Publications by authors named "Mikhail Shugay"

49 Publications

HLA binding of self-peptides is biased towards proteins with specific molecular functions.

bioRxiv 2021 Feb 17. Epub 2021 Feb 17.

Human leukocyte antigen (HLA) is highly polymorphic and plays a key role in guiding adaptive immune responses by presenting foreign and self peptides to T cells. Each HLA variant selects a minor fraction of peptides that match a certain motif required for optimal interaction with the peptide-binding groove. These restriction rules define the landscape of peptides presented to T cells. Given these limitations, one might suggest that the choice of peptides presented by HLA is non-random and there is preferential presentation of an array of peptides that is optimal for distinguishing self and foreign proteins. In this study we explore these preferences with a comparative analysis of self peptides enriched and depleted in HLA ligands. We show that HLAs exhibit preferences towards presenting peptides from certain proteins while disfavoring others with specific functions, and highlight differences between various HLA genes and alleles in those preferences. We link those differences to HLA anchor residue propensities and amino acid composition of preferentially presented proteins. The set of proteins that peptides presented by a given HLA are most likely to be derived from can be used to distinguish between class I and class II HLAs and HLA alleles. Our observations can be extrapolated to explain the protective effect of certain HLA alleles in infectious diseases, and we hypothesize that they can also explain susceptibility to certain autoimmune diseases and cancers. We demonstrate that these differences lead to differential presentation of HIV, influenza virus, SARS-CoV-1 and SARS-CoV-2 proteins by various HLA alleles. Finally, we show that the reported self peptidome preferences of distinct HLA variants can be compensated by combinations of HLA-A/HLA-B and HLA-A/HLA-C alleles in frequent haplotypes.
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http://dx.doi.org/10.1101/2021.02.16.431395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899460PMC
February 2021

A T cell repertoire timestamp is at the core of responsiveness to CTLA-4 blockade.

iScience 2021 Feb 27;24(2):102100. Epub 2021 Jan 27.

The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.

Biology of the response to anti-CTLA-4 involves the dynamics of specific T cell clones. Reasons for clinical success and failure of this treatment are still largely unknown. Here, we quantified the dynamics of the T cell receptor (TCR) repertoire, throughout 4 weeks involving treatment with anti-CTLA-4, in a syngeneic mouse model for colorectal cancer. These dynamics show an initial increase in clonality in tandem with a decrease in diversity, effects which gradually subside. Furthermore, response to treatment is tightly connected to the shared and public parts of the T cell repertoire. We were able to recognize time-dependent behaviors of specific TCR sequences and cell types and to show the response is dominated by specific motifs. We see that a single, specific time point might be useful to inform a physician of the true response to treatmentThe research further highlights the importance of temporal analyses of the immune response.
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http://dx.doi.org/10.1016/j.isci.2021.102100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876555PMC
February 2021

SARS-CoV-2 Epitopes Are Recognized by a Public and Diverse Repertoire of Human T Cell Receptors.

Immunity 2020 12 13;53(6):1245-1257.e5. Epub 2020 Nov 13.

National Research Center for Hematology, Moscow, Russia. Electronic address:

Understanding the hallmarks of the immune response to SARS-CoV-2 is critical for fighting the COVID-19 pandemic. We assessed antibody and T cell reactivity in convalescent COVID-19 patients and healthy donors sampled both prior to and during the pandemic. Healthy donors examined during the pandemic exhibited increased numbers of SARS-CoV-2-specific T cells, but no humoral response. Their probable exposure to the virus resulted in either asymptomatic infection without antibody secretion or activation of preexisting immunity. In convalescent patients, we observed a public and diverse T cell response to SARS-CoV-2 epitopes, revealing T cell receptor (TCR) motifs with germline-encoded features. Bulk CD4 and CD8 T cell responses to the spike protein were mediated by groups of homologous TCRs, some of them shared across multiple donors. Overall, our results demonstrate that the T cell response to SARS-CoV-2, including the identified set of TCRs, can serve as a useful biomarker for surveying antiviral immunity.
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http://dx.doi.org/10.1016/j.immuni.2020.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664363PMC
December 2020

T-cell tracking, safety, and effect of low-dose donor memory T-cell infusions after αβ T cell-depleted hematopoietic stem cell transplantation.

Bone Marrow Transplant 2021 Apr 17;56(4):900-908. Epub 2020 Nov 17.

Department of Hematopoietic Stem Cell Transplantation, Dmitriy Rogachev National Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia.

The delayed recovery of adaptive immunity underlies transplant-related mortality (TRM) after αβ T cell-depleted hematopoietic stem cell transplantation (HSCT). We tested the use of low-dose memory donor lymphocyte infusions (mDLIs) after engraftment of αβ T cell-depleted grafts.A cohort of 131 pediatric patients (median age 9 years) were grafted with αβ T cell-depleted products from either haplo (n = 79) or unrelated donors (n = 52). After engraftment, patients received mDLIs prepared by CD45RA depletion. Cell dose was escalated monthly from 25 × 10 to 100 × 10/kg (haplo) and from 100 × 10 to 300 × 10 /kg (MUD). In a subcohort of 16 patients, T-cell receptor (TCR) repertoire profiling with deep sequencing was used to track T-cell clones and to evaluate the contribution of mDLI to the immune repertoire.In total, 343 mDLIs were administered. The cumulative incidence (CI) of grades II and III de novo acute graft-versus-host disease (aGVHD) was 5% and 2%, respectively, and the CI of chronic graft-versus-host disease was 7%. Half of the patients with undetectable CMV-specific T cells before mDLI recovered CMV-specific T cells. TCR repertoire profiling confirmed that mDLI-derived T cells significantly contribute to the TCR repertoire up to 1 year after HSCT and include persistent, CMV-specific T-cell clones.
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http://dx.doi.org/10.1038/s41409-020-01128-2DOI Listing
April 2021

Benchmarking of T cell receptor repertoire profiling methods reveals large systematic biases.

Nat Biotechnol 2021 02 7;39(2):236-245. Epub 2020 Sep 7.

Sorbonne Université, INSERM, Immunology-Immunopathology-Immunotherapy (i3), Paris, France.

Monitoring the T cell receptor (TCR) repertoire in health and disease can provide key insights into adaptive immune responses, but the accuracy of current TCR sequencing (TCRseq) methods is unclear. In this study, we systematically compared the results of nine commercial and academic TCRseq methods, including six rapid amplification of complementary DNA ends (RACE)-polymerase chain reaction (PCR) and three multiplex-PCR approaches, when applied to the same T cell sample. We found marked differences in accuracy and intra- and inter-method reproducibility for T cell receptor α (TRA) and T cell receptor β (TRB) TCR chains. Most methods showed a lower ability to capture TRA than TRB diversity. Low RNA input generated non-representative repertoires. Results from the 5' RACE-PCR methods were consistent among themselves but differed from the RNA-based multiplex-PCR results. Using an in silico meta-repertoire generated from 108 replicates, we found that one genomic DNA-based method and two non-unique molecular identifier (UMI) RNA-based methods were more sensitive than UMI methods in detecting rare clonotypes, despite the better clonotype quantification accuracy of the latter.
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http://dx.doi.org/10.1038/s41587-020-0656-3DOI Listing
February 2021

CD4 T Cells Recognize Conserved Influenza A Epitopes through Shared Patterns of V-Gene Usage and Complementary Biochemical Features.

Cell Rep 2020 07;32(2):107885

Cardiff University, School of Medicine, Heath Park, Cardiff, UK. Electronic address:

T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8 T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4 T cells. Here, we investigate CD4 T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4 T cells in five HLA-DR1 subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.
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http://dx.doi.org/10.1016/j.celrep.2020.107885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370177PMC
July 2020

MHC-II alleles shape the CDR3 repertoires of conventional and regulatory naïve CD4 T cells.

Proc Natl Acad Sci U S A 2020 06 1;117(24):13659-13669. Epub 2020 Jun 1.

Department of Genomics of Adaptive Immunity, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, 117997, Russia;

T cell maturation and activation depend upon T cell receptor (TCR) interactions with a wide variety of antigenic peptides displayed in a given major histocompatibility complex (MHC) context. Complementarity-determining region 3 (CDR3) is the most variable part of the TCRα and -β chains, which govern interactions with peptide-MHC complexes. However, it remains unclear how the CDR3 landscape is shaped by individual MHC context during thymic selection of naïve T cells. We established two mouse strains carrying distinct allelic variants of and analyzed thymic and peripheral production and TCR repertoires of naïve conventional CD4 T (T) and naïve regulatory CD4 T (T) cells. Compared with tuberculosis-resistant C57BL/6 (H2-A) mice, the tuberculosis-susceptible H2-A mice had fewer CD4 T cells of both subsets in the thymus. In the periphery, this deficiency was only apparent for T and was compensated for by peripheral reconstitution for T We show that H2-A favors selection of a narrower and more convergent repertoire with more hydrophobic and strongly interacting amino acid residues in the middle of CDR3α and CDR3β, suggesting more stringent selection against a narrower peptide-MHC-II context. H2-A and H2-A mice have prominent reciprocal differences in CDR3α and CDR3β features, probably reflecting distinct modes of TCR fitting to MHC-II variants. These data reveal the mechanics and extent of how MHC-II shapes the naïve CD4 T cell CDR3 landscape, which essentially defines adaptive response to infections and self-antigens.
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http://dx.doi.org/10.1073/pnas.2003170117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306996PMC
June 2020

Comprehensive analysis of structural and sequencing data reveals almost unconstrained chain pairing in TCRαβ complex.

PLoS Comput Biol 2020 03 12;16(3):e1007714. Epub 2020 Mar 12.

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.

Antigen recognition by T-cells is guided by the T-cell receptor (TCR) heterodimer formed by α and β chains. A huge diversity of TCR sequences should be maintained by the immune system in order to be able to mount an effective response towards foreign pathogens, so, due to cooperative binding of α and β chains to the pathogen, any constraints on chain pairing can have a profound effect on immune repertoire structure, diversity and antigen specificity. By integrating available structural data and paired chain sequencing results we were able to show that there are almost no constraints on pairing in TCRαβ complexes, allowing naive T-cell repertoire to reach the highest possible diversity. Additional analysis reveals that the specific choice of contacting amino acids can still have a profound effect on complex conformation. Moreover, antigen-driven selection can distort the uniform landscape of chain pairing, while small, yet significant, differences in the pairing can be attributed to various specialized T-cell subsets such as MAIT and iNKT T-cells, as well as other TCR sets specific to certain antigens.
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http://dx.doi.org/10.1371/journal.pcbi.1007714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093030PMC
March 2020

The Interplay between CD27 and CD27 B Cells Ensures the Flexibility, Stability, and Resilience of Human B Cell Memory.

Cell Rep 2020 03;30(9):2963-2977.e6

Multifactorial Disease and Complex Phenotype Research Area, Bambino Gesù Children's Hospital, IRCSS, 00146 Rome, Italy.

Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27 and CD27 MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27 and CD27 MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27 into the CD27 MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27 MBCs transit to the more differentiated CD27 stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27 clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27 MBCs that expand and differentiate in response to change.
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http://dx.doi.org/10.1016/j.celrep.2020.02.022DOI Listing
March 2020

Benchmarking immunoinformatic tools for the analysis of antibody repertoire sequences.

Bioinformatics 2020 03;36(6):1731-1739

Institute of Biomedical Engineering and Medical Informatics, School of Life Sciences, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz 4132, Switzerland.

Summary: Antibody repertoires reveal insights into the biology of the adaptive immune system and empower diagnostics and therapeutics. There are currently multiple tools available for the annotation of antibody sequences. All downstream analyses such as choosing lead drug candidates depend on the correct annotation of these sequences; however, a thorough comparison of the performance of these tools has not been investigated. Here, we benchmark the performance of commonly used immunoinformatic tools, i.e. IMGT/HighV-QUEST, IgBLAST and MiXCR, in terms of reproducibility of annotation output, accuracy and speed using simulated and experimental high-throughput sequencing datasets.We analyzed changes in IMGT reference germline database in the last 10 years in order to assess the reproducibility of the annotation output. We found that only 73/183 (40%) V, D and J human genes were shared between the reference germline sets used by the tools. We found that the annotation results differed between tools. In terms of alignment accuracy, MiXCR had the highest average frequency of gene mishits, 0.02 mishit frequency and IgBLAST the lowest, 0.004 mishit frequency. Reproducibility in the output of complementarity determining three regions (CDR3 amino acids) ranged from 4.3% to 77.6% with preprocessed data. In addition, run time of the tools was assessed: MiXCR was the fastest tool for number of sequences processed per unit of time. These results indicate that immunoinformatic analyses greatly depend on the choice of bioinformatics tool. Our results support informed decision-making to immunoinformaticians based on repertoire composition and sequencing platforms.

Availability And Implementation: All tools utilized in the paper are free for academic use.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075533PMC
March 2020

An overview of immunoinformatics approaches and databases linking T cell receptor repertoires to their antigen specificity.

Immunogenetics 2020 02 18;72(1-2):77-84. Epub 2019 Nov 18.

Pirogov Russian Medical State University, Moscow, Russia.

Recent advances in molecular and bioinformatic methods have greatly improved our ability to study the formation of an adaptive immune response towards foreign pathogens, self-antigens, and cancer neoantigens. T cell receptors (TCR) are the key players in this process that recognize peptides presented by major histocompatibility complex (MHC). Owing to the huge diversity of both TCR sequence variants and peptides they recognize, accumulation and complex analysis of large amounts of TCR-antigen specificity data is required for understanding the structure and features of adaptive immune responses towards pathogens, vaccines, cancer, as well as autoimmune responses. In the present review, we summarize recent efforts on gathering and interpreting TCR-antigen specificity data and outline the critical role of tighter integration with other immunoinformatics data sources that include epitope MHC restriction, TCR repertoire structure models, and TCR/peptide/MHC structural data. We suggest that such integration can lead to the ability to accurately annotate individual TCR repertoires, efficiently estimate epitope and neoantigen immunogenicity, and ultimately, in silico identify TCRs specific to yet unstudied antigens and predict self-peptides related to autoimmunity.
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http://dx.doi.org/10.1007/s00251-019-01139-4DOI Listing
February 2020

sumrep: A Summary Statistic Framework for Immune Receptor Repertoire Comparison and Model Validation.

Front Immunol 2019 1;10:2533. Epub 2019 Nov 1.

Fred Hutchinson Cancer Research Center, Seattle, WA, United States.

The adaptive immune system generates an incredible diversity of antigen receptors for B and T cells to keep dangerous pathogens at bay. The DNA sequences coding for these receptors arise by a complex recombination process followed by a series of productivity-based filters, as well as affinity maturation for B cells, giving considerable diversity to the circulating pool of receptor sequences. Although these datasets hold considerable promise for medical and public health applications, the complex structure of the resulting adaptive immune receptor repertoire sequencing (AIRR-seq) datasets makes analysis difficult. In this paper we introduce sumrep, an R package that efficiently performs a wide variety of repertoire summaries and comparisons, and show how sumrep can be used to perform model validation. We find that summaries vary in their ability to differentiate between datasets, although many are able to distinguish between covariates such as donor, timepoint, and cell type for BCR and TCR repertoires. We show that deletion and insertion lengths resulting from V(D)J recombination tend to be more discriminative characterizations of a repertoire than summaries that describe the amino acid composition of the CDR3 region. We also find that state-of-the-art generative models excel at recapitulating gene usage and recombination statistics in a given experimental repertoire, but struggle to capture many physiochemical properties of real repertoires.
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http://dx.doi.org/10.3389/fimmu.2019.02533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838214PMC
November 2020

A Framework for Annotation of Antigen Specificities in High-Throughput T-Cell Repertoire Sequencing Studies.

Front Immunol 2019 26;10:2159. Epub 2019 Sep 26.

Genomics of Adaptive Immunity Department, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.

Recently developed molecular methods allow large-scale profiling of T-cell receptor (TCR) sequences that encode for antigen specificity and immunological memory of these cells. However, it is well-known that the even unperturbed TCR repertoire structure is extremely complex due to the high diversity of TCR rearrangements and multiple biases imprinted by VDJ rearrangement process. The latter gives rise to the phenomenon of "public" TCR clonotypes that can be shared across multiple individuals and non-trivial structure of the TCR similarity network. Here, we outline a framework for TCR sequencing data analysis that can control for these biases in order to infer TCRs that are involved in response to antigens of interest. We apply two previously published methods, ALICE and TCRNET, to detect groups of homologous TCRs that are enriched in samples of interest. Using an example dataset of donors with known HLA haplotype and CMV status, we demonstrate that by applying HLA restriction rules and matching against a database of TCRs with known antigen specificity, it is possible to robustly detect motifs of epitope-specific responses in individual repertoires. We also highlight potential shortcomings of TCR clustering methods and demonstrate that highly expanded TCRs should be individually assessed to get the full picture of antigen-specific response.
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http://dx.doi.org/10.3389/fimmu.2019.02159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6775185PMC
October 2020

VDJdb in 2019: database extension, new analysis infrastructure and a T-cell receptor motif compendium.

Nucleic Acids Res 2020 01;48(D1):D1057-D1062

Pirogov Russian Medical State University, Moscow, Russia.

Here, we report an update of the VDJdb database with a substantial increase in the number of T-cell receptor (TCR) sequences and their cognate antigens. The update further provides a new database infrastructure featuring two additional analysis modes that facilitate database querying and real-world data analysis. The increased yield of TCR specificity identification methods and the overall increase in the number of studies in the field has allowed us to expand the database more than 5-fold. Furthermore, several new analysis methods are included. For example, batch annotation of TCR repertoire sequencing samples allows for annotating large datasets on-line. Using recently developed bioinformatic methods for TCR motif mining, we have built a reduced set of high-quality TCR motifs that can be used for both training TCR specificity predictors and matching against TCRs of interest. These additions enhance the versatility of the VDJdb in the task of exploring T-cell antigen specificities. The database is available at https://vdjdb.cdr3.net.
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http://dx.doi.org/10.1093/nar/gkz874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943061PMC
January 2020

Detecting T cell receptors involved in immune responses from single repertoire snapshots.

PLoS Biol 2019 06 13;17(6):e3000314. Epub 2019 Jun 13.

Laboratoire de physique théorique, CNRS, Sorbonne Université, Université Paris-Diderot, and École normale supérieure (PSL University), Paris, France.

Hypervariable T cell receptors (TCRs) play a key role in adaptive immunity, recognizing a vast diversity of pathogen-derived antigens. Our ability to extract clinically relevant information from large high-throughput sequencing of TCR repertoires (RepSeq) data is limited, because little is known about TCR-disease associations. We present Antigen-specific Lymphocyte Identification by Clustering of Expanded sequences (ALICE), a statistical approach that identifies TCR sequences actively involved in current immune responses from a single RepSeq sample and apply it to repertoires of patients with a variety of disorders - patients with autoimmune disease (ankylosing spondylitis [AS]), under cancer immunotherapy, or subject to an acute infection (live yellow fever [YF] vaccine). We validate the method with independent assays. ALICE requires no longitudinal data collection nor large cohorts, and it is directly applicable to most RepSeq datasets. Its results facilitate the identification of TCR variants associated with diseases and conditions, which can be used for diagnostics and rational vaccine design.
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http://dx.doi.org/10.1371/journal.pbio.3000314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592544PMC
June 2019

Comparative Analysis of B-Cell Receptor Repertoires Induced by Live Yellow Fever Vaccine in Young and Middle-Age Donors.

Front Immunol 2018 9;9:2309. Epub 2018 Oct 9.

Adaptive Immunity Group, Central European Institute of Technology, Brno, Czechia.

Age-related changes can significantly alter the state of adaptive immune system and often lead to attenuated response to novel pathogens and vaccination. In present study we employed 5'RACE UMI-based full length and nearly error-free immunoglobulin profiling to compare plasma cell antibody repertoires in young (19-26 years) and middle-age (45-58 years) individuals vaccinated with a live yellow fever vaccine, modeling a newly encountered pathogen. Our analysis has revealed age-related differences in the responding antibody repertoire ranging from distinct IGH CDR3 repertoire properties to differences in somatic hypermutation intensity and efficiency and antibody lineage tree structure. Overall, our findings suggest that younger individuals respond with a more diverse antibody repertoire and employ a more efficient somatic hypermutation process than elder individuals in response to a newly encountered pathogen.
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http://dx.doi.org/10.3389/fimmu.2018.02309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189279PMC
October 2019

High-resolution repertoire analysis reveals a major bystander activation of Tfh and Tfr cells.

Proc Natl Acad Sci U S A 2018 09 29;115(38):9604-9609. Epub 2018 Aug 29.

Immunology-Immunopathology-Immunotherapy Laboratory, UMR_S 959, INSERM, Sorbonne Université, F-75005 Paris, France;

T follicular helper (Tfh) and regulatory (Tfr) cells are terminally differentiated cells found in germinal centers (GCs), specialized secondary lymphoid organ structures dedicated to antibody production. As such, follicular T (Tfol) cells are supposed to be specific for immunizing antigens, which has been reported for Tfh cells but is debated for Tfr cells. Here, we used high-throughput T cell receptor (TCR) sequencing to analyze the repertoires of Tfh and Tfr cells, at homeostasis and after immunization with self- or foreign antigens. We observed that, whatever the conditions, Tfh and Tfr cell repertoires are less diverse than those of effector T cells and Treg cells of the same tissues; surprisingly, these repertoires still represent thousands of different sequences, even after immunization with a single antigen that induces a 10-fold increase in Tfol cell numbers. Thorough analysis of the sharing and network of TCR sequences revealed that a specific response to the immunizing antigen can only, but hardly, be detected in Tfh cells immunized with a foreign antigen and Tfr cells immunized with a self-antigen. These antigen-specific responses are obscured by a global stimulation of Tfh and Tfr cells that appears to be antigen-independent. Altogether, our results suggest a major bystander Tfol cell activation during the immune response in the GCs.
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http://dx.doi.org/10.1073/pnas.1808594115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156623PMC
September 2018

Exploring the pre-immune landscape of antigen-specific T cells.

Genome Med 2018 08 25;10(1):68. Epub 2018 Aug 25.

Department of Genomics of Adaptive Immunity, IBCH RAS, Moscow, Russia.

Background: Adaptive immune responses to newly encountered pathogens depend on the mobilization of antigen-specific clonotypes from a vastly diverse pool of naive T cells. Using recent advances in immune repertoire sequencing technologies, models of the immune receptor rearrangement process, and a database of annotated T cell receptor (TCR) sequences with known specificities, we explored the baseline frequencies of T cells specific for defined human leukocyte antigen (HLA) class I-restricted epitopes in healthy individuals.

Methods: We used a database of TCR sequences with known antigen specificities and a probabilistic TCR rearrangement model to estimate the baseline frequencies of TCRs specific to distinct antigens epitopespecificT-cells. We verified our estimates using a publicly available collection of TCR repertoires from healthy individuals. We also interrogated a database of immunogenic and non-immunogenic peptides is used to link baseline T-cell frequencies with epitope immunogenicity.

Results: Our findings revealed a high degree of variability in the prevalence of T cells specific for different antigens that could be explained by the physicochemical properties of the corresponding HLA class I-bound peptides. The occurrence of certain rearrangements was influenced by ancestry and HLA class I restriction, and umbilical cord blood samples contained higher frequencies of common pathogen-specific TCRs. We also identified a quantitative link between specific T cell frequencies and the immunogenicity of cognate epitopes presented by defined HLA class I molecules.

Conclusions: Our results suggest that the population frequencies of specific T cells are strikingly non-uniform across epitopes that are known to elicit immune responses. This inference leads to a new definition of epitope immunogenicity based on specific TCR frequencies, which can be estimated with a high degree of accuracy in silico, thereby providing a novel framework to integrate computational and experimental genomics with basic and translational research efforts in the field of T cell immunology.
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http://dx.doi.org/10.1186/s13073-018-0577-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109350PMC
August 2018

The Changing Landscape of Naive T Cell Receptor Repertoire With Human Aging.

Front Immunol 2018 24;9:1618. Epub 2018 Jul 24.

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.

Human aging is associated with a profound loss of thymus productivity, yet naïve T lymphocytes still maintain their numbers by division in the periphery for many years. The extent of such proliferation may depend on the cytokine environment, including IL-7 and T-cell receptor (TCR) "tonic" signaling mediated by self pMHCs recognition. Additionally, intrinsic properties of distinct subpopulations of naïve T cells could influence the overall dynamics of aging-related changes within the naïve T cell compartment. Here, we investigated the differences in the architecture of TCR beta repertoires for naïve CD4, naïve CD8, naïve CD4CD25CD31 (enriched with recent thymic emigrants, RTE), and mature naïve CD4CD25CD31 peripheral blood subsets between young and middle-age/old healthy individuals. In addition to observing the accumulation of clonal expansions (as was shown previously), we reveal several notable changes in the characteristics of T cell repertoire. We observed significant decrease of CDR3 length, NDN insert, and number of non-template added N nucleotides within TCR beta CDR3 with aging, together with a prominent change of physicochemical properties of the central part of CDR3 loop. These changes were similar across CD4, CD8, RTE-enriched, and mature CD4 subsets of naïve T cells, with minimal or no difference observed between the latter two subsets for individuals of the same age group. We also observed an increase in "publicity" (fraction of shared clonotypes) of CD4, but not CD8 naïve T cell repertoires. We propose several explanations for these phenomena built upon previous studies of naïve T-cell homeostasis, and call for further studies of the mechanisms causing the observed changes and of consequences of these changes in respect of the possible holes formed in the landscape of naïve T cell TCR repertoire.
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http://dx.doi.org/10.3389/fimmu.2018.01618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066563PMC
July 2018

CD8+ T cells with characteristic T cell receptor beta motif are detected in blood and expanded in synovial fluid of ankylosing spondylitis patients.

Rheumatology (Oxford) 2018 06;57(6):1097-1104

Molecular Technologies Department, Translational Medicine Institute, Pirogov Russian National Research Medical University, Moscow, Russia.

Objective: The risk of AS is associated with genomic variants related to antigen presentation and specific cytokine signalling pathways, suggesting the involvement of cellular immunity in disease initiation/progression. The aim of the present study was to explore the repertoire of TCR sequences in healthy donors and AS patients to uncover AS-linked TCR variants.

Methods: Using quantitative molecular-barcoded 5'-RACE, we performed deep TCR β repertoire profiling of peripheral blood (PB) and SF samples for 25 AS patients and 108 healthy donors. AS-linked TCR variants were identified using a new computational approach that relies on a probabilistic model of the VDJ rearrangement process.

Results: Using the donor-agnostic probabilistic model, we reveal a TCR β motif characteristic for PB of AS patients, represented by eight highly homologous amino acid sequence variants. Some of these variants were previously reported in SF and PB of patients with ReA and in PB of AS patients. We demonstrate that identified AS-linked clones have a CD8+ phenotype, present at relatively low frequencies in PB, and are significantly enriched in matched SF samples of AS patients.

Conclusion: Our results suggest the involvement of a particular antigen-specific subset of CD8+ T cells in AS pathogenesis, confirming and expanding earlier findings. The high similarity of the clonotypes with the ones found in ReA implies common mechanisms for the initiation of the diseases.
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http://dx.doi.org/10.1093/rheumatology/kex517DOI Listing
June 2018

Comparative analysis of murine T-cell receptor repertoires.

Immunology 2018 02 27;153(2):133-144. Epub 2017 Nov 27.

Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia.

For understanding the rules and laws of adaptive immunity, high-throughput profiling of T-cell receptor (TCR) repertoires becomes a powerful tool. The structure of TCR repertoires is instructive even before the antigen specificity of each particular receptor becomes available. It embodies information about the thymic and peripheral selection of T cells; the readiness of an adaptive immunity to withstand new challenges; the character, magnitude and memory of immune responses; and the aetiological and functional proximity of T-cell subsets. Here, we describe our current analytical approaches for the comparative analysis of murine TCR repertoires, and show several examples of how these approaches can be applied for particular experimental settings. We analyse the efficiency of different metrics used for estimation of repertoire diversity, repertoire overlap, V-gene and J-gene segments usage similarity, and amino acid composition of CDR3. We discuss basic differences of these metrics and their advantages and limitations in different experimental models, and we provide guidelines for choosing an efficient way to lead a comparative analysis of TCR repertoires. Applied to the various known and newly developed mouse models, such analysis should allow us to disentangle multiple sophisticated puzzles in adaptive immunity.
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http://dx.doi.org/10.1111/imm.12857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765371PMC
February 2018

VDJdb: a curated database of T-cell receptor sequences with known antigen specificity.

Nucleic Acids Res 2018 01;46(D1):D419-D427

Pirogov Russian National Research Medical University, Moscow 117997, Russia.

The ability to decode antigen specificities encapsulated in the sequences of rearranged T-cell receptor (TCR) genes is critical for our understanding of the adaptive immune system and promises significant advances in the field of translational medicine. Recent developments in high-throughput sequencing methods (immune repertoire sequencing technology, or RepSeq) and single-cell RNA sequencing technology have allowed us to obtain huge numbers of TCR sequences from donor samples and link them to T-cell phenotypes. However, our ability to annotate these TCR sequences still lags behind, owing to the enormous diversity of the TCR repertoire and the scarcity of available data on T-cell specificities. In this paper, we present VDJdb, a database that stores and aggregates the results of published T-cell specificity assays and provides a universal platform that couples antigen specificities with TCR sequences. We demonstrate that VDJdb is a versatile instrument for the annotation of TCR repertoire data, enabling a concatenated view of antigen-specific TCR sequence motifs. VDJdb can be accessed at https://vdjdb.cdr3.net and https://github.com/antigenomics/vdjdb-db.
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http://dx.doi.org/10.1093/nar/gkx760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753233PMC
January 2018

Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries.

BMC Genomics 2017 06 5;18(1):440. Epub 2017 Jun 5.

Pirogov Russian National Research Medical University, Moscow, Russia.

Background: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps.

Results: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification.

Conclusion: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.
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http://dx.doi.org/10.1186/s12864-017-3815-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460480PMC
June 2017

A high-throughput assay for quantitative measurement of PCR errors.

Sci Rep 2017 06 2;7(1):2718. Epub 2017 Jun 2.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia.

The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.
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http://dx.doi.org/10.1038/s41598-017-02727-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457411PMC
June 2017

MAGERI: Computational pipeline for molecular-barcoded targeted resequencing.

PLoS Comput Biol 2017 05 5;13(5):e1005480. Epub 2017 May 5.

Shemyakin-Ovchinnikov Institute of bioorganic chemistry RAS, Miklukho-Maklaya 16/10, Moscow, Russia.

Unique molecular identifiers (UMIs) show outstanding performance in targeted high-throughput resequencing, being the most promising approach for the accurate identification of rare variants in complex DNA samples. This approach has application in multiple areas, including cancer diagnostics, thus demanding dedicated software and algorithms. Here we introduce MAGERI, a computational pipeline that efficiently handles all caveats of UMI-based analysis to obtain high-fidelity mutation profiles and call ultra-rare variants. Using an extensive set of benchmark datasets including gold-standard biological samples with known variant frequencies, cell-free DNA from tumor patient blood samples and publicly available UMI-encoded datasets we demonstrate that our method is both robust and efficient in calling rare variants. The versatility of our software is supported by accurate results obtained for both tumor DNA and viral RNA samples in datasets prepared using three different UMI-based protocols.
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http://dx.doi.org/10.1371/journal.pcbi.1005480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5419444PMC
May 2017

Single-cell analysis of glandular T cell receptors in Sjögren's syndrome.

JCI Insight 2016 Jun;1(8)

Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation (OMRF), Oklahoma City, Oklahoma, USA; Department of Pathology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, Oklahoma, USA.

CD4 T cells predominate in salivary gland (SG) inflammatory lesions in Sjögren's syndrome (SS). However, their antigen specificity, degree of clonal expansion, and relationship to clinical disease features remain unknown. We used multiplex reverse-transcriptase PCR to amplify paired T cell receptor α (TCRα) and β transcripts of single CD4CD45RA T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype. TCRα and β sequences were obtained from a median of 91 SG and 107 PB cells per subject. The degree of clonal expansion and frequency of cells expressing two productively rearranged α genes were increased in SG versus PB. Expanded clones from SG exhibited complementary-determining region 3 (CDR3) sequence similarity both within and among subjects, suggesting antigenic selection and shared antigen recognition. CDR3 similarities were shared among expanded clones from individuals discordant for canonical Ro and La autoantibodies, suggesting recognition of alternative SG antigen(s). The extent of SG clonal expansion correlated with reduced saliva production and increased SG fibrosis, linking expanded SG T cells with glandular dysfunction. Knowledge of paired TCRα and β sequences enables further work toward identification of target antigens and development of novel therapies.
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http://dx.doi.org/10.1172/jci.insight.85609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922426PMC
June 2016

VDJviz: a versatile browser for immunogenomics data.

BMC Genomics 2016 06 13;17:453. Epub 2016 Jun 13.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997, Moscow, Russia.

Background: The repertoire of T- and B-cell receptor sequences encodes the antigen specificity of adaptive immunity system, determines its present state and guides its ability to mount effective response against encountered antigens in future. High throughput sequencing of immune repertoires (Rep-Seq) is a promising technique that allows to profile millions of antigen receptors of an individual in a single experiment. While a substantial number of tools for mapping and assembling Rep-Seq data were published recently, the field still lacks an intuitive and flexible tool that can be used by researchers with little or no computational background for in-depth analysis of immune repertoire profiles.

Results: Here we report VDJviz, a web tool that can be used to browse, analyze and perform quality control of Rep-Seq results generated by various pre-processing software. On a set of real data examples we show that VDJviz can be used to explore key repertoire characteristics such as spectratype, repertoire clonality, V-(D)-J recombination patterns and to identify shared clonotypes. We also demonstrate the utility of VDJviz in detection of critical Rep-Seq biases such as artificial repertoire diversity and cross-sample contamination.

Conclusions: VDJviz is a versatile and lightweight tool that can be easily employed by biologists, immunologists and immunogeneticists for routine analysis and quality control of Rep-Seq data. The software is freely available for non-commercial purposes, and can be downloaded from: https://github.com/antigenomics/vdjviz .
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http://dx.doi.org/10.1186/s12864-016-2799-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907000PMC
June 2016

Dynamics of Individual T Cell Repertoires: From Cord Blood to Centenarians.

J Immunol 2016 06 13;196(12):5005-13. Epub 2016 May 13.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow 117997, Russia; Pirogov Russian National Research Medical University, Moscow 117997, Russia; Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic; and

The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRβ repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity.
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http://dx.doi.org/10.4049/jimmunol.1600005DOI Listing
June 2016