaptive step finding for Ki67 hotspot detection in adrenal cortical cancer

BACKGROUND: In prognosis and therapeutics of adrenal cortical carcinoma (ACC), the selection of the most active areas in proliferative rate (hotspots) within a slide and objective quantification of immunohistochemical Ki67 Labelling Index (LI) are of critical importa

Next-generation antigen receptor sequencing of paired diagnosis and relapse samples of B-cell acute lymphoblastic leukemia: Clonal evolution and implications for minimal residual disease target selection

Antigen receptor gene rearrangements are frequently applied as molecular targets for detection of minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukemia patients. Since such targets may be lost at relapse, appropriate selection of antigen receptor genes as MRD-PCR target is critical. Recently, next-generation sequencing (NGS) – much more sensitive and quantitative than classical PCR-heteroduplex approaches – has been introduced for identification of MRD-PCR targets. We evaluated 42 paired diagnosis-relapse samples by NGS (IGH, IGK, TRG, TRD, and TRB) to evaluate clonal evolution patterns and to design an algorithm for selection of antigen receptor gene rearrangements most likely to remain stable at relapse. Overall, only 393 out of 1446 (27%) clonal rearrangements were stable between diagnosis and relapse. If only index clones with a frequency >5% at diagnosis were taken into account, this number increased to 65%; including only index clones with an absolute read count >10,000, indicating truly major clones, further increased the stability to 84%. Over 90% of index clones at relapse were also present as index clone at diagnosis. Our data provide detailed information about the stability of antigen receptor gene rearrangements, based on which we propose an algorithm for selecting stable MRD-PCR targets, successful in >97% of patients

Identification of CVID patients with defects in immune repertoire formation or specification

Common variable immune deficiency disorder (CVID) is the most clinically relevant cause of antibody failure. It is a highly heterogeneous disease with different underlying etiologies. CVID has been associated with a quantitative B cell defect, however, little is known about the quality of B cells present. Here, we studied the naïve and antigen selected B-cell receptor (BCR) repertoire in 33 CVID patients using next generation sequencing, to investigate B cells quality. Analysis for each individual patient revealed whether they have a defect in immune repertoire formation [V(D)J recombination] or specification (somatic hypermutation, subclass distribution, or selection). The naïve BCR repertoire was normal in most of the patients, although alterations in repertoire diversity and the junctions were found in a limited number of patients indicating possible defects in early B-cell development or V(D)J recombination in these patients. In contrast, major differences were found in the antigen selected BCR repertoire. Here, most patients (15/17) showed a reduced frequency of somatic hypermutation (SHM), changes in subclass distribution and/or minor alterations in antigen selection. Together these data show that in our CVID cohort only a small number of patients have a defect in formation of the naïve BCR repertoire, whereas the clear majority of patients have disturbances in their antigen selected repertoire, suggesting a defect in repertoire specification in the germinal centers of these patients. This highlights that CVID patients not only have a quantitative B cell defect, but that also the quality of, especially post germinal center B cells, is impaired

Antigen receptor galaxy: A user-friendly, web-based tool for analysis and visualization of T and B cell receptor repertoire data

Antigen Receptor Galaxy (ARGalaxy) is a Web-based tool for analyses and visualization of TCR and BCR sequencing data of 13 species. ARGalaxy consists of four parts: The demultiplex tool, the international ImMunoGeneTics information system (IMGT) concatenate tool, the immune repertoire pipeline, and the somatic hypermutation (SHM) and class switch recombination (CSR) pipeline. Together they allow the analysis of all different aspects of the immune repertoire. All pipelines can be run independently or combined, depending on the available data and the question of interest. The demultiplex tool allows data trimming and demultiplexing, whereas with the concatenate tool multiple IMGT/HighV-QUEST output files can be merged into a single file. The immune repertoire pipeline is an extended version of our previously published ImmunoGlobulin Galaxy (IGGalaxy) virtual machine that was developed to visualize V(D)J gene usage. It allows analysis of both BCR and TCR rearrangements, visualizes CDR3 characteristics (length and amino acid usage) and junction characteristics, and calculates the diversity of the immune repertoire. Finally, ARGalaxy includes the newly developed SHM and CSR pipeline to analyze SHM and/or CSR in BCR rearrangements. It analy

Strategies for B-cell receptor repertoire analysis in primary immunodeficiencies: from severe combined immunodeficiency to common variable immunodeficiency

The antigen receptor repertoires of B and T cells form the basis of the adaptive immune response. The repertoires should be sufficiently diverse to recognize all possible pathogens. However, careful selection is needed to prevent responses to self or harmless antigens. Limited antigen receptor repertoire diversity leads to immunodeficiency, whereas unselected or misdirected repertoires can result in autoimmunity. The antigen receptor repertoire harbors information about abnormalities in many immunological disorders. Recent developments in next generation sequencing allow the analysis of the antigen receptor repertoire in much greater detail than ever before. Analyzing the antigen receptor repertoire in patients with mutations in genes responsible for the generation of the antigen receptor repertoire will give new insights into repertoire formation and selection. In this perspective we describe strategies and considerations for analysis of the naive and antigen selected B-cell repertoires in primary immunodeficiency (PID) patients with a focus on severe combined immunodeficiency (SCID) and common variable immunodeficiency (CVID)

Public Clonotypes and Convergent Recombination Characterize the Naïve CD8+ T-Cell Receptor Repertoire of Extremely Preterm Neonates

Respiratory support improvements have aided survival of premature neonates, but infection susceptibility remains a predominant problem. We previously reported that neonatal mice have a rapidly evolving T-cell receptor (TCR) repertoire that impairs CD8+ T cell immunity. To understand the impact of prematurity on the human CD8+ TCR repertoire, we performed next-generation sequencing of the complementarity-determining region 3 (CDR3) from the rearranged TCR variable beta (Vβ) in sorted, naïve CD8+ T cells from extremely preterm neonates (23–27 weeks gestation), term neonates (37–41 weeks gestation), children (16–56 months), and adults (25–50 years old). Strikingly, preterm neonates had an increased frequency of public clonotypes shared between unrelated individuals. Public clonotypes identified in preterm infants were encoded by germline gene sequences, and some of these clonotypes persisted into adulthood. The preterm neonatal naïve CD8+ TCR repertoire exhibited convergent recombination, characterized by different nucleotide sequences encoding the same amino acid CDR3 sequence. As determined by Pielou’s evenness and iChao1 metrics, extremely preterm neonates have less clonality, and a much lower bound for the number of unique TCR within an individual preterm neonate, which indicates a less rich and diverse repertoire, as compared to term neonates, children, and adults. This suggests that T cell selection in the preterm neonate may be less stringent or different. Our analysis is the first to compare the TCR repertoire of naïve CD8+ T cells between viable preterm neonates and term neonates. We find preterm neonates have a repertoire immaturity which potentially contributes to their increased infection susceptibility. A developmentally regulated, evenly distributed repertoire in preterm neonates may lead to the inclusion of public TCR CDR3β sequences that overlap between unrelated individuals in the preterm repertoire