T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5'D2-RS - 3'D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response.

• Keywords
• NGS - next generation sequencing, T cell, TRB repertoire, Thymus, VDJ recombination,
• MeSH
• Apoptosis * MeSH
• Clone Cells MeSH
• Chromosome Aberrations MeSH
• Genes, T-Cell Receptor beta * MeSH
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Memory T Cells MeSH
• V(D)J Recombination * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND AND AIMS: Intestinal inflammation in inflammatory bowel diseases [IBD] is thought to be T cell mediated and therefore dependent on the interaction between the T cell receptor [TCR] and human leukocyte antigen [HLA] proteins expressed on antigen presenting cells. The collection of all TCRs in one individual, known as the TCR repertoire, is characterised by enormous diversity and inter-individual variability. It was shown that healthy monozygotic [MZ] twins are more similar in their TCR repertoire than unrelated individuals. Therefore MZ twins, concordant or discordant for IBD, may be useful to identify disease-related and non-genetic factors in the TCR repertoire which could potentially be used as disease biomarkers. METHODS: Employing unique molecular barcoding that can distinguish between polymerase chain reaction [PCR] artefacts and true sequence variation, we performed deep TCRα and TCRβ repertoire profiling of the peripheral blood of 28 MZ twin pairs from Denmark and Germany, 24 of whom were discordant and four concordant for IBD. RESULTS: We observed disease- and smoking-associated traits such as sharing, diversity and abundance of specific clonotypes in the TCR repertoire of IBD patients, and particularly in patients with active disease, compared with their healthy twins. CONCLUSIONS: Our findings identified TCR repertoire features specific for smokers and IBD patients, particularly when signs of disease activity were present. These findings are a first step towards the application of TCR repertoire analyses as a valuable tool to characterise inflammatory bowel diseases and to identify potential biomarkers and true disease causes.

• Keywords
• T cell receptor [TCR] repertoire, inflammatory bowel diseases [IBD], monozygotic twins,
• MeSH
• C-Reactive Protein analysis   MeSH
• Crohn Disease * diagnosis   immunology   physiopathology   MeSH
• Adult MeSH
• Twins, Monozygotic MeSH
• Feces MeSH
• Genes, T-Cell Receptor alpha * MeSH
• Genes, T-Cell Receptor beta * MeSH
• Smoking immunology   MeSH
• Leukocyte L1 Antigen Complex analysis   MeSH
• Humans MeSH
• Patient Acuity MeSH
• Receptors, Antigen, T-Cell, alpha-beta blood   MeSH
• Sequence Analysis, DNA MeSH
• Colitis, Ulcerative * diagnosis   immunology   physiopathology   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Twin Study MeSH
• Geographicals
• Denmark MeSH
• Germany MeSH
• Names of Substances
• C-Reactive Protein MeSH
• Leukocyte L1 Antigen Complex MeSH
• Receptors, Antigen, T-Cell, alpha-beta 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

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.

• Keywords
• RepSeq, T cell receptor, high-throughput sequencing, twins, vaccination,
• MeSH
• Antigens, Viral immunology   MeSH
• Tissue Donors MeSH
• Twins, Monozygotic MeSH
• Immunization methods   MeSH
• Humans MeSH
• Receptors, Antigen, T-Cell immunology   MeSH
• T-Lymphocytes immunology   MeSH
• Yellow Fever Vaccine immunology   MeSH
• Vaccination 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
• Receptors, Antigen, T-Cell MeSH
• Yellow Fever Vaccine MeSH

• MeSH
• Gene Regulatory Networks * MeSH
• RNA * MeSH
• Sequence Analysis, RNA MeSH
• Publication type
• Journal Article MeSH
• Comment MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA * 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