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ɑ/β T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) ɑ and β chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies. Here, we present TIRTL-seq (T hroughput- I ntensive R apid T CR L ibrary seq uencing ) , a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well. The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired ɑβTCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is ∼$200. We benchmarked TIRTL-seq against state-of-the-art 5'RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. The sequences of both ɑ and β TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire.

Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection 1,2 , yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of samples from patients with MIS-C to identify a distinct set of host proteins targeted by patient autoantibodies including a particular autoreactive epitope within SNX8, a protein involved in regulating an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed antibody responses from patients with MIS-C to the complete SARS-CoV-2 proteome and found enriched reactivity against a distinct domain of the SARS-CoV-2 nucleocapsid protein. The immunogenic regions of the viral nucleocapsid and host SNX8 proteins bear remarkable sequence similarity. Consequently, we found that many children with anti-SNX8 autoantibodies also have cross-reactive T cells engaging both the SNX8 and the SARS-CoV-2 nucleocapsid protein epitopes. Together, these findings suggest that patients with MIS-C develop a characteristic immune response to the SARS-CoV-2 nucleocapsid protein that is associated with cross-reactivity to the self-protein SNX8, demonstrating a mechanistic link between the infection and the inflammatory syndrome, with implications for better understanding a range of post-infectious autoinflammatory diseases., (© 2024. The Author(s).)

Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 T cell responses to this fusion in FLC patients and evaluate fusion-specific T cell receptors (TCRs) for use in cellular immunotherapies. We observe that fusion-specific CD8 T cells are rare and that FLC patient TCR repertoires lack large clusters of related TCR sequences characteristic of potent antigen-specific responses, potentially explaining why endogenous immune responses are insufficient to clear FLC tumors. Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity in vivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC., Competing Interests: Declaration of interests J.C.C., A.M.K., A.E.Z., S.E.S., and P.G.T. have a patent application for TCRs for treating FLC. J.C.C. has additional patent applications in the field of immunotherapy. A.W.P. is a member of the scientific advisory board (SAB) of Bioinformatics Solutions Inc. (Canada), shareholder and SAB member of Evaxion Biotech (Denmark), consultant for Grey Wolf Therapeutics (UK), and cofounder of Resseptor Therapeutics (Melbourne). S.G. is a co-inventor on patent applications in the fields of cell/gene therapy for cancer, consultant of TESSA Therapeutics, member of the Data and Safety Monitoring Board of Immatics, and SAB member of Be Biopharma and has received honoraria from Tidal, Catamaran Bio, and Sanofi within the last 2 years. S.E.S. is a cofounder, stockholder, and paid consultant for Gliknik Inc., serves on the SAB and holds stock options for Virion Inc., and receives royalties from the Mayo Clinic for licensed IP surrounding manipulation of the PD-1:PD-L1 pathway for cancer treatment. P.G.T. is on the SAB of Immunoscape and Shennon Bio, received personal fees and research support from Elevate Bio, and consulted for 10×, Illumina, Pfizer, Cytoagents, and JNJ., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

CD8 + T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8 + T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M1 58-66 (A2/M1 58 ) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M1 58 + CD8 + T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8 + T cells, which was linked to highly functional public T cell receptor (TCR)αβ signatures. Suboptimal TCRαβ signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8 + T cell responses toward viral infections., (© 2023. The Author(s).)

High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4 + and CD8 + T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people., (© 2023. The Author(s).)

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vaccination. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (∼26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and peptide-HLA tetramer-specific CD4 + /CD8 + T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vaccinated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response., Competing Interests: Declaration of interests The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines, which list F.K. as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. F.K. has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. H.A.M. and B.Y.C. are currently consulting for Ena Respiratory. B.W.T. has received research funding from MSD, Seqirus, and Sanofi and is on the advisory board for Moderna, CSL-Behring, and Takeda., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: One of the current hypotheses to explain the proinflammatory immune response in IBD is a dysregulated T cell reaction to yet unknown intestinal antigens. As such, it may be possible to identify disease-associated T cell clonotypes by analysing the peripheral and intestinal T-cell receptor (TCR) repertoire of patients with IBD and controls., Design: We performed bulk TCR repertoire profiling of both the TCR alpha and beta chains using high-throughput sequencing in peripheral blood samples of a total of 244 patients with IBD and healthy controls as well as from matched blood and intestinal tissue of 59 patients with IBD and disease controls. We further characterised specific T cell clonotypes via single-cell RNAseq., Results: We identified a group of clonotypes, characterised by semi-invariant TCR alpha chains, to be significantly enriched in the blood of patients with Crohn's disease (CD) and particularly expanded in the CD8 + T cell population. Single-cell RNAseq data showed an innate-like phenotype of these cells, with a comparable gene expression to unconventional T cells such as mucosal associated invariant T and natural killer T (NKT) cells, but with distinct TCRs., Conclusions: We identified and characterised a subpopulation of unconventional Crohn-associated invariant T (CAIT) cells. Multiple evidence suggests these cells to be part of the NKT type II population. The potential implications of this population for CD or a subset thereof remain to be elucidated, and the immunophenotype and antigen reactivity of CAIT cells need further investigations in future studies., Competing Interests: Competing interests: ER, AF, PB and IZM hold a pending patent application based on the results described in this manuscript (EP21217058.3)., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27-). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products., Significance: Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients. This article is highlighted in the In This Issue feature, p. 2007., (©2022 The Authors; Published by the American Association for Cancer Research.)

The development and implementation of vaccines have been growing exponentially, remaining one of the major successes of healthcare over the last century. Nowadays, active regular immunizations prevent epidemics of many viral diseases, including tick-borne encephalitis (TBE). Along with the generation of virus-specific antibodies, a highly effective vaccine should induce T cell responses providing long-term immune defense. In this study, we performed longitudinal high-throughput T cell receptor (TCR) sequencing to characterize changes in individual T cell repertoires of 11 donors immunized with an inactivated TBE vaccine. After two-step immunization, we found significant clonal expansion of both CD4 + and CD8 + T cells, ranging from 302 to 1706 vaccine-associated TCRβ clonotypes in different donors. We detected several waves of T cell clonal expansion generated by distinct groups of vaccine-responding clones. Both CD4 + and CD8 + vaccine-responding T cell clones formed 17 motifs in TCRβ sequences shared by donors with identical HLA alleles. Our results indicate that TBE vaccination leads to a robust T cell response due to the production of a variety of T cell clones with a memory phenotype, which recognize a large set of epitopes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sycheva, Komech, Pogorelyy, Minervina, Urazbakhtin, Salnikova, Vorovitch, Kopantzev, Zvyagin, Komkov, Mamedov and Lebedev.)

The current strategy to detect immunodominant T cell responses focuses on the antigen, employing large peptide pools to screen for functional cell activation. However, these approaches are labor and sample intensive and scale poorly with increasing size of the pathogen peptidome. T cell receptors (TCRs) recognizing the same epitope frequently have highly similar sequences, and thus, the presence of large sequence similarity clusters in the TCR repertoire likely identify the most public and immunodominant responses. Here, we perform a meta-analysis of large, publicly available single-cell and bulk TCR datasets from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals to identify public CD4 + responses. We report more than 1,200 αβTCRs forming six prominent similarity clusters and validate histocompatibility leukocyte antigen (HLA) restriction and epitope specificity predictions for five clusters using transgenic T cell lines. Collectively, these data provide information on immunodominant CD4 + T cell responses to SARS-CoV-2 and demonstrate the utility of the reverse epitope discovery approach., Competing Interests: Declaration of interests P.B. and A.S. are consultants of Miltenyi Biotec, who own IP rights concerning parts of the ARTE technology. P.G.T. has consulted and/or received honoraria and travel support from Illumina, Johnson and Johnson, and 10X Genomics. P.G.T. serves on the Scientific Advisory Board of Immunoscape and Cytoagents. The authors have applied for patents covering some aspects of these studies., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4 + and CD8 + T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαβ repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαβ motifs in unvaccinated seroconverted children after their first virus encounter., Competing Interests: Declaration of interests SARS-CoV-2 serological assays (US Provisional Application #62/994252, 63/018457, 63/020503, and 63/024436) and NDV-based SARS-CoV-2 vaccines (US Provisional Application #63/251020) list F.K. as a co-inventor. F.A. is also a co-inventor of the serological assay patents. Patent applications were submitted by the Icahn School of Medicine at Mount Sinai. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. F.K. has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Third Rock Ventures, Seqirus, and Avimex. F.K. laboratory collaborates with Pfizer on the animal models of SARS-CoV-2. P.G.T. is on the SAB of Immunoscape and Cytoagents, has consulted for JNJ, received travel support and/or honoraria from Illumina, 10X Genomics, and has patents on TCR discovery and expression. P.S.P. received research grants from Astra Zeneca and Pfizer and has served on advisory boards for Sanofi Pasteur and Seqirus., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although mRNA vaccine efficacy against severe coronavirus disease 2019 remains high, variant emergence has prompted booster immunizations. However, the effects of repeated exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens on memory T cells are poorly understood. Here, we utilize major histocompatibility complex multimers with single-cell RNA sequencing to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two or three antigen exposures, including vaccination, primary infection and breakthrough infection. Exposure order determined the distribution between spike-specific and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7 - CD45RA + effectors. In contrast, individuals after breakthrough infection mount vigorous non-spike-specific responses. Analysis of over 4,000 epitope-specific T cell antigen receptor (TCR) sequences demonstrates that all exposures elicit diverse repertoires characterized by shared TCR motifs, confirmed by monoclonal TCR characterization, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and current vaccination protocols continue to expand and differentiate spike-specific memory., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4 + T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4 + T (T FH ) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node T FH . Mining of the responding T FH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1 ∗ 04-restricted response to S 167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific T FH cells peak one week after the second immunization, S-specific T FH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust T FH cell responses play in establishing long-term immunity by this efficacious human vaccine., Competing Interests: Declaration of interests The Ellebedy laboratory received funding under sponsored research agreements that are unrelated to the data presented in the current study from Emergent BioSolutions and from AbbVie. A.H.E. has received consulting payments from Mubadala Investment Company, InBios International, and Fimbrion Therapeutics and is the founder of ImmuneBio Consulting. P.G.T. has consulted and/or received honoraria and travel support from Illumina, Johnson and Johnson, and 10X Genomics. P.G.T. serves on the Scientific Advisory Board of Immunoscape and Cytoagents. The authors have applied for patents covering some aspects of these studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: AM, EK, AT, MB, ER, IM, AF, GE, DC, TM, YL, MP No competing interests declared, AW Senior editor, eLife, (© 2021, Minervina et al.)

Immune repertoires provide a unique fingerprint reflecting the immune history of individuals, with potential applications in precision medicine. However, the question of how personal that information is and how it can be used to identify individuals has not been explored. Here, we show that individuals can be uniquely identified from repertoires of just a few thousands lymphocytes. We present "Immprint," a classifier using an information-theoretic measure of repertoire similarity to distinguish pairs of repertoire samples coming from the same versus different individuals. Using published T-cell receptor repertoires and statistical modeling, we tested its ability to identify individuals with great accuracy, including identical twins, by computing false positive and false negative rates < 10-6 from samples composed of 10,000 T-cells. We verified through longitudinal datasets that the method is robust to acute infections and that the immune fingerprint is stable for at least three years. These results emphasize the private and personal nature of repertoire data., Competing Interests: The authors have declared that no competing interests exist.

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., (© European Crohn’s and Colitis Organisation (ECCO) 2019.)

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 H2-A and analyzed thymic and peripheral production and TCR repertoires of naïve conventional CD4 + T (T conv ) and naïve regulatory CD4 + T (T reg ) cells. Compared with tuberculosis-resistant C57BL/6 (H2-A b ) mice, the tuberculosis-susceptible H2-A j mice had fewer CD4 + T cells of both subsets in the thymus. In the periphery, this deficiency was only apparent for T conv and was compensated for by peripheral reconstitution for T reg We show that H2-A j 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 j and H2-A b 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., Competing Interests: The authors declare no competing interest.

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., (Copyright © 2020 Yuzhakova, Volchkova, Pogorelyy, Serebrovskaya, Shagina, Bryushkova, Nakonechnaya, Izosimova, Zavyalova, Karabut, Izraelson, Samoylenko, Zagainov, Chudakov, Zagaynova and Sharonov.)

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., Competing Interests: AM, MP, EK, VK, PB, ER, AF, DC, IM, YL, TM No competing interests declared, AW Senior editor, eLife, (© 2020, Minervina et al.)

Recently developed molecular methods allow large-scale profiling of T-cell receptor (TCR) sequences that encode for antigen specificity and immunological memory of these cells. However, it is well-known that the even unperturbed TCR repertoire structure is extremely complex due to the high diversity of TCR rearrangements and multiple biases imprinted by VDJ rearrangement process. The latter gives rise to the phenomenon of "public" TCR clonotypes that can be shared across multiple individuals and non-trivial structure of the TCR similarity network. Here, we outline a framework for TCR sequencing data analysis that can control for these biases in order to infer TCRs that are involved in response to antigens of interest. We apply two previously published methods, ALICE and TCRNET, to detect groups of homologous TCRs that are enriched in samples of interest. Using an example dataset of donors with known HLA haplotype and CMV status, we demonstrate that by applying HLA restriction rules and matching against a database of TCRs with known antigen specificity, it is possible to robustly detect motifs of epitope-specific responses in individual repertoires. We also highlight potential shortcomings of TCR clustering methods and demonstrate that highly expanded TCRs should be individually assessed to get the full picture of antigen-specific response., (Copyright © 2019 Pogorelyy and Shugay.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

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.

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 CD4 + CD25 - CD31 + (enriched with recent thymic emigrants, RTE), and mature naïve CD4 + CD25 - CD31 - 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.

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.

Autologous hematopoietic stem cell transplantation (HSCT), a safer type of HSCT than allogeneic HSCT, is a promising therapy for patients with severe autoimmune diseases (ADs). Despite the long history of medical practice, structural changes in the adaptive immune system as a result of autologous HSCT in patients with various types of ADs remain poorly understood. In this study, we used high-throughput sequencing to investigate the structural changes in the peripheral blood T-cell repertoire in adult patients with ankylosing spondylitis (AS) during two years after autologous HSCT. The implementation of unique molecular identifiers allowed us to substantially reduce the impact of the biases occurring during the preparation of libraries, to carry out a comparative analysis of the various properties of the T-cell repertoire between different time points, and to track the dynamics of both distinct T-cell clonotypes and T-cell subpopulations. In the first year of the reconstitution, clonal diversity of the T-cell repertoire remained lower than the initial one in both patients. During the second year after HSCT, clonal diversity continued to increase and reached a normal value in one of the patients. The increase in the diversity was associated with the emergence of a large number of low-frequency clonotypes, which were not identified before HSCT. Efficiency of clonotypes detection after HSCT was dependent on their abundance in the initial repertoire. Almost all of the 100 most abundant clonotypes observed before HSCT were detected 2 years after transplantation and remained highly abundant irrespective of their CD4+ or CD8+ phenotype. A total of up to 25% of peripheral blood T cells 2 years after HSCT were represented by clonotypes from the initial repertoire.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 ., Competing Interests: MP, AM, DC, IM, YL, TM No competing interests declared, AW Reviewing editor, eLife, (© 2018, Pogorelyy et al.)

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.

The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRβ repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Despite the growing number of immune repertoire sequencing studies, the field still lacks software for analysis and comprehension of this high-dimensional data. Here we report VDJtools, a complementary software suite that solves a wide range of T cell receptor (TCR) repertoires post-analysis tasks, provides a detailed tabular output and publication-ready graphics, and is built on top of a flexible API. Using TCR datasets for a large cohort of unrelated healthy donors, twins, and multiple sclerosis patients we demonstrate that VDJtools greatly facilitates the analysis and leads to sound biological conclusions. VDJtools software and documentation are available at https://github.com/mikessh/vdjtools.

Background: The Immunoglobulins (IG) and the T cell receptors (TR) play the key role in antigen recognition during the adaptive immune response. Recent progress in next-generation sequencing technologies has provided an opportunity for the deep T cell receptor repertoire profiling. However, a specialised software is required for the rational analysis of massive data generated by next-generation sequencing., Results: Here we introduce tcR, a new R package, representing a platform for the advanced analysis of T cell receptor repertoires, which includes diversity measures, shared T cell receptor sequences identification, gene usage statistics computation and other widely used methods. The tool has proven its utility in recent research studies., Conclusions: tcR is an R package for the advanced analysis of T cell receptor repertoires after primary TR sequences extraction from raw sequencing reads. The stable version can be directly installed from The Comprehensive R Archive Network ( http://cran.r-project.org/mirrors.html ). The source code and development version are available at tcR GitHub ( http://imminfo.github.io/tcr/ ) along with the full documentation and typical usage examples.

Retroelement activity is a common source of polymorphisms in human genome. The mechanism whereby retroelements contribute to the intraindividual genetic heterogeneity by inserting into the DNA of somatic cells is gaining increasing attention. Brain tissues are suspected to accumulate genetic heterogeneity as a result of the retroelements somatic activity. This study aims to expand our understanding of the role retroelements play in generating somatic mosaicism of neural tissues. Whole-genome Alu and L1 profiling of genomic DNA extracted from the cerebellum, frontal cortex, subventricular zone, dentate gyrus, and the myocardium revealed hundreds of somatic insertions in each of the analyzed tissues. Interestingly, the highest concentration of such insertions was detected in the dentate gyrus-the hotspot of adult neurogenesis. Insertions of retroelements and their activity could produce genetically diverse neuronal subsets, which can be involved in hippocampal-dependent learning and memory.

Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some α but not β J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.

The relationship between maternal and child immunity has been actively studied in the context of complications during pregnancy, autoimmune diseases, and haploidentical transplantation of hematopoietic stem cells and solid organs. Here, we have for the first time used high-throughput Illumina HiSeq sequencing to perform deep quantitative profiling of T cell receptor (TCR) repertoires for peripheral blood samples of three mothers and their six children. Advanced technology allowed accurate identification of 5 × 10(5) to 2 × 10(6) TCR beta clonotypes per individual. We performed comparative analysis of these TCR repertoires with the aim of revealing characteristic features that distinguish related mother-child pairs, such as relative TCR beta variable segment usage frequency and relative overlap of TCR beta complementarity-determining region 3 (CDR3) repertoires. We show that thymic selection essentially and similarly shapes the initial output of the TCR recombination machinery in both related and unrelated pairs, with minor effect from inherited differences. The achieved depth of TCR profiling also allowed us to test the hypothesis that mature T cells transferred across the placenta during pregnancy can expand and persist as functional microchimeric clones in their new host, using characteristic TCR beta CDR3 variants as clonal identifiers.