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Asset Tag: immunology

September 22, 2019

Long reads: their purpose and place.

In recent years long-read technologies have moved from being a niche and specialist field to a point of relative maturity likely to feature frequently in the genomic landscape. Analogous to next generation sequencing, the cost of sequencing using long-read technologies has materially dropped whilst the instrument throughput continues to increase. Together these changes present the prospect of sequencing large numbers of individuals with the aim of fully characterizing genomes at high resolution. In this article, we will endeavour to present an introduction to long-read technologies showing: what long reads are; how they are distinct from short reads; why long reads are useful and how they are being used. We will highlight the recent developments in this field, and the applications and potential of these technologies in medical research, and clinical diagnostics and therapeutics.

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September 22, 2019

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo.

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September 22, 2019

Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques.

Pig-tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) studies. Their infectability with minimally modified HIV makes them a uniquely valuable animal model to mimic human infection with HIV and progression to acquired immunodeficiency syndrome (AIDS). However, variation in the pig-tailed macaque major histocompatibility complex (MHC) and the impact of individual transcripts on the pathogenesis of HIV and other infectious diseases is understudied compared to that of rhesus and cynomolgus macaques. In this study, we used Pacific Biosciences single-molecule real-time circular consensus sequencing to describe full-length MHC class I (MHC-I) transcripts for 194 pig-tailed macaques from three breeding centers. We then used the full-length sequences to infer Mane-A and Mane-B haplotypes containing groups of MHC-I transcripts that co-segregate due to physical linkage. In total, we characterized full-length open reading frames (ORFs) for 313 Mane-A, Mane-B, and Mane-I sequences that defined 86 Mane-A and 106 Mane-B MHC-I haplotypes. Pacific Biosciences technology allows us to resolve these Mane-A and Mane-B haplotypes to the level of synonymous allelic variants. The newly defined haplotypes and transcript sequences containing full-length ORFs provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS-like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also benefits transplant research by allowing researchers to more specifically match haplotypes between donors and recipients to the level of nonsynonymous allelic variation, thus reducing the risk of graft-versus-host disease.

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September 22, 2019

Reduction of nonspecificity motifs in synthetic antibody libraries.

Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on the surface of yeast, identifying both individual amino acids and motifs within the complementarity-determining regions which contribute to the phenotype. We find enrichment of glycine, valine, and arginine as both individual amino acids and as a part of motifs, and additionally enrichment of motifs containing tryptophan. Insertion of any of these motifs into the complementarity-determining region H3 of a “clean” antibody increased its nonspecificity, with greatest increases in antibodies containing Trp or Val motifs. We next applied these rules to the creation of a synthetic diversity library based on natural frameworks with significantly decreased incorporation of such motifs and demonstrated its ability to isolate binders to a wide panel of antigens. This work both provides a greater understanding of the drivers of nonspecificity and provides design rules to increase efficiency in the isolation of antibodies with drug-like properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

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September 22, 2019

Predicting an HLA-DPB1 expression marker based on standard DPB1 genotyping: Linkage analysis of over 32,000 samples.

The risk of acute graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation is increased with donor-recipient HLA-DPB1 allele mismatching. The single-nucleotide polymorphism (SNP) rs9277534 within the 3′ untranslated region (UTR) correlates with HLA-DPB1 allotype expression and serves as a marker for permissive HLA-DPB1 mismatches. Since rs9277534 is not routinely typed, we analyzed 32,681 samples of mostly European ancestry to investigate if the rs9277534 allele can be reliably imputed from standard DPB1 genotyping. We confirmed the previously-defined linkages between rs9277534 and 18 DPB1 alleles and established additional linkages for 46 DPB1 alleles. Based on these linkages, the rs9277534 allele could be predicted for 99.6% of the samples based on DPB1 genotypes (99.99% concordance). We demonstrate that 100% prediction accuracy could be achieved if the prediction utilized exon 3 sequence information. DPB1 genotyping based on exon 2 data alone allows no unambiguous rs9277534 allele prediction but was estimated to maintain 99% accuracy for samples of European descent. We conclude that DPB1 genotyping is sufficient to infer the DPB1 expression marker rs9277534 with high accuracy. This information could be used to select donors with permissive HLA-DPB1 mismatches without directly screening for rs9277534. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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September 22, 2019

An ancient integration in a plant NLR is maintained as a trans-species polymorphism

Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens. Nucleotide-binding leucine-rich repeat (NLR) proteins are one class of cytoplasmic immune receptors whose genes commonly show signatures of adaptive evolution. While it is known that balancing selection contributes to maintaining high intraspecific allelic diversity, the evolutionary mechanism that influences the transmission of alleles during speciation remains unclear. The barley Mla locus has over 30 described alleles conferring isolate-specific resistance to barley powdery mildew and contains three NLR families (RGH1, RGH2, and RGH3). We discovered (using sequence capture and RNAseq) the presence of a novel integrated Exo70 domain in RGH2 in the Mla3 haplotype. Allelic variation across barley accessions includes presence/absence of the integrated domain in RGH2. Expanding our search to several Poaceae species, we found shared interspecific conservation in the RGH2-Exo70 integration. We hypothesise that balancing selection has maintained allelic variation at Mla as a trans-species polymorphism over 24 My, thus contributing to and preserving interspecific allelic diversity during speciation.

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September 22, 2019

Pathogen-specific binding soluble Down syndrome cell adhesion molecule (Dscam) regulates phagocytosis via membrane-bound Dscam in crab.

The Down syndrome cell adhesion molecule (Dscam) gene is an extraordinary example of diversity that can produce thousands of isoforms and has so far been found only in insects and crustaceans. Cumulative evidence indicates that Dscam may contribute to the mechanistic foundations of specific immune responses in insects. However, the mechanism and functions of Dscam in relation to pathogens and immunity remain largely unknown. In this study, we identified the genome organization and alternative Dscam exons from Chinese mitten crab, Eriocheir sinensis. These variants, designated EsDscam, potentially produce 30,600 isoforms due to three alternatively spliced immunoglobulin (Ig) domains and a transmembrane domain. EsDscam was significantly upregulated after bacterial challenge at both mRNA and protein levels. Moreover, bacterial specific EsDscam isoforms were found to bind specifically with the original bacteria to facilitate efficient clearance. Furthermore, bacteria-specific binding of soluble EsDscam via the complete Ig1-Ig4 domain significantly enhanced elimination of the original bacteria via phagocytosis by hemocytes; this function was abolished by partial Ig1-Ig4 domain truncation. Further studies showed that knockdown of membrane-bound EsDscam inhibited the ability of EsDscam with the same extracellular region to promote bacterial phagocytosis. Immunocytochemistry indicated colocalization of the soluble and membrane-bound forms of EsDscam at the hemocyte surface. Far-Western and coimmunoprecipitation assays demonstrated homotypic interactions between EsDscam isoforms. This study provides insights into a mechanism by which soluble Dscam regulates hemocyte phagocytosis via bacteria-specific binding and specific interactions with membrane-bound Dscam as a phagocytic receptor.

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September 22, 2019

Somatic hypermutation of T cell receptor a chain contributes to selection in nurse shark thymus.

Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on a chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRa was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRa utilizes SHM to broaden diversification of the primary aß T cell repertoire in sharks, the first reported use in vertebrates.© 2018, Ott et al.

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September 22, 2019

Genomic architecture of haddock (Melanogrammus aeglefinus) shows expansions of innate immune genes and short tandem repeats.

Increased availability of genome assemblies for non-model organisms has resulted in invaluable biological and genomic insight into numerous vertebrates, including teleosts. Sequencing of the Atlantic cod (Gadus morhua) genome and the genomes of many of its relatives (Gadiformes) demonstrated a shared loss of the major histocompatibility complex (MHC) II genes 100 million years ago. An improved version of the Atlantic cod genome assembly shows an extreme density of tandem repeats compared to other vertebrate genome assemblies. Highly contiguous assemblies are therefore needed to further investigate the unusual immune system of the Gadiformes, and whether the high density of tandem repeats found in Atlantic cod is a shared trait in this group.Here, we have sequenced and assembled the genome of haddock (Melanogrammus aeglefinus) – a relative of Atlantic cod – using a combination of PacBio and Illumina reads. Comparative analyses reveal that the haddock genome contains an even higher density of tandem repeats outside and within protein coding sequences than Atlantic cod. Further, both species show an elevated number of tandem repeats in genes mainly involved in signal transduction compared to other teleosts. A characterization of the immune gene repertoire demonstrates a substantial expansion of MCHI in Atlantic cod compared to haddock. In contrast, the Toll-like receptors show a similar pattern of gene losses and expansions. For the NOD-like receptors (NLRs), another gene family associated with the innate immune system, we find a large expansion common to all teleosts, with possible lineage-specific expansions in zebrafish, stickleback and the codfishes.The generation of a highly contiguous genome assembly of haddock revealed that the high density of short tandem repeats as well as expanded immune gene families is not unique to Atlantic cod – but possibly a feature common to all, or most, codfishes. A shared expansion of NLR genes in teleosts suggests that the NLRs have a more substantial role in the innate immunity of teleosts than other vertebrates. Moreover, we find that high copy number genes combined with variable genome assembly qualities may impede complete characterization of these genes, i.e. the number of NLRs in different teleost species might be underestimates.

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September 22, 2019

The antibody loci of the domestic goat (Capra hircus).

The domestic goat (Capra hircus) is an important ruminant species both as a source of antibody-based reagents for research and biomedical applications and as an economically important animal for agriculture, particularly for developing nations that maintain most of the global goat population. Characterization of the loci encoding the goat immune repertoire would be highly beneficial for both vaccine and immune reagent development. However, in goat and other species whose reference genomes were generated using short-read sequencing technologies, the immune loci are poorly assembled as a result of their repetitive nature. Our recent construction of a long-read goat genome assembly (ARS1) has facilitated characterization of all three antibody loci with high confidence and comparative analysis to cattle. We observed broad similarity of goat and cattle antibody-encoding loci but with notable differences that likely influence formation of the functional antibody repertoire. The goat heavy-chain locus is restricted to only four functional and nearly identical IGHV genes, in contrast to the ten observed in cattle. Repertoire analysis indicates that light-chain usage is more balanced in goats, with greater representation of kappa light chains (~ 20-30%) compared to that in cattle (~ 5%). The present study represents the first characterization of the goat antibody loci and will help inform future investigations of their antibody responses to disease and vaccination.

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September 22, 2019

Discovery of gorilla MHC-C expressing C1 ligand for KIR.

In comparison to humans and chimpanzees, gorillas show low diversity at MHC class I genes (Gogo), as reflected by an overall reduced level of allelic variation as well as the absence of a functionally important sequence motif that interacts with killer cell immunoglobulin-like receptors (KIR). Here, we use recently generated large-scale genomic sequence data for a reassessment of allelic diversity at Gogo-C, the gorilla orthologue of HLA-C. Through the combination of long-range amplifications and long-read sequencing technology, we obtained, among the 35 gorillas reanalyzed, three novel full-length genomic sequences including a coding region sequence that has not been previously described. The newly identified Gogo-C*03:01 allele has a divergent recombinant structure that sets it apart from other Gogo-C alleles. Domain-by-domain phylogenetic analysis shows that Gogo-C*03:01 has segments in common with Gogo-B*07, the additional B-like gene that is present on some gorilla MHC haplotypes. Identified in ~ 50% of the gorillas analyzed, the Gogo-C*03:01 allele exclusively encodes the C1 epitope among Gogo-C allotypes, indicating its important function in controlling natural killer cell (NK cell) responses via KIR. We further explored the hypothesis whether gorillas experienced a selective sweep which may have resulted in a general reduction of the gorilla MHC class I repertoire. Our results provide little support for a selective sweep but rather suggest that the overall low Gogo class I diversity can be best explained by drastic demographic changes gorillas experienced in the ancient and recent past.

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September 22, 2019

Genetic and molecular basis of the immune system in the brachiopod Lingula anatina.

The extension of comparative immunology to non-model systems, such as mollusks and annelids, has revealed an unexpected diversity in the complement of immune receptors and effectors among evolutionary lineages. However, several lophotrochozoan phyla remain unexplored mainly due to the lack of genomic resources. The increasing accessibility of high-throughput sequencing technologies offers unique opportunities for extending genome-wide studies to non-model systems. As a result, the genome-based study of the immune system in brachiopods allows a better understanding of the alternative survival strategies developed by these immunologically neglected phyla. Here we present a detailed overview of the molecular components of the immune system identified in the genome of the brachiopod Lingula anatina. Our findings reveal conserved intracellular signaling pathways as well as unique strategies for pathogen detection and killing in brachiopods. Copyright © 2017 Elsevier Ltd. All rights reserved.

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