An important challenge in vaccine development is to figure out why a vaccine succeeds in some individuals and fails in others. Although antibody repertoires hold the key to answering this question, ...there have been very few personalized immunogenomics studies so far aimed at revealing how variations in immunoglobulin genes affect a vaccine response. We conducted an immunosequencing study of 204 calves vaccinated against bovine respiratory disease (BRD) with the goal to reveal variations in immunoglobulin genes and somatic hypermutations that impact the efficacy of vaccine response. Our study represents the largest longitudinal personalized immunogenomics study reported to date across all species, including humans. To analyze the generated data set, we developed an algorithm for identifying variations of the immunoglobulin genes (as well as frequent somatic hypermutations) that affect various features of the antibody repertoire and titers of neutralizing antibodies. In contrast to relatively short human antibodies, cattle have a large fraction of ultralong antibodies that have opened new therapeutic opportunities. Our study reveals that ultralong antibodies are a key component of the immune response against the costliest disease of beef cattle in North America. The detected variants of the cattle immunoglobulin genes, which are implicated in the success/failure of the BRD vaccine, have the potential to direct the selection of individual cattle for ongoing breeding programs.
Unexpected allergic reactions to peanut are the most common cause of fatal food-related anaphylaxis. Mechanisms underlying the variable severity of peanut-allergic reactions remain unclear.
We sought ...to expand mechanistic understanding of reaction severity in peanut allergy.
We performed an integrated transcriptomic and epigenomic study of peanut-allergic children as they reacted in vivo during double-blind, placebo-controlled peanut challenges. We integrated whole-blood transcriptome and CD4+ T-cell epigenome profiles to identify molecular signatures of reaction severity (ie, how severely a peanut-allergic child reacts when exposed to peanut). A threshold-weighted reaction severity score was calculated for each subject based on symptoms experienced during peanut challenge and the eliciting dose. Through linear mixed effects modeling, network construction, and causal mediation analysis, we identified genes, CpGs, and their interactions that mediate reaction severity. Findings were replicated in an independent cohort.
We identified 318 genes with changes in expression during the course of reaction associated with reaction severity, and 203 CpG sites with differential DNA methylation associated with reaction severity. After replicating these findings in an independent cohort, we constructed interaction networks with the identified peanut severity genes and CpGs. These analyses and leukocyte deconvolution highlighted neutrophil-mediated immunity. We identified NFKBIA and ARG1 as hubs in the networks and 3 groups of interacting key node CpGs and peanut severity genes encompassing immune response, chemotaxis, and regulation of macroautophagy. In addition, we found that gene expression of PHACTR1 and ZNF121 causally mediates the association between methylation at corresponding CpGs and reaction severity, suggesting that methylation may serve as an anchor upon which gene expression modulates reaction severity.
Our findings enhance current mechanistic understanding of the genetic and epigenetic architecture of reaction severity in peanut allergy.
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Fumarates are effective immunomodulators in multiple sclerosis but their mechanism of action remains elusive. Ntranos et al. show that the immunomodulatory effect of fumarates is due to epigenetic ...regulation of brain-homing T cells. Treatment with fumarates leads to hypermethylation of microRNA-21, preventing its upregulation in encephalitogenic T-helper and T-cytotoxic cells.
Abstract
Cell-permeable formulations of metabolites, such as fumaric acid esters, have been used as highly effective immunomodulators in patients with multiple sclerosis and yet their mechanism of action remains elusive. Since fumaric acid esters are metabolites, and cell metabolism is highly intertwined with the epigenetic regulation of gene expression, we investigated whether this metabolic-epigenetic interplay could be leveraged for therapeutic purposes. To this end we recruited 47 treatment-naïve and 35 fumaric acid ester-treated patients with multiple sclerosis, as well as 16 glatiramer acetate-treated patients as a non-metabolite treatment control. Here we identify a significant immunomodulatory effect of fumaric acid esters on the expression of the brain-homing chemokine receptor CCR6 in CD4 and CD8 T cells of patients with multiple sclerosis, which include T helper-17 and T cytotoxic-17 cells. We report differences in DNA methylation of CD4 T cells isolated from untreated and treated patients with multiple sclerosis, using the Illumina EPIC 850K BeadChip. We first demonstrate that Krebs cycle intermediates, such as fumaric acid esters, have a significantly higher impact on epigenome-wide DNA methylation changes in CD4 T cells compared to amino-acid polymers such as glatiramer acetate. We then define a fumaric acid ester treatment-specific hypermethylation effect on microRNA MIR-21, which is critical for the differentiation of T helper-17 cells. This hypermethylation effect was attributed to the subpopulation of T helper-17 cells using a decomposition analysis and was further validated in an independent prospective cohort of seven patients before and after treatment with fumaric acid esters. In vitro treatment of CD4 and CD8 T cells with fumaric acid esters supported a direct and dose-dependent effect on DNA methylation at the MIR-21 promoter. Finally, the upregulation of miR-21 transcripts and CCR6 expression was inhibited if CD4 or CD8 T cells stimulated under T helper-17 or T cytotoxic-17 polarizing conditions were treated with fumaric acid esters in vitro. These data collectively define a direct link between fumaric acid ester treatment and hypermethylation of the MIR-21 locus in both CD4 and CD8 T cells and suggest that the immunomodulatory effect of fumaric acid esters in multiple sclerosis is at least in part due to the epigenetic regulation of the brain-homing CCR6+ CD4 and CD8 T cells.
Sensitization to human leukocyte antigens (HLA) is a persistent problem in heart transplant (HT) candidates. We sought to characterize the anti-HLA antibody and circulating B cell repertoire in a ...cohort of highly sensitized HT candidates.
We assessed immunoglobulin G (IgG) and immunoglobulin M (IgM) anti-HLA antibodies using Luminex single antigen bead assays in a cohort of 11 highly sensitized (HS; calculated panel reactive antibody ≥ 90%) and 3 mildly sensitized (MS) candidates. We also performed B cell receptor repertoire sequencing (BCRseq) in HS candidates and 33 non-candidate controls. HLA antibody strength was measured by mean fluorescence intensity (MFI).
We found that IgM anti-HLA antibodies were present in all HS candidates, but with a lower breadth and strength as compared to IgG. When anti-HLA IgG specificities intersected with IgM, binding strength was higher. In contrast, there were IgM but no intersecting IgG specificities for the MS group. In four candidates in the HS group, IgG anti-HLA antibodies decreased in both breadth and strength after HT, but the decrease in strength was smaller if the IgG possessed a specificity that intersected with pre-transplant IgM. BCRseq revealed larger B cell clonotypes in HS candidates but similar diversity as compared to controls.
IgM marks IgG anti-HLA antibodies with higher strength before HT and persistence after HT. The presence of IgM intersecting IgG for an anti-HLA specificity may be a useful approach to determine which donor HLA should be avoided for a sensitized candidate.
Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we ...present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.
High-throughput sequencing of adaptive immune receptor repertoires (AIRR, i.e., IG and TR) has revolutionized the ability to carry out large-scale experiments to study the adaptive immune response. ...Since the method was first introduced in 2009, AIRR sequencing (AIRR-Seq) has been applied to survey the immune state of individuals, identify antigen-specific or immune-state-associated signatures of immune responses, study the development of the antibody immune response, and guide the development of vaccines and antibody therapies. Recent advancements in the technology include sequencing at the single-cell level and in parallel with gene expression, which allows the introduction of multi-omics approaches to understand in detail the adaptive immune response. Analyzing AIRR-seq data can prove challenging even with high-quality sequencing, in part due to the many steps involved and the need to parameterize each step. In this chapter, we outline key factors to consider when preprocessing raw AIRR-Seq data and annotating the genetic origins of the rearranged receptors. We also highlight a number of common difficulties with common AIRR-seq data processing and provide strategies to address them.
The zebrafish
lens opaque (
lop) mutant was previously isolated in a genetic screen and shown to lack rod and cone photoreceptors and exhibit lens opacity, or cataract, at 7 days post-fertilization ...(dpf). In this manuscript, we provide four different lines of evidence demonstrating that the
lop phenotype results from a defect in the
cdipt (
phosphatidylinositol (PI) synthase; CDP-diacylglycerol-inositol 3-phosphatidyltransferase) gene. First, DNA sequence analysis revealed that the
lop mutant contained a missense mutation in the
lop open reading frame, which yields a nonconservative amino acid substitution (Ser-111-Cys) within the PI synthase catalytic domain. Second, morpholino-mediated knockdown of the
cdipt-encoded PI synthase protein phenocopied the
cdipt
lop/lop
mutant, with abnormal lens epithelial and secondary fiber cell morphologies and reduced numbers of photoreceptors. Third, microinjection of
in vitro transcribed, wild-type
cdipt mRNA into 1–4 cell stage
cdipt
lop/lop
embryos significantly reduced the percentage of larvae displaying lens opacity at 7 dpf. Fourth, a
cdipt retroviral-insertion allele,
cdipt
hi559
, exhibited similar lens and retinal abnormalities and failed to complement the
cdipt
lop
mutant phenotype.
To determine the initial cellular defects associated with the
cdipt mutant, we examined homozygous
cdipt
hi559/hi559
mutants prior to gross lens opacification at 6 dpf. The
cdipt
hi559/hi559
mutants first exhibited photoreceptor layer disruption and photoreceptor cell death at 3 and 4 dpf, respectively, followed by lens dismorphogenesis by 5 dpf. RT-PCR revealed that the
cdipt gene is maternally expressed and continues to be transcribed throughout development and into adulthood, in a wide variety of tissues. Using an anti-zebrafish PI synthase polyclonal antiserum, we localized the protein throughout the developing eye, including the photoreceptor layer and lens cortical secondary fiber cells. As expected, the polyclonal antiserum revealed that the PI synthase protein was reduced in amount in both the
cdipt
lop/lop
and
cdipt
hi559/hi559
mutants. Furthermore, we used a heterologous yeast phenotypic complementation assay to confirm that the wild-type zebrafish
cdipt allele encodes functional PI synthase activity. Taken together, the
cdipt-encoded PI synthase is required for survival of photoreceptor cells and lens epithelial and secondary cortical fiber cells. These zebrafish
cdipt alleles represent excellent
in vivo genetic tools to study the role of phosphatidylinositol and its phosphorylated derivatives in lens and photoreceptor development and maintenance.
► The lens opaque mutation is a missense change in PI (phosphatidylinositol) synthase. ► PI synthase is required for photoreceptor cell viability. ► PI synthase is necessary to maintain lens epithelial cells. ► PI synthase is required for lens cortical secondary fiber differentiation.
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