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

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.

• Keywords
• CDR3 properties, TCR repertoire, helper CD4+ subsets, human, immunology, inflammation, plasticity of CD4+ subsets,
• MeSH
• Cell Lineage immunology   MeSH
• CD4-Positive T-Lymphocytes immunology   MeSH
• Humans MeSH
• Receptors, Antigen, T-Cell, alpha-beta immunology   MeSH
• T-Lymphocyte Subsets immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Receptors, Antigen, T-Cell, alpha-beta MeSH

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 in situ 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.

• Keywords
• TCR repertoire, clonal expansions, immunoglobulin repertoire, tumour clonality, tumour heterogeneity,
• Publication type
• Journal Article 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

BACKGROUND: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps. RESULTS: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification. CONCLUSION: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.

• Keywords
• High-throughput sequencing, PCR, Targeted resequencing, Unique molecular identifiers,
• MeSH
• DNA Primers genetics   MeSH
• ErbB Receptors genetics   MeSH
• Gene Library * MeSH
• Polymerase Chain Reaction methods   MeSH
• Sequence Analysis, DNA MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA Primers MeSH
• ErbB Receptors MeSH

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

• MeSH
• Databases, Genetic MeSH
• Humans MeSH
• Biomarkers, Tumor blood   genetics   MeSH
• Neoplasms genetics   MeSH
• RNA, Viral genetics   MeSH
• Sequence Analysis, DNA methods   MeSH
• Sequence Analysis, RNA methods   MeSH
• Software * MeSH
• Computational Biology methods   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
• Biomarkers, Tumor MeSH
• RNA, Viral MeSH

High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.

• MeSH
• Immunoglobulin Light Chains genetics   MeSH
• Humans MeSH
• Mutation MeSH
• Mice MeSH
• Quality Control MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA methods   MeSH
• Immunoglobulin Heavy Chains genetics   MeSH
• High-Throughput Nucleotide Sequencing methods   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
• Immunoglobulin Light Chains MeSH
• Immunoglobulin Heavy Chains MeSH

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 .

• Keywords
• B-cell, Browser, High-throughput sequencing, Immunology, Repertoire sequencing, T-cell,
• MeSH
• B-Lymphocytes immunology   metabolism   MeSH
• Genomics methods   standards   MeSH
• Complementarity Determining Regions genetics   MeSH
• Web Browser MeSH
• Clonal Evolution genetics   MeSH
• Humans MeSH
• Cluster Analysis MeSH
• Software * MeSH
• T-Lymphocytes immunology   metabolism   MeSH
• V(D)J Recombination * MeSH
• Computational Biology methods   standards   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Complementarity Determining Regions MeSH

CD4+ T cells predominate in salivary gland (SG) inflammatory lesions in Sjögren's syndrome (SS). However, their antigen specificity, degree of clonal expansion, and relationship to clinical disease features remain unknown. We used multiplex reverse-transcriptase PCR to amplify paired T cell receptor α (TCRα) and β transcripts of single CD4+CD45RA- T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype. TCRα and β sequences were obtained from a median of 91 SG and 107 PB cells per subject. The degree of clonal expansion and frequency of cells expressing two productively rearranged α genes were increased in SG versus PB. Expanded clones from SG exhibited complementary-determining region 3 (CDR3) sequence similarity both within and among subjects, suggesting antigenic selection and shared antigen recognition. CDR3 similarities were shared among expanded clones from individuals discordant for canonical Ro and La autoantibodies, suggesting recognition of alternative SG antigen(s). The extent of SG clonal expansion correlated with reduced saliva production and increased SG fibrosis, linking expanded SG T cells with glandular dysfunction. Knowledge of paired TCRα and β sequences enables further work toward identification of target antigens and development of novel therapies.

• Publication type
• Journal Article MeSH

• Keywords
• TCR profiling, minor lymphocyte subsets, unique molecular barcodes,
• MeSH
• Humans MeSH
• Lymphocytes metabolism   MeSH
• Lymphocyte Count * MeSH
• Gene Expression Profiling * MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Comment MeSH
• Editorial MeSH