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
• dospělí MeSH
• Jurkat buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• počítačová simulace MeSH
• receptory antigenů T-buněk alfa-beta genetika   MeSH
• receptory antigenů T-buněk genetika   MeSH
• reprodukovatelnost výsledků MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zkreslení výsledků (epidemiologie) MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• receptory antigenů T-buněk alfa-beta MeSH
• receptory antigenů T-buněk MeSH

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.

• Klíčová slova
• COVID-19, RepSeq, SARS-CoV-2, TCR, computational biology, human, immunology, inflammation, systems biology,
• MeSH
• COVID-19 imunologie   patofyziologie   MeSH
• genová knihovna MeSH
• imunologická paměť * MeSH
• lidé MeSH
• longitudinální studie MeSH
• mapování epitopu MeSH
• receptory antigenů T-buněk chemie   genetika   MeSH
• SARS-CoV-2 fyziologie   MeSH
• sekvence aminokyselin MeSH
• stupeň závažnosti nemoci MeSH
• T-lymfocyty imunologie   MeSH
• testování histokompatibility MeSH
• zkřížené reakce MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• receptory antigenů T-buněk MeSH

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.

• Klíčová slova
• TCR, computational biology, human, immunology, inflammation, single-cell, systems biology, vaccination, yellow fever,
• MeSH
• časové faktory MeSH
• dospělí MeSH
• epitopy imunologie   MeSH
• fenotyp MeSH
• imunologická paměť MeSH
• lidé MeSH
• podskupiny lymfocytů imunologie   fyziologie   MeSH
• receptory antigenů T-buněk genetika   imunologie   fyziologie   MeSH
• T-lymfocyty imunologie   fyziologie   virologie   MeSH
• transkriptom MeSH
• vakcína proti žluté zimnici imunologie   farmakologie   MeSH
• virus žluté zimnice imunologie   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• žlutá zimnice imunologie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epitopy MeSH
• receptory antigenů T-buněk MeSH
• vakcína proti žluté zimnici 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
• adaptivní imunita genetika   MeSH
• antigeny virové MeSH
• antigeny MeSH
• hypervariabilní oblasti genetika   fyziologie   MeSH
• imunoterapie MeSH
• lidé MeSH
• receptory antigenů T-buněk imunologie   metabolismus   fyziologie   MeSH
• sekvenční analýza DNA metody   MeSH
• shluková analýza MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny virové MeSH
• antigeny MeSH
• hypervariabilní oblasti MeSH
• receptory antigenů T-buněk MeSH