Publications by authors named "Dmitriy M Chudakov"

95 Publications

Longitudinal high-throughput TCR repertoire profiling reveals the dynamics of T-cell memory formation after mild COVID-19 infection.

Elife 2021 01 5;10. Epub 2021 Jan 5.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russian Federation.

COVID-19 is a global pandemic caused by the SARS-CoV-2 coronavirus. T cells play a key role in the adaptive antiviral immune response by killing infected cells and facilitating the selection of virus-specific antibodies. However, neither the dynamics and cross-reactivity of the SARS-CoV-2-specific T-cell response nor the diversity of resulting immune memory is well understood. In this study, we use longitudinal high-throughput T-cell receptor (TCR) sequencing to track changes in the T-cell repertoire following two mild cases of COVID-19. In both donors, we identified CD4 and CD8 T-cell clones with transient clonal expansion after infection. We describe characteristic motifs in TCR sequences of COVID-19-reactive clones and show preferential occurrence of these motifs in publicly available large dataset of repertoires from COVID-19 patients. We show that in both donors, the majority of infection-reactive clonotypes acquire memory phenotypes. Certain T-cell clones were detected in the memory fraction at the pre-infection time point, suggesting participation of pre-existing cross-reactive memory T cells in the immune response to SARS-CoV-2.
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http://dx.doi.org/10.7554/eLife.63502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806265PMC
January 2021

Adoptive Immunotherapy Based on Chain-Centric TCRs in Treatment of Infectious Diseases.

iScience 2020 Dec 24;23(12):101854. Epub 2020 Nov 24.

Federal State Budgetary Institution "N. N. Blokhin National Medical Research Center of Oncology" оf the Ministry of Health of the Russian Federation, 115478 Moscow, Russia.

Complications after vaccination, lack of vaccines against certain infections, and the emergence of antibiotic-resistant microorganisms point to the need for alternative ways of protection and treatment of infectious diseases. Here, we proposed a therapeutic approach to control salmonellosis based on adoptive cell therapy. We showed that the T cell receptor (TCR) repertoire of salmonella-specific memory cells contains 20% of TCR variants with the dominant-active α-chain. Transduction of intact T lymphocytes with the dominant salmonella-specific TCRα led to their enhanced proliferation in response to salmonella. Adoptive transfer of transduced T cells resulted in a significant decrease in bacterial loads in mice infected with salmonella before or after the adoptive transfer. We demonstrated that adoptive immunotherapy based on T cells, transduced with dominant-specific TCRα could be successfully applied for treatment and prevention of infectious diseases and represent a useful addition to vaccination and existing therapeutic strategies.
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http://dx.doi.org/10.1016/j.isci.2020.101854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721641PMC
December 2020

Functionally specialized human CD4 T-cell subsets express physicochemically distinct TCRs.

Elife 2020 12 8;9. Epub 2020 Dec 8.

Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russian Federation.

The organizational integrity of the adaptive immune system is determined by functionally discrete subsets of CD4 T cells, but it has remained unclear to what extent lineage choice is influenced by clonotypically expressed T-cell receptors (TCRs). To address this issue, we used a high-throughput approach to profile the αβ TCR repertoires of human naive and effector/memory CD4 T-cell subsets, irrespective of antigen specificity. Highly conserved physicochemical and recombinatorial features were encoded on a subset-specific basis in the effector/memory compartment. Clonal tracking further identified forbidden and permitted transition pathways, mapping effector/memory subsets related by interconversion or ontogeny. Public sequences were largely confined to particular effector/memory subsets, including regulatory T cells (Tregs), which also displayed hardwired repertoire features in the naive compartment. Accordingly, these cumulative repertoire portraits establish a link between clonotype fate decisions in the complex world of CD4 T cells and the intrinsic properties of somatically rearranged TCRs.
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http://dx.doi.org/10.7554/eLife.57063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773335PMC
December 2020

Two subsets of stem-like CD8 memory T cell progenitors with distinct fate commitments in humans.

Nat Immunol 2020 12 12;21(12):1552-1562. Epub 2020 Oct 12.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8 memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8 memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8 memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.
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http://dx.doi.org/10.1038/s41590-020-0791-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610790PMC
December 2020

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

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

Measuring Intratumoral Heterogeneity of Immune Repertoires.

Front Oncol 2020 8;10:512. Epub 2020 May 8.

Laboratory of Genomics of Antitumor Adaptive Immunity, Privolzhsky Research Medical University, Nizhny Novgorod, Russia.

There is considerable clinical and fundamental value in measuring the clonal heterogeneity of T and B cell expansions in tumors and tumor-associated lymphoid structures-along with the associated heterogeneity of the tumor neoantigen landscape-but such analyses remain challenging to perform. Here, we propose a straightforward approach to analyze the heterogeneity of immune repertoires between different tissue sections in a quantitative and controlled way, based on a beta-binomial noise model trained on control replicates obtained at the level of single-cell suspensions. This approach allows to identify local clonal expansions with high accuracy. We reveal proliferation of clonal T cells in a mouse model of melanoma, and analyze heterogeneity of immunoglobulin repertoires between sections of a metastatically-infiltrated lymph node in human melanoma and primary human colon tumor. On the latter example, we demonstrate the importance of training the noise model on datasets with depth and content that is comparable to the samples being studied. Altogether, we describe here the crucial basic instrumentarium needed to facilitate proper experimental setup planning in the rapidly evolving field of intratumoral immune repertoires, from the wet lab to bioinformatics analysis.
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http://dx.doi.org/10.3389/fonc.2020.00512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227437PMC
May 2020

RNA-Seq-Based TCR Profiling Reveals Persistently Increased Intratumoral Clonality in Responders to Anti-PD-1 Therapy.

Front Oncol 2020 28;10:385. Epub 2020 Apr 28.

Laboratory of Genomics of Antitumor Adaptive Immunity, Privolzhsky Research Medical University, Nizhny Novgorod, Russia.

Substantial effort is being invested in the search for peripheral or intratumoral T cell receptor (TCR) repertoire features that could predict the response to immunotherapy. Here we demonstrate the utility of MiXCR software for TCR and immunoglobulin repertoire extraction from RNA-Seq data obtained from sorted tumor-infiltrating T and B cells. We use this approach to extract TCR repertoires from RNA-Seq data obtained from sorted tumor-infiltrating CD4 and CD8 T cells in an HKP1 (Krasp53) syngeneic mouse model of lung cancer after anti-PD-1 treatment. For both subsets, we demonstrate decreased TCR diversity in response to therapy. At a later time point, repertoire diversity is restored in progressing disease but remains decreased in responders to therapy in both CD4 and CD8 subsets. These observations complement previous studies and suggest that stably increased intratumoral CD4 and CD8 T cell clonality after anti-PD-1/PD-L1 therapy could serve as a predictor of long-term response.
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http://dx.doi.org/10.3389/fonc.2020.00385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199218PMC
April 2020

TCRs with segment TRAV9-2 or a CDR3 histidine are overrepresented among nickel-specific CD4+ T cells.

Allergy 2020 10 13;75(10):2574-2586. Epub 2020 May 13.

Department of Chemical and Product Safety, German Federal Institute for Risk Assessment, Berlin, Germany.

Background: Nickel is the most frequent cause of T cell-mediated allergic contact dermatitis worldwide. In vitro, CD4+ T cells from all donors respond to nickel but the involved αβ T cell receptor (TCR) repertoire has not been comprehensively analyzed.

Methods: We introduce CD154 (CD40L) upregulation as a fast, unbiased, and quantitative method to detect nickel-specific CD4+ T cells ex vivo in blood of clinically characterized allergic and non allergic donors. Naïve (CCR7+ CD45RA+) and memory (not naïve) CD154+ CD4+ T cells were analyzed by flow cytometry after 5 hours of stimulation with 200 µmol/L NiSO ., TCR α- and β-chains of sorted nickel-specific and control cells were studied by high-throughput sequencing.

Results: Stimulation of PBMCs with NiSO induced CD154 expression on ~0.1% (mean) of naïve and memory CD4+ T cells. In allergic donors with recent positive patch test, memory frequencies further increased ~13-fold and were associated with markers of in vivo activation. CD154 expression was TCR-mediated since single clones could be specifically restimulated. Among nickel-specific CD4+ T cells of allergic and non allergic donors, TCRs expressing the α-chain segment TRAV9-2 or a histidine in their α- or β-chain complementarity determining region 3 (CDR3) were highly overrepresented.

Conclusions: Induced CD154 expression represents a reliable method to study nickel-specific CD4+ T cells. TCRs with particular features respond in all donors, while strongly increased blood frequencies indicate nickel allergy for some donors. Our approach may be extended to other contact allergens for the further development of diagnostic and predictive in vitro tests.
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http://dx.doi.org/10.1111/all.14322DOI Listing
October 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

Primary and secondary anti-viral response captured by the dynamics and phenotype of individual T cell clones.

Elife 2020 02 21;9. Epub 2020 Feb 21.

Laboratoire de physique de l'École normale supérieure, ENS, PSL, Sorbonne Université, Université de Paris, and CNRS, Paris, France.

The diverse repertoire of T-cell receptors (TCR) plays a key role in the adaptive immune response to infections. Using TCR alpha and beta repertoire sequencing for T-cell subsets, as well as single-cell RNAseq and TCRseq, we track the concentrations and phenotypes of individual T-cell clones in response to primary and secondary yellow fever immunization - the model for acute infection in humans - showing their large diversity. We confirm the secondary response is an order of magnitude weaker, albeit ∼10 days faster than the primary one. Estimating the fraction of the T-cell response directed against the single immunodominant epitope, we identify the sequence features of TCRs that define the high precursor frequency of the two major TCR motifs specific for this particular epitope. We also show the consistency of clonal expansion dynamics between bulk alpha and beta repertoires, using a new methodology to reconstruct alpha-beta pairings from clonal trajectories.
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http://dx.doi.org/10.7554/eLife.53704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060039PMC
February 2020

B cells, plasma cells and antibody repertoires in the tumour microenvironment.

Nat Rev Immunol 2020 05 27;20(5):294-307. Epub 2020 Jan 27.

Laboratory of Genomics of Antitumor Adaptive Immunity, Privolzhsky Research Medical University, Nizhny Novgorod, Russia.

Recent data show that B cells and plasma cells located in tumours or in tumour-draining lymph nodes can have important roles in shaping antitumour immune responses. In tumour-associated tertiary lymphoid structures, T cells and B cells interact and undergo cooperative selection, specialization and clonal expansion. Importantly, B cells can present cognate tumour-derived antigens to T cells, with the functional consequences of such interactions being shaped by the B cell phenotype. Furthermore, the isotype and specificity of the antibodies produced by plasma cells can drive distinct immune responses. Here we summarize our current knowledge of the roles of B cells and antibodies in the tumour microenvironment. Moreover, we discuss the potential of using immunoglobulin repertoires as a source of tumour-specific receptors for immunotherapy or as biomarkers to predict the efficacy of immunotherapeutic interventions.
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http://dx.doi.org/10.1038/s41577-019-0257-xDOI Listing
May 2020

CD4 T Follicular Helper Cells in Human Tonsils and Blood Are Clonally Convergent but Divergent from Non-Tfh CD4 Cells.

Cell Rep 2020 01;30(1):137-152.e5

Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7FZ, UK. Electronic address:

T follicular helper (Tfh) cells are fundamental for B cell selection and antibody maturation in germinal centers. Circulating Tfh (cTfh) cells constitute a minor proportion of the CD4 T cells in peripheral blood, but their clonotypic relationship to Tfh populations resident in lymph nodes and the extent to which they differ from non-Tfh CD4 cells have been unclear. Using donor-matched blood and tonsil samples, we investigate T cell receptor (TCR) sharing between tonsillar Tfh cells and peripheral Tfh and non-Tfh cell populations. TCR transcript sequencing reveals considerable clonal overlap between peripheral and tonsillar Tfh cell subsets as well as a clear distinction between Tfh and non-Tfh cells. Furthermore, influenza-specific cTfh cell clones derived from blood can be found in the repertoire of tonsillar Tfh cells. Therefore, human blood samples can be used to gain insight into the specificity of Tfh responses occurring in lymphoid tissues, provided that cTfh subsets are studied.
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http://dx.doi.org/10.1016/j.celrep.2019.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029615PMC
January 2020

CXCR3 Identifies Human Naive CD8 T Cells with Enhanced Effector Differentiation Potential.

J Immunol 2019 12 18;203(12):3179-3189. Epub 2019 Nov 18.

Laboratory of Translational Immunology, Humanitas Clinical and Research Center, 20089 Rozzano, Milan, Italy;

In mice, the ability of naive T (T) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8 T cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3 T cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide-HLA class I Ags. Moreover, CXCR3 T cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3 T cells were transcriptionally equivalent to murine CXCR3 T cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8 T cells.
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http://dx.doi.org/10.4049/jimmunol.1901072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900484PMC
December 2019

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

PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice.

Nat Commun 2019 03 29;10(1):1415. Epub 2019 Mar 29.

The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.
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http://dx.doi.org/10.1038/s41467-019-09180-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440946PMC
March 2019

Memory CD4 T cells are generated in the human fetal intestine.

Nat Immunol 2019 03 21;20(3):301-312. Epub 2019 Jan 21.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands.

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4 T cell compartment in the human fetal intestine. We identified 22 CD4 T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4 T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4 T cells. Imaging mass cytometry indicated that memory-like CD4 T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4 T cells in the human fetal intestine that is consistent with exposure to foreign antigens.
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http://dx.doi.org/10.1038/s41590-018-0294-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420108PMC
March 2019

Precise tracking of vaccine-responding T cell clones reveals convergent and personalized response in identical twins.

Proc Natl Acad Sci U S A 2018 12 20;115(50):12704-12709. Epub 2018 Nov 20.

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

T cell receptor (TCR) repertoire data contain information about infections that could be used in disease diagnostics and vaccine development, but extracting that information remains a major challenge. Here we developed a statistical framework to detect TCR clone proliferation and contraction from longitudinal repertoire data. We applied this framework to data from three pairs of identical twins immunized with the yellow fever vaccine. We identified 600 to 1,700 responding TCRs in each donor and validated them using three independent assays. While the responding TCRs were mostly private, albeit with higher overlap between twins, they could be well-predicted using a classifier based on sequence similarity. Our method can also be applied to samples obtained postinfection, making it suitable for systematic discovery of new infection-specific TCRs in the clinic.
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http://dx.doi.org/10.1073/pnas.1809642115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294963PMC
December 2018

Reply to "Evaluation of immune repertoire inference methods from RNA-seq data".

Nat Biotechnol 2018 11;36(11):1035-1036

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

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http://dx.doi.org/10.1038/nbt.4296DOI Listing
November 2018

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

CD49b defines functionally mature Treg cells that survey skin and vascular tissues.

J Exp Med 2018 11 24;215(11):2796-2814. Epub 2018 Oct 24.

Howard Hughes Medical Institute, Immunology Program, and Ludwig Center, Memorial Sloan Kettering Cancer Center, New York, NY

Regulatory T (Treg) cells prevent autoimmunity by limiting immune responses and inflammation in the secondary lymphoid organs and nonlymphoid tissues. While unique subsets of Treg cells have been described in some nonlymphoid tissues, their relationship to Treg cells in secondary lymphoid organs and circulation remains unclear. Furthermore, it is possible that Treg cells from similar tissue types share largely similar properties. We have identified a short-lived effector Treg cell subset that expresses the α integrin, CD49b, and exhibits a unique tissue distribution, being abundant in peripheral blood, vasculature, skin, and skin-draining lymph nodes, but uncommon in the intestines and in viscera-draining lymph nodes. CD49b Treg cells, which display superior functionality revealed by in vitro and in vivo assays, appear to develop after multiple rounds of cell division and TCR-dependent activation. Accordingly, single-cell RNA-seq analysis placed these cells at the apex of the Treg developmental trajectory. These results shed light on the identity and development of a functionally potent subset of mature effector Treg cells that recirculate through and survey peripheral tissues.
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http://dx.doi.org/10.1084/jem.20181442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219731PMC
November 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

Optimized Peptide-MHC Multimer Protocols for Detection and Isolation of Autoimmune T-Cells.

Front Immunol 2018 29;9:1378. Epub 2018 Jun 29.

Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, United Kingdom.

Peptide-MHC (pMHC) multimers have become the "gold standard" for the detection and isolation of antigen-specific T-cells but recent evidence shows that normal use of these reagents can miss fully functional T-cells that bear T-cell receptors (TCRs) with low affinity for cognate antigen. This issue is particularly pronounced for anticancer and autoimmune T-cells as self-reactive T-cell populations are enriched for low-affinity TCRs due to the removal of cells with higher affinity receptors by immune tolerance mechanisms. Here, we stained a wide variety of self-reactive human T-cells using regular pMHC staining and an optimized technique that included: (i) protein kinase inhibitor (PKI), to prevent TCR triggering and internalization, and (ii) anti-fluorochrome antibody, to reduce reagent dissociation during washing steps. Lymphocytes derived from the peripheral blood of type 1 diabetes patients were stained with pMHC multimers made with epitopes from preproinsulin (PPI), insulin-β chain, glutamic acid decarboxylase 65 (GAD65), or glucose-6-phospate catalytic subunit-related protein (IGRP) presented by disease-risk allelles HLA A*02:01 or HLA*24:02. Samples from ankylosing spondylitis patients were stained with a multimerized epitope from vasoactive intestinal polypeptide receptor 1 (VIPR1) presented by HLA B*27:05. Optimized procedures stained an average of 40.5-fold ( = 0.01, range between 1.4 and 198) more cells than could be detected without the inclusion of PKI and cross-linking anti-fluorochrome antibody. Higher order pMHC dextramers recovered more cells than pMHC tetramers in parallel assays, and standard staining protocols with pMHC tetramers routinely recovered less cells than functional assays. HLA A*02:01-restricted PPI-specific and HLA B*27:05-restricted VIPR1-specific T-cell clones generated using the optimized procedure could not be stained by standard pMHC tetramer staining. However, these clones responded well to exogenously supplied peptide and endogenously processed and presented epitopes. We also showed that anti-fluorochrome antibody-conjugated magnetic beads enhanced staining of self-reactive T-cells that could not be stained using standard protocols, thus enabling rapid isolation of autoimmune T-cells. We, therefore, conclude that regular pMHC tetramer staining is generally unsuitable for recovering self-reactive T-cells from clinical samples and recommend the use of the optimized protocols described herein.
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http://dx.doi.org/10.3389/fimmu.2018.01378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034003PMC
June 2018

Human liver infiltrating γδ T cells are composed of clonally expanded circulating and tissue-resident populations.

J Hepatol 2018 09 18;69(3):654-665. Epub 2018 May 18.

Cancer Immunology and Immunotherapy Centre, Institute of Immunology and Immunotherapy, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Electronic address:

Background & Aims: γδ T cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human γδ T cells is primarily based on peripheral blood subsets, while the immunobiology of tissue-associated subsets remains largely unclear. Therefore, we aimed to elucidate the T cell receptor (TCR) diversity, immunophenotype and function of γδ T cells in the human liver.

Methods: We characterised the TCR repertoire, immunophenotype and function of human liver infiltrating γδ T cells, by TCR sequencing analysis, flow cytometry, in situ hybridisation and immunohistochemistry. We focussed on the predominant tissue-associated Vδ2 γδ subset, which is implicated in liver immunopathology.

Results: Intrahepatic Vδ2 γδ T cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T cells were predominantly CD27 effector lymphocytes, whereas naïve CD27, TCR-diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RA Vδ2 γδ effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver Vδ2 γδ T cell pool also included a phenotypically distinct CD45RA effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating Vδ2 γδ cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli.

Conclusion: These findings suggest that the ability of Vδ2 γδ T cells to undergo clonotypic expansion and differentiation is crucial in permitting access to solid tissues, such as the liver, which results in functionally distinct peripheral and liver-resident memory γδ T cell subsets. They also highlight the inherent functional plasticity within the Vδ2 γδ T cell compartment and provide information that could be used for the design of cellular therapies that suppress liver inflammation or combat liver cancer.

Lay Summary: γδ T cells are frequently enriched in many solid tissues, however the immunobiology of such tissue-associated subsets in humans has remained unclear. We show that intrahepatic γδ T cells are enriched for clonally expanded effector T cells, whereas naïve γδ T cells are largely excluded. Moreover, whereas a distinct proportion of circulating T cell clonotypes was present in both the liver tissue and peripheral blood, a functionally and clonotypically distinct population of liver-resident γδ T cells was also evident. Our findings suggest that factors triggering γδ T cell clonal selection and differentiation, such as infection, can drive enrichment of γδ T cells into liver tissue, allowing the development of functionally distinct tissue-restricted memory populations specialised in local hepatic immunosurveillance.
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http://dx.doi.org/10.1016/j.jhep.2018.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6089840PMC
September 2018

The human Vδ2 T-cell compartment comprises distinct innate-like Vγ9 and adaptive Vγ9 subsets.

Nat Commun 2018 05 2;9(1):1760. Epub 2018 May 2.

Cancer Immunology and Immunotherapy Centre, Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, UK.

Vδ2 T cells form the predominant human γδ T-cell population in peripheral blood and mediate T-cell receptor (TCR)-dependent anti-microbial and anti-tumour immunity. Here we show that the Vδ2 compartment comprises both innate-like and adaptive subsets. Vγ9 Vδ2 T cells display semi-invariant TCR repertoires, featuring public Vγ9 TCR sequences equivalent in cord and adult blood. By contrast, we also identify a separate, Vγ9 Vδ2 T-cell subset that typically has a CD27CCR7CD28IL-7Rα naive-like phenotype and a diverse TCR repertoire, however in response to viral infection, undergoes clonal expansion and differentiation to a CD27CD45RACXCR1granzymeA/B effector phenotype. Consistent with a function in solid tissue immunosurveillance, we detect human intrahepatic Vγ9 Vδ2 T cells featuring dominant clonal expansions and an effector phenotype. These findings redefine human γδ T-cell subsets by delineating the Vδ2 T-cell compartment into innate-like (Vγ9) and adaptive (Vγ9) subsets, which have distinct functions in microbial immunosurveillance.
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http://dx.doi.org/10.1038/s41467-018-04076-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932074PMC
May 2018

Method for identification of condition-associated public antigen receptor sequences.

Elife 2018 03 13;7. Epub 2018 Mar 13.

Laboratoire de Physique Theorique, CNRS, Sorbonne University, École Normale Supérieure, Paris, France.

Diverse repertoires of hypervariable immunoglobulin receptors (TCR and BCR) recognize antigens in the adaptive immune system. The development of immunoglobulin receptor repertoire sequencing methods makes it possible to perform repertoire-wide disease association studies of antigen receptor sequences. We developed a statistical framework for associating receptors to disease from only a small cohort of patients, with no need for a control cohort. Our method successfully identifies previously validated Cytomegalovirus and type one diabetes responsive TCR[Formula: see text] sequences .
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http://dx.doi.org/10.7554/eLife.33050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873893PMC
March 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

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
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