Publications by authors named "Ivan V Zvyagin"

20 Publications

  • Page 1 of 1

Surface NKG2C Identifies Differentiated αβT-Cell Clones Expanded in Peripheral Blood.

Front Immunol 2020 16;11:613882. Epub 2021 Feb 16.

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

T cells that express CD56 in peripheral blood of healthy humans represent a heterogeneous and poorly studied subset. In this work, we analyzed this subset for NKG2C expression. In both CD56 and CD56 subsets most of the NKG2C T cells had a phenotype of highly differentiated CD8 TEMRA cells. The CD56NKG2C T cells also expressed a number of NK cell receptors, such as NKG2D, CD16, KIR2DL2/DL3, and maturation marker CD57 more often than the CD56NKG2CCD3 cells. TCR β-chain repertoire of the CD3CD56NKG2C cell fraction was limited by the prevalence of one or several clonotypes which can be found within the most abundant clonotypes in total or CD8 T cell fraction TCRβ repertoire. Thus, NKG2C expression in highly differentiated CD56 T cells was associated with the most expanded αβ T cell clones. NKG2C T cells produced almost no IFN-γ in response to stimulation with HCMV pp65-derived peptides. This may be partially due to the high content of CD45RACD57 cells in the fraction. CD3NKG2C cells showed signs of activation, and the frequency of this T-cell subset in HCMV-positive individuals was positively correlated with the frequency of NKG2C NK cells that may imply a coordinated in a certain extent development of the NKG2C T and NK cell subsets under HCMV infection.
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http://dx.doi.org/10.3389/fimmu.2020.613882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921799PMC
June 2021

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

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

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

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

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

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

Quantitative profiling reveals minor changes of T cell receptor repertoire in response to subunit inactivated influenza vaccine.

Vaccine 2018 03 15;36(12):1599-1605. Epub 2018 Feb 15.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Miklukho-Maklaya 16/10, 117997 Moscow, Russia; Pirogov Russian National Research Medical University, 117997 Moscow, Russia. Electronic address:

Vaccination against influenza is widely used to protect against seasonal flu epidemic although its effectiveness is debated. Here we performed deep quantitative T cell receptor repertoire profiling in peripheral blood of a healthy volunteer in response to trivalent subunit influenza vaccine. We did not observe significant rebuilding of peripheral blood T cell receptors composition in response to vaccination. However, we found several clonotypes in memory T cell fraction that were undetectable before the vaccination and had a maximum concentration at day 45 after vaccine administration. These cells were found in lower concentration in the course of repertoire monitoring for two years period. Our observation suggests a potential for recruitment of only a limited number of new T cells after each seasonal influenza vaccination.
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http://dx.doi.org/10.1016/j.vaccine.2018.02.027DOI Listing
March 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

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

VDJtools: Unifying Post-analysis of T Cell Receptor Repertoires.

PLoS Comput Biol 2015 Nov 25;11(11):e1004503. Epub 2015 Nov 25.

Shemyakin-Ovchinnikov Institute of bioorganic chemistry RAS, Moscow, Russia.

Despite the growing number of immune repertoire sequencing studies, the field still lacks software for analysis and comprehension of this high-dimensional data. Here we report VDJtools, a complementary software suite that solves a wide range of T cell receptor (TCR) repertoires post-analysis tasks, provides a detailed tabular output and publication-ready graphics, and is built on top of a flexible API. Using TCR datasets for a large cohort of unrelated healthy donors, twins, and multiple sclerosis patients we demonstrate that VDJtools greatly facilitates the analysis and leads to sound biological conclusions. VDJtools software and documentation are available at https://github.com/mikessh/vdjtools.
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http://dx.doi.org/10.1371/journal.pcbi.1004503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659587PMC
November 2015

tcR: an R package for T cell receptor repertoire advanced data analysis.

BMC Bioinformatics 2015 May 28;16:175. Epub 2015 May 28.

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

Background: The Immunoglobulins (IG) and the T cell receptors (TR) play the key role in antigen recognition during the adaptive immune response. Recent progress in next-generation sequencing technologies has provided an opportunity for the deep T cell receptor repertoire profiling. However, a specialised software is required for the rational analysis of massive data generated by next-generation sequencing.

Results: Here we introduce tcR, a new R package, representing a platform for the advanced analysis of T cell receptor repertoires, which includes diversity measures, shared T cell receptor sequences identification, gene usage statistics computation and other widely used methods. The tool has proven its utility in recent research studies.

Conclusions: tcR is an R package for the advanced analysis of T cell receptor repertoires after primary TR sequences extraction from raw sequencing reads. The stable version can be directly installed from The Comprehensive R Archive Network ( http://cran.r-project.org/mirrors.html ). The source code and development version are available at tcR GitHub ( http://imminfo.github.io/tcr/ ) along with the full documentation and typical usage examples.
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http://dx.doi.org/10.1186/s12859-015-0613-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445501PMC
May 2015

Distinctive properties of identical twins' TCR repertoires revealed by high-throughput sequencing.

Proc Natl Acad Sci U S A 2014 Apr 7;111(16):5980-5. Epub 2014 Apr 7.

Department of Genomics and Postgenomic Technologies, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.

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 α but not β J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.
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http://dx.doi.org/10.1073/pnas.1319389111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000852PMC
April 2014

Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling.

Front Immunol 2013 23;4:456. Epub 2013 Dec 23.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science , Moscow , Russia ; CEITEC, Masaryk University , Brno , Czech Republic.

High-throughput sequencing has the power to reveal the nature of adaptive immunity as represented by the full complexity of T-cell receptor (TCR) and antibody (IG) repertoires, but is at present severely compromised by the quantitative bias, bottlenecks, and accumulated errors that inevitably occur in the course of library preparation and sequencing. Here we report an optimized protocol for the unbiased preparation of TCR and IG cDNA libraries for high-throughput sequencing, starting from thousands or millions of live cells in an investigated sample. Critical points to control are revealed, along with tips that allow researchers to minimize quantitative bias, accumulated errors, and cross-sample contamination at each stage, and to enhance the subsequent bioinformatic analysis. The protocol is simple, reliable, and can be performed in 1-2 days.
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http://dx.doi.org/10.3389/fimmu.2013.00456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3870325PMC
January 2014

Pairing of T-cell receptor chains via emulsion PCR.

Eur J Immunol 2013 Sep 26;43(9):2507-15. Epub 2013 Jun 26.

Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia.

Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains--both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.
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http://dx.doi.org/10.1002/eji.201343453DOI Listing
September 2013

Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms.

Eur J Immunol 2012 Nov 24;42(11):3073-83. Epub 2012 Sep 24.

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

The TCR repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of next generation sequencing applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.
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http://dx.doi.org/10.1002/eji.201242517DOI Listing
November 2012

Quantitative tracking of T cell clones after haematopoietic stem cell transplantation.

EMBO Mol Med 2011 Apr 4;3(4):201-7. Epub 2011 Mar 4.

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

Autologous haematopoietic stem cell transplantation is highly efficient for the treatment of systemic autoimmune diseases, but its consequences for the immune system remain poorly understood. Here, we describe an optimized RNA-based technology for unbiased amplification of T cell receptor beta-chain libraries and use it to perform the first detailed, quantitative tracking of T cell clones during 10 months after transplantation. We show that multiple clones survive the procedure, contribute to the immune response to activated infections, and form a new skewed and stable T cell receptor repertoire.
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http://dx.doi.org/10.1002/emmm.201100129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3377069PMC
April 2011

Contribution of functional KIR3DL1 to ankylosing spondylitis.

Cell Mol Immunol 2010 Nov 6;7(6):471-6. Epub 2010 Sep 6.

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

Increasing evidence points to a role for killer immunoglobulin-like receptors (KIRs) in the development of autoimmune diseases. In particular, a positive association of KIR3DS1 (activating receptor) and a negative association of KIR3DL1 (inhibitory receptor) alleles with ankylosing spondylitis (AS) have been reported by several groups. However, none of the studies analyzed these associations in the context of functionality of polymorphic KIR3DL1. To better understand how the KIR3DL1/3DS1 genes determine susceptibility to AS, we analyzed the frequencies of alleles and genotypes encoding functional (KIR3DL1*F) and non-functional (KIR3DL1*004) receptors. We genotyped 83 AS patients and 107 human leukocyte antigen (HLA)-B27-positive healthy controls from the Russian Caucasian population using a two-stage sequence-specific primer PCR, which distinguishes KIR3DS1, KIR3DL1*F and KIR3DL1*004 alleles. For the patients carrying two functional KIR3DL1 alleles, those alleles were additionally genotyped to identify KIR3DL1*005 and KIR3DL1*007 alleles, which are functional but are expressed at low levels. KIR3DL1 was negatively associated with AS at the expense of KIR3DL1*F but not of KIR3DL1*004. This finding indicates that the inhibitory KIR3DL1 receptor protects against the development of AS and is not simply a passive counterpart of the segregating KIR3DS1 allele encoding the activating receptor. However, analysis of genotype frequencies indicates that the presence of KIR3DS1 is a more important factor for AS susceptibility than the absence of KIR3DL1*F. The activation of either natural killer (NK) or T cells via the KIR3DS1 receptor can be one of the critical events in AS development, while the presence of the functional KIR3DL1 receptor has a protective effect. Nevertheless, even individuals with a genotype that carried two inhibitory KIR3DL1 alleles expressed at high levels could develop AS.
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http://dx.doi.org/10.1038/cmi.2010.42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002958PMC
November 2010

Universal and rapid method for purification of GFP-like proteins by the ethanol extraction.

Protein Expr Purif 2009 May 3;65(1):108-13. Epub 2008 Dec 3.

Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Nauki Prosp. 6, 142290 Pushchino, Moscow Region, Russia.

GFP-like fluorescent proteins (FPs) are crucial in biological and biomedical studies. The majority of FP purification techniques either include multiple time-consuming chromatography steps with a low yield of the desired product or require prior protein modification (addition of special tags). In the present work, we propose an alternative ethanol extraction-based technique previously used for GFP purification and then modified for diverse FPs originated from different sources. The following recombinant FPs were expressed using Escherichia coli M15 (pREP4) strain as a host transformed with pQE30 plasmid bearing one of the target FP genes: TagCFP, TagGFP, TagYFP, TagRFP, TurboGFP, TurboRFP, Dendra2, TurboFP602 and KillerRed. Despite their diversity, all tested recombinant FPs were successfully purified and yielded a highly homogeneous product. The method is easily scalable for purification of any amount of protein and requires no expensive reagents and equipment.
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http://dx.doi.org/10.1016/j.pep.2008.11.008DOI Listing
May 2009