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.

• MeSH
• Adult MeSH
• Jurkat Cells MeSH
• Middle Aged MeSH
• Humans MeSH
• Computer Simulation MeSH
• Receptors, Antigen, T-Cell, alpha-beta genetics   MeSH
• Receptors, Antigen, T-Cell genetics   MeSH
• Reproducibility of Results MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Bias MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Receptors, Antigen, T-Cell, alpha-beta MeSH
• Receptors, Antigen, T-Cell MeSH

A balanced immune response is a cornerstone of healthy aging. Here, we uncover distinctive features of the long-lived blind mole-rat (Spalax spp.) adaptive immune system, relative to humans and mice. The T-cell repertoire remains diverse throughout the Spalax lifespan, suggesting a paucity of large long-lived clones of effector-memory T cells. Expression of master transcription factors of T-cell differentiation, as well as checkpoint and cytotoxicity genes, remains low as Spalax ages. The thymus shrinks as in mice and humans, while interleukin-7 and interleukin-7 receptor expression remains high, potentially reflecting the sustained homeostasis of naive T cells. With aging, immunoglobulin hypermutation level does not increase and the immunoglobulin-M repertoire remains diverse, suggesting shorter B-cell memory and sustained homeostasis of innate-like B cells. The Spalax adaptive immune system thus appears biased towards sustained functional and receptor diversity over specialized, long-lived effector-memory clones-a unique organizational strategy that potentially underlies this animal's extraordinary longevity and healthy aging.

• MeSH
• Adaptive Immunity MeSH
• Immunoglobulins metabolism   MeSH
• Interleukin-7 metabolism   MeSH
• Humans MeSH
• Mole Rats MeSH
• Mice MeSH
• Spalax * genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Immunoglobulins MeSH
• Interleukin-7 MeSH

In mice, the ability of naive T (TN) 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+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN 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+ TN 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+ TN cells were transcriptionally equivalent to murine CXCR3+ TN 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.

• MeSH
• Lymphocyte Activation immunology   MeSH
• Biomarkers MeSH
• Cell Differentiation immunology   MeSH
• CD8-Positive T-Lymphocytes immunology   metabolism   MeSH
• Adult MeSH
• Immunophenotyping MeSH
• Immunologic Memory MeSH
• Cells, Cultured MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Mice MeSH
• Receptors, CXCR3 metabolism   MeSH
• Aged MeSH
• T-Lymphocyte Subsets immunology   metabolism   MeSH
• Age Factors MeSH
• Animals MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Mice MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Biomarkers MeSH
• CXCR3 protein, human MeSH Browser
• Receptors, CXCR3 MeSH

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.

• MeSH
• Adaptive Immunity genetics   MeSH
• Antigens, Viral MeSH
• Antigens MeSH
• Complementarity Determining Regions genetics   physiology   MeSH
• Immunotherapy MeSH
• Humans MeSH
• Receptors, Antigen, T-Cell immunology   metabolism   physiology   MeSH
• Sequence Analysis, DNA methods   MeSH
• Cluster Analysis MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Viral MeSH
• Antigens MeSH
• Complementarity Determining Regions MeSH
• Receptors, Antigen, T-Cell MeSH

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.

• Keywords
• age, immunoglobulin repertoire, plasma cell, vaccination, yellow fever,
• MeSH
• Immunity, Active * genetics   MeSH
• B-Lymphocytes immunology   metabolism   MeSH
• Adult MeSH
• Immunoglobulin Constant Regions genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Antibodies, Viral immunology   MeSH
• Receptors, Antigen, B-Cell genetics   metabolism   MeSH
• Somatic Hypermutation, Immunoglobulin MeSH
• Immunoglobulin Heavy Chains genetics   MeSH
• Yellow Fever Vaccine immunology   MeSH
• Vaccination MeSH
• Yellow Fever prevention & control   MeSH
• Animals MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Immunoglobulin Constant Regions MeSH
• Antibodies, Viral MeSH
• Receptors, Antigen, B-Cell MeSH
• Immunoglobulin Heavy Chains MeSH
• Yellow Fever Vaccine MeSH

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.

• Keywords
• Antigen, Immune repertoire, Immunogenicity, T cell receptor,
• MeSH
• Epitopes immunology   MeSH
• Humans MeSH
• Histocompatibility Antigens Class I immunology   MeSH
• Peptides immunology   MeSH
• Receptors, Antigen, T-Cell immunology   MeSH
• Models, Statistical MeSH
• T-Lymphocytes immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epitopes MeSH
• Histocompatibility Antigens Class I MeSH
• Peptides MeSH
• Receptors, Antigen, T-Cell MeSH

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.

• Keywords
• T cell, T-cell receptor repertoires, aging, diversity, functional T-cell subsets,
• MeSH
• Immunity, Cellular physiology   MeSH
• Complementarity Determining Regions genetics   immunology   MeSH
• Mice MeSH
• T-Lymphocyte Subsets cytology   immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Complementarity Determining Regions MeSH

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.

• MeSH
• Single-Cell Analysis MeSH
• Molecular Sequence Annotation * MeSH
• Antigens chemistry   immunology   metabolism   MeSH
• Databases, Protein * MeSH
• Major Histocompatibility Complex genetics   immunology   MeSH
• Protein Interaction Domains and Motifs MeSH
• Internet MeSH
• Humans MeSH
• Macaca mulatta MeSH
• Models, Molecular MeSH
• Mice MeSH
• Receptors, Antigen, T-Cell chemistry   immunology   metabolism   MeSH
• Protein Structure, Secondary MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Sequence Alignment MeSH
• Software * MeSH
• T-Lymphocytes cytology   immunology   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens MeSH
• Receptors, Antigen, T-Cell MeSH

• MeSH
• Algorithms MeSH
• Databases, Genetic MeSH
• Humans MeSH
• Melanoma chemistry   genetics   mortality   MeSH
• Mice MeSH
• Receptors, Antigen, T-Cell chemistry   genetics   MeSH
• Receptors, Antigen * analysis   genetics   metabolism   MeSH
• RNA analysis   genetics   MeSH
• Sequence Analysis, RNA methods   MeSH
• Gene Expression Profiling methods   MeSH
• Computational Biology methods   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Receptors, Antigen, T-Cell MeSH
• Receptors, Antigen * MeSH
• RNA MeSH

The diversity of T-cell receptors recognizing foreign pathogens is generated through a highly stochastic recombination process, making the independent production of the same sequence rare. Yet unrelated individuals do share receptors, which together constitute a "public" repertoire of abundant clonotypes. The TCR repertoire is initially formed prenatally, when the enzyme inserting random nucleotides is downregulated, producing a limited diversity subset. By statistically analyzing deep sequencing T-cell repertoire data from twins, unrelated individuals of various ages, and cord blood, we show that T-cell clones generated before birth persist and maintain high abundances in adult organisms for decades, slowly decaying with age. Our results suggest that large, low-diversity public clones are created during pre-natal life, and survive over long periods, providing the basis of the public repertoire.

• MeSH
• T-Cell Antigen Receptor Specificity genetics   MeSH
• Twins, Monozygotic genetics   MeSH
• Genetic Variation genetics   MeSH
• Gene Rearrangement, T-Lymphocyte genetics   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Receptors, Antigen, T-Cell physiology   MeSH
• Recombination, Genetic MeSH
• Base Sequence MeSH
• Aging genetics   immunology   MeSH
• Gene Expression Regulation, Developmental genetics   immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Receptors, Antigen, T-Cell MeSH