IGHV allele similarity clustering improves genotype inference from adaptive immune receptor repertoire sequencing data.

In adaptive immune receptor repertoire analysis, determining the germline variable (V) allele associated with each T- and B-cell receptor sequence is a crucial step. This process is highly impacted by allele annotations. Aligning sequences, assigning them to specific germline alleles, and inferring individual genotypes are challenging when the repertoire is highly mutated, or sequence reads do not cover the whole V region. Here, we propose an alternative naming scheme for the V alleles, as well as a novel method to infer individual genotypes. We demonstrate the strengths of the two by comparing their outcomes to other genotype inference methods. We validate the genotype approach with independent genomic long-read data. The naming scheme is compatible with current annotation tools and pipelines. Analysis results can be converted from the proposed naming scheme to the nomenclature determined by the International Union of Immunological Societies (IUIS). Both the naming scheme and the genotype procedure are implemented in a freely available R package (PIgLET https://bitbucket.org/yaarilab/piglet). To allow researchers to further explore the approach on real data and to adapt it for their uses, we also created an interactive website (https://yaarilab.github.io/IGHV_reference_book).

AIRR community curation and standardised representation for immunoglobulin and T cell receptor germline sets.

Analysis of an individual's immunoglobulin or T cell receptor gene repertoire can provide important insights into immune function. High-quality analysis of adaptive immune receptor repertoire sequencing data depends upon accurate and relatively complete germline sets, but current sets are known to be incomplete. Established processes for the review and systematic naming of receptor germline genes and alleles require specific evidence and data types, but the discovery landscape is rapidly changing. To exploit the potential of emerging data, and to provide the field with improved state-of-the-art germline sets, an intermediate approach is needed that will allow the rapid publication of consolidated sets derived from these emerging sources. These sets must use a consistent naming scheme and allow refinement and consolidation into genes as new information emerges. Name changes should be minimised, but, where changes occur, the naming history of a sequence must be traceable. Here we outline the current issues and opportunities for the curation of germline IG/TR genes and present a forward-looking data model for building out more robust germline sets that can dovetail with current established processes. We describe interoperability standards for germline sets, and an approach to transparency based on principles of findability, accessibility, interoperability, and reusability.

AIRR-C IG Reference Sets: curated sets of immunoglobulin heavy and light chain germline genes.

Introduction: Analysis of an individual's immunoglobulin (IG) gene repertoire requires the use of high-quality germline gene reference sets. When sets only contain alleles supported by strong evidence, AIRR sequencing (AIRR-seq) data analysis is more accurate and studies of the evolution of IG genes, their allelic variants and the expressed immune repertoire is therefore facilitated. Methods: The Adaptive Immune Receptor Repertoire Community (AIRR-C) IG Reference Sets have been developed by including only human IG heavy and light chain alleles that have been confirmed by evidence from multiple high-quality sources. To further improve AIRR-seq analysis, some alleles have been extended to deal with short 3' or 5' truncations that can lead them to be overlooked by alignment utilities. To avoid other challenges for analysis programs, exact paralogs (e.g. IGHV1-69*01 and IGHV1-69D*01) are only represented once in each set, though alternative sequence names are noted in accompanying metadata. Results and discussion: The Reference Sets include less than half the previously recognised IG alleles (e.g. just 198 IGHV sequences), and also include a number of novel alleles: 8 IGHV alleles, 2 IGKV alleles and 5 IGLV alleles. Despite their smaller sizes, erroneous calls were eliminated, and excellent coverage was achieved when a set of repertoires comprising over 4 million V(D)J rearrangements from 99 individuals were analyzed using the Sets. The version-tracked AIRR-C IG Reference Sets are freely available at the OGRDB website (https://ogrdb.airr-community.org/germline_sets/Human) and will be regularly updated to include newly observed and previously reported sequences that can be confirmed by new high-quality data.

Immunoglobulin genes, reproductive isolation and vertebrate speciation.

Reproductive isolation drives the formation of new species, and many genes contribute to this through Dobzhansky-Muller incompatibilities (DMIs). These incompatibilities occur when gene divergence affects loci encoding interacting products such as receptors and their ligands. We suggest here that the nature of vertebrate immunoglobulin (IG) genes must make them prone to DMIs. The genes of these complex loci form functional genes through the process of recombination, giving rise to a repertoire of heterodimeric receptors of incredible diversity. This repertoire, within individuals and within species, must defend against pathogens but must also avoid pathogenic self-reactivity. We suggest that this avoidance of autoimmunity is only achieved through a coordination of evolution between heavy- and light-chain genes, and between these genes and the rest of the genome. Without coordinated evolution, the hybrid offspring of two diverging populations will carry a heavy burden of DMIs, resulting in a loss of fitness. Critical incompatibilities could manifest as incompatibilities between a mother and her divergent offspring. During fetal development, biochemical differences between the parents of hybrid offspring could make their offspring a target of the maternal immune system. This hypothesis was conceived in the light of recent insights into the population genetics of IG genes. This has suggested that antibody genes are probably as susceptible to evolutionary forces as other parts of the genome. Further repertoire studies in human and nonhuman species should now help determine whether antibody genes have been part of the evolutionary forces that drive the development of species.

Social comparisons: A potential mechanism linking problematic social media use with depression.

Background and aims: The majority of Australians are regular users of social media, especially young adults. Of concern, is that a minority of people appear to use social media in an addictive or problematic way which is associated with negative psychological outcomes such as depression. Social comparisons, where users compare themselves to others on social media, have also been linked with depression. Therefore, the key aim of the study was to determine whether social comparisons mediate the relationship between Problematic Social Media Use (PSMU) and depression. Method: In a two-part study 144 participants (65 females) answered a series of self-report questions assessing factors relating to PSMU and then came into the lab to view a series of social media images, (pre-tested to be upward or downward comparisons). Results: Females used social media more problematically, liked more upward than downward comparison images and compared themselves more negatively to others on social media than did males. Higher PSMU scores were associated with depression and low self-esteem and comparing oneself more negatively to others on social media. Finally, focusing on upward comparisons and a tendency to make negative comparisons to others on social media partially mediated the association between PSMU and depression. Discussion and conclusions: Social comparisons may function as a mechanism linking PSMU with negative psychological outcomes. Clinical interventions for individuals with PSMU which reduce the focus on upward social comparisons may also reduce negative psychological outcomes such as depression.

A BALB/c IGHV Reference Set, Defined by Haplotype Analysis of Long-Read VDJ-C Sequences From F1 (BALB/c x C57BL/6) Mice.

The immunoglobulin genes of inbred mouse strains that are commonly used in models of antibody-mediated human diseases are poorly characterized. This compromises data analysis. To infer the immunoglobulin genes of BALB/c mice, we used long-read SMRT sequencing to amplify VDJ-C sequences from F1 (BALB/c x C57BL/6) hybrid animals. Strain variations were identified in the Ighm and Ighg2b genes, and analysis of VDJ rearrangements led to the inference of 278 germline IGHV alleles. 169 alleles are not present in the C57BL/6 genome reference sequence. To establish a set of expressed BALB/c IGHV germline gene sequences, we computationally retrieved IGHV haplotypes from the IgM dataset. Haplotyping led to the confirmation of 162 BALB/c IGHV gene sequences. A musIGHV398 pseudogene variant also appears to be present in the BALB/cByJ substrain, while a functional musIGHV398 gene is highly expressed in the BALB/cJ substrain. Only four of the BALB/c alleles were also observed in the C57BL/6 haplotype. The full set of inferred BALB/c sequences has been used to establish a BALB/c IGHV reference set, hosted at https://ogrdb.airr-community.org. We assessed whether assemblies from the Mouse Genome Project (MGP) are suitable for the determination of the genes of the IGH loci. Only 37 (43.5%) of the 85 confirmed IMGT-named BALB/c IGHV and 33 (42.9%) of the 77 confirmed non-IMGT IGHV were found in a search of the MGP BALB/cJ genome assembly. This suggests that current MGP assemblies are unsuitable for the comprehensive documentation of germline IGHVs and more efforts will be needed to establish strain-specific reference sets.

T cell receptor beta germline variability is revealed by inference from repertoire data.

BACKGROUND: T and B cell receptor (TCR, BCR) repertoires constitute the foundation of adaptive immunity. Adaptive immune receptor repertoire sequencing (AIRR-seq) is a common approach to study immune system dynamics. Understanding the genetic factors influencing the composition and dynamics of these repertoires is of major scientific and clinical importance. The chromosomal loci encoding for the variable regions of TCRs and BCRs are challenging to decipher due to repetitive elements and undocumented structural variants. METHODS: To confront this challenge, AIRR-seq-based methods have recently been developed for B cells, enabling genotype and haplotype inference and discovery of undocumented alleles. However, this approach relies on complete coverage of the receptors' variable regions, whereas most T cell studies sequence a small fraction of that region. Here, we adapted a B cell pipeline for undocumented alleles, genotype, and haplotype inference for full and partial AIRR-seq TCR data sets. The pipeline also deals with gene assignment ambiguities, which is especially important in the analysis of data sets of partial sequences. RESULTS: From the full and partial AIRR-seq TCR data sets, we identified 39 undocumented polymorphisms in T cell receptor Beta V (TRBV) and 31 undocumented 5 ' UTR sequences. A subset of these inferences was also observed using independent genomic approaches. We found that a single nucleotide polymorphism differentiating between the two documented T cell receptor Beta D2 (TRBD2) alleles is strongly associated with dramatic changes in the expressed repertoire. CONCLUSIONS: We reveal a rich picture of germline variability and demonstrate how a single nucleotide polymorphism dramatically affects the composition of the whole repertoire. Our findings provide a basis for annotation of TCR repertoires for future basic and clinical studies.

Germline immunoglobulin genes: disease susceptibility genes hidden in plain sight?

Immunoglobulin genes are rarely considered as disease susceptibility genes despite their obvious and central contributions to immune function. This appears to be a consequence of historical views on antibody repertoire formation that no longer stand, and of difficulties that until recently surrounded the documentation of the suite of antibody genes in any individual. If these important genes are to be accessible to GWAS studies, allelic variation within the human population needs to be better documented, and a curated set of genomic variations associated with antibody genes needs to be formulated. Repertoire studies arising from the COVID-19 pandemic provide an opportunity to meet these needs, and may provide insights into the profound variability that is seen in outcomes to this infection.

VDJbase: an adaptive immune receptor genotype and haplotype database.

VDJbase is a publicly available database that offers easy searching of data describing the complete sets of gene sequences (genotypes and haplotypes) inferred from adaptive immune receptor repertoire sequencing datasets. VDJbase is designed to act as a resource that will allow the scientific community to explore the genetic variability of the immunoglobulin (Ig) and T cell receptor (TR) gene loci. It can also assist in the investigation of Ig- and TR-related genetic predispositions to diseases. Our database includes web-based query and online tools to assist in visualization and analysis of the genotype and haplotype data. It enables users to detect those alleles and genes that are significantly over-represented in a particular population, in terms of genotype, haplotype and gene expression. The database website can be freely accessed at https://www.vdjbase.org/, and no login is required. The data and code use creative common licenses and are freely downloadable from https://bitbucket.org/account/user/yaarilab/projects/GPHP.

A comparison of immunoglobulin IGHV, IGHD and IGHJ genes in wild-derived and classical inbred mouse strains.

The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus. We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflects differences in subspecies origin. To investigate this hypothesis, we conducted high-throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ) and diversity (IGHD) genes in four inbred wild-derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ and PWD/PhJ) and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline IGHV sequences were inferred in the wild-derived strains, including 247 not curated in the international ImMunoGeneTics information system. By contrast, 83/84 inferred NOD IGHV genes had previously been observed in C57BL/6 mice. Variability among the strains examined was observed for only a single IGHJ gene, involving a description of a novel allele. By contrast, unexpected variation was found in the IGHD gene loci, with four previously unreported IGHD gene sequences being documented. Very few IGHV sequences of C57BL/6 and BALB/c mice were shared with strains representing major subspecies, suggesting that their IGH loci may be complex mosaics of genes of disparate origins. This suggests a similar level of diversity is likely present in the IGH loci of other classical inbred strains. This must now be documented if we are to properly understand interstrain variation in models of antibody-mediated disease.

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.

Mosaic deletion patterns of the human antibody heavy chain gene locus shown by Bayesian haplotyping.

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.

A Proposed New Nomenclature for the Immunoglobulin Genes of Mus musculus.

Mammalian immunoglobulin (IG) genes are found in complex loci that contain hundreds of highly similar pseudogenes, functional genes and repetitive elements, which has made their investigation particularly challenging. High-throughput sequencing has provided new avenues for the investigation of these loci, and has recently been applied to study the IG genes of important inbred mouse strains, revealing unexpected differences between their IG loci. This demonstrated that the structural differences are of such magnitude that they call into question the merits of the current mouse IG gene nomenclatures. Three nomenclatures for the mouse IG heavy chain locus (Igh) are presently in use, and they are all positional nomenclatures using the C57BL/6 genome reference sequence as their template. The continued use of these nomenclatures requires that genes of other inbred strains be confidently identified as allelic variants of C57BL/6 genes, but this is clearly impossible. The unusual breeding histories of inbred mouse strains mean that, regardless of the genetics of wild mice, no single ancestral origin for the IG loci exists for laboratory mice. Here we present a general discussion of the challenges this presents for any IG nomenclature. Furthermore, we describe principles that could be followed in the formulation of a solution to these challenges. Finally, we propose a non-positional nomenclature that accords with the guidelines of the International Mouse Nomenclature Committee, and outline strategies that can be adopted to meet the nomenclature challenges if three systems are to give way to a new one.

Immunoglobulin Light Chain Gene Rearrangements, Receptor Editing and the Development of a Self-Tolerant Antibody Repertoire.

Discussion of the antibody repertoire usually emphasizes diversity, but a conspicuous feature of the light chain repertoire is its lack of diversity. The diversity of reported allelic variants of germline light chain genes is also limited, even in well-studied species. In this review, the implications of this lack of diversity are considered. We explore germline and rearranged light chain genes in a variety of species, with a particular focus on human and mouse genes. The importance of the number, organization and orientation of the genes for the control of repertoire development is discussed, and we consider how primary rearrangements and receptor editing together shape the expressed light chain repertoire. The resulting repertoire is dominated by just a handful of IGKV and IGLV genes. It has been hypothesized that an important function of the light chain is to guard against self-reactivity, and the role of secondary rearrangements in this process could explain the genomic organization of the light chain genes. It could also explain why the light chain repertoire is so limited. Heavy and light chain genes may have co-evolved to ensure that suitable light chain partners are usually available for each heavy chain that forms early in B cell development. We suggest that the co-evolved loci of the house mouse often became separated during the inbreeding of laboratory mice, resulting in new pairings of loci that are derived from different sub-species of the house mouse. A resulting vulnerability to self-reactivity could explain at least some mouse models of autoimmune disease.

On being the right size: antibody repertoire formation in the mouse and human.

The immune systems of all mammals include populations of B cells producing antibodies with incredibly diverse specificities. Repertoire diversity has been described as the "miracle of immunology," and it was long thought to be the result of essentially stochastic processes. Recently, however, analysis of high throughput gene sequencing data has shown that hard-wired biases in these processes result in antibody repertoires that are broadly predictable. The repertoires of mice and humans are both predictable, but they are strikingly different. In this review, features of the naïve antibody repertoires of the two species are contrasted. We show that the mouse repertoire includes a conspicuous population of public clonotypes that are shared by different individuals of an inbred strain. These clonotypes are the result of gene rearrangements that involve little gene processing. By skewing repertoire formation toward such sequences, which probably target commonly encountered pathogens, it may be that the relatively small mouse repertoire is appropriate and effective despite its size. Species like the mouse face challenges that are a direct consequence of their small body sizes and the limitations this places on the antibody arsenal-particularly early in ontogeny. We propose that it is the differences in the naïve repertoires of mice and humans, and the differences in the ways these repertoires are used, which ensure that the very different biological needs of the two species are met. The processes that contribute to repertoire formation may appear to be stochastic, but in both species, evolution has left little to chance.

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).

Functionalized Triblock Copolymer Vectors for the Treatment of Acute Lymphoblastic Leukemia.

The chemotherapeutic Parthenolide is an exciting new candidate for the treatment of acute lymphoblastic leukemia, but like many other small-molecule drugs, it has low aqueous solubility. As a consequence, Parthenolide can only be administered clinically in the presence of harmful cosolvents. Accordingly, we describe the synthesis, characterization, and testing of a range of biocompatible triblock copolymer micelles as particle-based delivery vectors for the hydrophobic drug Parthenolide. The drug-loaded particles are produced via an emulsion-to-micelle transition method, and the effects of introducing anionic and cationic surface charges on stability, drug sequestration, biocompatibility, and efficacy are investigated. Significantly, we demonstrate high levels of efficacy in the organic solvent-free systems against human mesenchymal stem cells and primary T-acute lymphoblastic leukemia patient cells, highlighting the effectiveness of the delivery vectors for the treatment of acute lymphoblastic leukemia.