High-throughput sequencing of immune repertoires in multiple sclerosis.
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ABSTRACT:
SUBMITTER: Lossius A
PROVIDER: S-EPMC4818741 | biostudies-other | 2016 Apr
REPOSITORIES: biostudies-other
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Project description:BackgroundRegulatory T (Treg) cells are important immune cells that are regulated by adaptive immunity in the composition of Treg-cell subsets and T-cell receptors (TCRs). Treg cells are related to most autoimmune diseases, such as rheumatoid arthritis (RA). In traditional Chinese medicine (TCM), RA is typically attributed to kidney deficiency (KD) associated with the immunosenescence that causes immune dysfunction and the impaired function of Treg cells. So far, however, no mechanism related to KD and immune repertoires has been identified in RA.MethodsFlow cytometry and high-throughput Treg-cell receptor sequencing were used to investigate the amount of different Treg-cell subsets and the diversity of TCRs between RA patients and healthy subjects, as well as between KD RA and non-KD RA patients. RT-qPCR was used to validate the high-throughput sequencing results.ResultsThe data showed that the amount of naïve Treg cells in KD patients was less than in non-KD RA patients (P = 0.004) with no significant differences observed between other subsets. In the TCR of Treg cells, the length of complementarity determining region 3 (CDR3) was low and clonotypes increased in the KD group compared with the non-KD group. The diversity and abundance of Treg TCRs were low, as determined by the Hill number. In addition, several V(D)J combinations, such as T-cell receptor beta variable 7-2 (TRBV7-2), TRBV11-1, TRBV13, TRBV15, and TRBJ2-3, varied significantly between the two groups, indicating that KD causes Treg dysfunction. RT-qPCR shows that FOXP3 expression in peripheral blood Treg is lower in KD than in non-KD.ConclusionThe results demonstrate the close correlation between KD and immune repertoires in RA and provide a new evaluation method for RA in TCM.
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Project description:Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some ? but not ? J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.
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Project description:BackgroundDeep sequencing of lymphocyte receptor repertoires has made it possible to comprehensively profile the clonal composition of lymphocyte populations. This opens the door for novel approaches to diagnose and prognosticate diseases with a driving immune component by identifying repertoire sequence patterns associated with clinical phenotypes. Indeed, recent studies support the feasibility of this, demonstrating an association between repertoire-level summary statistics (e.g., diversity) and patient outcomes for several diseases. In our own prior work, we have shown that six codons in VH4-containing genes in B cells from the cerebrospinal fluid of patients with relapsing remitting multiple sclerosis (RRMS) have higher replacement mutation frequencies than observed in healthy controls or patients with other neurological diseases. However, prior methods to date have been limited to focusing on repertoire-level summary statistics, ignoring the vast amounts of information in the millions of individual immune receptors comprising a repertoire. We have developed a novel method that addresses this limitation by using innovative approaches for accommodating the extraordinary sequence diversity of immune receptors and widely used machine learning approaches. We applied our method to RRMS, an autoimmune disease that is notoriously difficult to diagnose.ResultsWe use the biochemical features encoded by the complementarity determining region 3 of each B cell receptor heavy chain in every patient repertoire as input to a detector function, which is fit to give the correct diagnosis for each patient using maximum likelihood optimization methods. The resulting statistical classifier assigns patients to one of two diagnosis categories, RRMS or other neurological disease, with 87% accuracy by leave-one-out cross-validation on training data (N = 23) and 72% accuracy on unused data from a separate study (N = 102).ConclusionsOur method is the first to apply statistical learning to immune repertoires to aid disease diagnosis, learning repertoire-level labels from the set of individual immune repertoire sequences. This method produced a repertoire-based statistical classifier for diagnosing RRMS that provides a high degree of diagnostic capability, rivaling the accuracy of diagnosis by a clinical expert. Additionally, this method points to a diagnostic biochemical motif in the antibodies of RRMS patients, which may offer insight into the disease process.
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Project description:UnlabelledDriven by dramatic technological improvements, large-scale characterization of lymphocyte receptor repertoires via high-throughput sequencing is now feasible. Although promising, the high germline and somatic diversity, especially of B-cell immunoglobulin repertoires, presents challenges for analysis requiring the development of specialized computational pipelines. We developed the REpertoire Sequencing TOolkit (pRESTO) for processing reads from high-throughput lymphocyte receptor studies. pRESTO processes raw sequences to produce error-corrected, sorted and annotated sequence sets, along with a wealth of metrics at each step. The toolkit supports multiplexed primer pools, single- or paired-end reads and emerging technologies that use single-molecule identifiers. pRESTO has been tested on data generated from Roche and Illumina platforms. It has a built-in capacity to parallelize the work between available processors and is able to efficiently process millions of sequences generated by typical high-throughput projects.Availability and implementationpRESTO is freely available for academic use. The software package and detailed tutorials may be downloaded from http://clip.med.yale.edu/presto.
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Project description:ILC2s have the unique ability to produce cytokines such as constitutive production as well as rapid and abundant production during inflammation. We found that the unique type 2 cytokine prodution from ILC2s is regulated by post-transcriptional regulation. Here we identified the genes involved in the post-transcriptional regulation of type 2 cytokine production in ILC2s by comparing the gene expression between multiple immune cells.
2021-09-30 | GSE184841 | GEO
Project description:The humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. Assessment of the baseline antibody repertoires of unimmunized model animals will be useful as a benchmark for future immunization experiments. We characterized the heavy chain and kappa light chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. Intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. Multiple differences were found in both the heavy and kappa chain repertoires between OmniRats and humans including gene segment usage, CDR3 length distributions, class switch recombination, somatic hypermutation levels and in features of V(D)J recombination. The Inference and Generation of Repertoires (IGoR) software tool was used to model recombination in VH regions which allowed for the quantification of some of these differences. Diversity estimates of the OmniRat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. Despite variation between the species repertoires, a high frequency of OmniRat clonotypes were also found in the human repertoire. These data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies.
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Project description:We analyze and assess BCR repertoires of SLE patients before and after high dose glucocorticoid therapy to address two fundamental questions: (1) After the treatment, how the BCR repertoire of SLE patient change on the clone level? (2) How to screen putative autoantibody clone set from BCR repertoire of SLE patients? The PBMCs of two SLE patients (P1 and P2) at different time points were collected, and DNA of these samples were extracted. High-throughput sequencing technology was applied in detection of BCR repertoire. Finally, we used bioinformatic methodology to analyse sequence data. We found that these two patients lost some IGHV3 family genes usage after treatment compared with before treatment. For pairing of IGHV-IGHJ gene, no significant change was shown for each patient. In addition, analyses of the composition of H-CDR3 showed overall AA compositions of H-CDR3 at three time points in each SLE patients were very similar, and the results of H-CDR3 AA usage that had the same length (14 AA) and the same position were similar. Antinuclear antibody tests of SLE patients showed that level of some antinuclear antibodies reduced after treatment; however, there was no sign that the percentage of autoantibody clones in BCR repertoires would reduce. High dose glucocorticoid treatment in short term will have little impact on composition of BCR repertoire of SLE patient. Treatment can reduce the amount of autoantibody in the protein level, but may not reduce the percentage of autoantibody clones in BCR repertoire in the clonal level.
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Project description:BackgroundT cells are key regulators of immunity and one of the cells recruited in atherosclerosis and participated in various stages of the development of atherosclerosis. Characterizing T-cell receptor (TCR) repertoires is a priority of great scientific interest and potential clinical utility for the early diagnosis, risk stratification and prognostic evaluation of acute myocardial infarction (AMI).MethodsThe TCR repertoires in 21 subjects including 7 patients with non-ST-segment elevation myocardial infarction (NSTEMI), 6 patients with ST-segment elevation myocardial infarction (STEMI) and 8 subjects with normal coronary artery (NCA) as control were characterized by using high-throughput sequencing. Bioinformatics analysis were performed.ResultsPatients with NSTEMI displayed more diverse TCR sequences than NCA controls, but they had lower percentage of top 200 TCR sequences. However, no significant differences were observed between the patients with STEMI and NCA controls, but STEMI group had lower percentage of top 200 TCR sequences. T cells from patients with AMI and NCA controls showed a differential V and J gene usage, especially, significant difference was observed in frequencies of V gene (TRBV2, TRBV29-1, TRBV30 and TRBV12-3) and J gene (TRBJ2-1) usage. Furthermore, significantly differences in average overlap was observed in groups of AMI and NCA control. The results showed that patients with AMI had distinct TCR repertoires which revealed the association between cardiovascular condition and T-cell clonotypes.ConclusionsOur findings revealed the differences of TCR repertoires between patients with AMI and NCA controls, which might be potential biomarkers for evaluating risk stratification or diagnosis of acute coronary syndrome.
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Project description:Natively paired sequencing (NPS) of B cell receptors [variable heavy (VH) and light (VL)] and T cell receptors (TCRb and TCRa) is essential for the understanding of adaptive immunity in health and disease. Despite many recent technical advances, determining the VH:VL or TCRb:a repertoire with high accuracy and throughput remains challenging. We discovered that the recently engineered xenopolymerase, RTX, is exceptionally resistant to cell lysate inhibition in single-cell emulsion droplets. We capitalized on the characteristics of this enzyme to develop a simple, rapid, and inexpensive in-droplet overlap extension reverse transcription polymerase chain reaction method for NPS not requiring microfluidics or other specialized equipment. Using this technique, we obtained high yields (5000 to >20,000 per sample) of paired VH:VL or TCRb:a clonotypes at low cost. As a demonstration, we performed NPS on peripheral blood plasmablasts and T follicular helper cells following seasonal influenza vaccination and discovered high-affinity influenza-specific antibodies and TCRb:a.
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