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T cells recognize a wide range of pathogens using surface receptors that interact directly with peptides presented on major histocompatibility complexes (MHC) encoded by the HLA loci in humans. Understanding the association between T cell receptors (TCR) and HLA alleles is an important step towards predicting TCR-antigen specificity from sequences. Here we analyze the TCR alpha and beta repertoires of large cohorts of HLA-typed donors to systematically infer such associations, by looking for overrepresentation of TCRs in individuals with a common allele.TCRs, associated with a specific HLA allele, exhibit sequence similarities that suggest prior antigen exposure. Immune repertoire sequencing has produced large numbers of datasets, however the HLA type of the corresponding donors is rarely available. Using our TCR-HLA associations, we trained a computational model to predict the HLA type of individuals from their TCR repertoire alone. We propose an iterative procedure to refine this model by using data from large cohorts of untyped individuals, by recursively typing them using the model itself. The resulting model shows good predictive performance, even for relatively rare HLA alleles., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: PGT has consulted or received travel support from JNJ, Pfizer, Illumina, 10X Genomics, Merck, and PACT Pharma and served on the SAB of Immunoscape, Shennon Bio, and Cytoagents., (Copyright: © 2025 Ruiz Ortega et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 αβTCR 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.

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.)

T cell receptor (TCR) repertoire diversity enables the orchestration of antigen-specific immune responses against the vast space of possible pathogens. Identifying TCR/antigen binding pairs from the large TCR repertoire and antigen space is crucial for biomedical research. Here, we introduce copepodTCR , an open-access tool for the design and interpretation of high-throughput experimental assays to determine TCR specificity. copepodTCR implements a combinatorial peptide pooling scheme for efficient experimental testing of T cell responses against large overlapping peptide libraries, useful for "deorphaning" TCRs of unknown specificity. The scheme detects experimental errors and, coupled with a hierarchical Bayesian model for unbiased results interpretation, identifies the response-eliciting peptide for a TCR of interest out of hundreds of peptides tested using a simple experimental set-up. We experimentally validated our approach on a library of 253 overlapping peptides covering the SARS-CoV-2 spike protein. We provide experimental guides for efficient design of larger screens covering thousands of peptides which will be crucial for the identification of antigen-specific T cells and their targets from limited clinical material., Competing Interests: Competing interests PGT has consulted for Pfizer, JNJ, Cytoagents, 10X, and Illumina, and serves on the SAB for Shennon Bio and Immunoscape. PGT, AAM, and MVP have patents related to TCR amplification, cloning, and applications thereof. The authors declare no other competing interests.

T cells recognize a wide range of pathogens using surface receptors that interact directly with pep-tides presented on major histocompatibility complexes (MHC) encoded by the HLA loci in humans. Understanding the association between T cell receptors (TCR) and HLA alleles is an important step towards predicting TCR-antigen specificity from sequences. Here we analyze the TCR alpha and beta repertoires of large cohorts of HLA-typed donors to systematically infer such associations, by looking for overrepresentation of TCRs in individuals with a common allele.TCRs, associated with a specific HLA allele, exhibit sequence similarities that suggest prior antigen exposure. Immune repertoire sequencing has produced large numbers of datasets, however the HLA type of the corresponding donors is rarely available. Using our TCR-HLA associations, we trained a computational model to predict the HLA type of individuals from their TCR repertoire alone. We propose an iterative procedure to refine this model by using data from large cohorts of untyped individuals, by recursively typing them using the model itself. The resulting model shows good predictive performance, even for relatively rare HLA alleles.

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.)

Spondyloarthritis (SpA) comprises a number of inflammatory rheumatic diseases with overlapping clinical manifestations. Strong association with several HLA-I alleles and T cell infiltration into an inflamed joint suggest involvement of T cells in SpA pathogenesis. In this study, we performed high-throughput T cell repertoire profiling of synovial fluid (SF) and peripheral blood (PB) samples collected from a large cohort of SpA patients. We showed that synovial fluid is enriched with expanded T cell clones that are shared between patients with similar HLA genotypes and persist during recurrent synovitis. Using an algorithm for identification of TCRs involved in immune response we discovered several antigen-driven CD8+ clonal groups associated with risk HLA-B*27 or HLA-B*38 alleles. We further show that these clonal groups were enriched in SF and had higher frequency in PB of SpA patients vs healthy donors, implying their relevance to SpA pathogenesis. Several of the groups were shared among patients with different SpAs that suggests a common immunopathological mechanism of the diseases. In summary, our results provide evidence for the role of specific CD8+ T cell clones in pathogenesis of SpA., Competing Interests: EL is a member of the Speakers bureau of Janssen. TK is a member of Speakers bureau of: Pfizer, MSD, Novartis, AbbVie, Janssen, Lilly, Celgene, JSC BIOCAD, and Novartis-Sandoz. SE is a member of the Speaker’s bureau of KRKKA, MCB and JSC BIOCAD. SL and IZ provide scientific advice for JSC BIOCAD. The remaining 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 Komech, Koltakova, Barinova, Minervina, Salnikova, Shmidt, Korotaeva, Loginova, Erdes, Bogdanova, Shugay, Lukyanov, Lebedev and Zvyagin.)

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.)

Although mRNA vaccine efficacy against severe COVID-19 remains high, variant emergence and breakthrough infections have changed vaccine policy to include booster immunizations. However, the effect of diverse and repeated antigen exposures on SARS-CoV-2 memory T cells is poorly understood. Here, we utilize DNA-barcoded MHC-multimers combined with scRNAseq and scTCRseq to capture the ex vivo profile of SARS-CoV-2-responsive T cells within a cohort of individuals with one, two, or three antigen exposures, including vaccination, primary infection, and breakthrough infection. We found that the order of exposure determined the relative distribution between spike- and non-spike-specific responses, with vaccination after infection leading to further expansion of spike-specific T cells and differentiation to a CCR7-CD45RA+ effector phenotype. In contrast, individuals experiencing a breakthrough infection mount vigorous non-spike-specific responses. In-depth analysis of over 4,000 epitope-specific T cell receptor sequences demonstrates that all types of exposures elicit diverse repertoires characterized by shared, dominant TCR motifs, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and that current vaccination protocols continue to expand and differentiate spike-specific memory responses.

The amount of scientific data and level of public sharing produced as a consequence of the COVID-19 pandemic, as well as the speed at which these data were produced, far exceeds any previous effort against a specific disease condition. This unprecedented situation allows for development and application of new research approaches. One of the major technical hurdles in immunology is the characterization of HLA-antigen-T cell receptor (TCR) specificities. Most approaches aim to identify reactive T cells starting from known antigens using functional assays. However, the need for a reverse approach identifying the antigen specificity of orphan TCRs is increasing. Utilizing large public single-cell gene expression and TCR datasets, we identified highly public CD4 + T cell responses to SARS-CoV-2, covering >75% of the analysed population. We performed an integrative meta-analysis to deeply characterize these clonotypes by TCR sequence, gene expression, HLA-restriction, and antigen-specificity, identifying strong and public CD4 + immunodominant responses with confirmed specificity. CD4 + COVID-enriched clonotypes show T follicular helper functional features, while clonotypes depleted in SARS-CoV-2 individuals preferentially had a central memory phenotype. In total we identify more than 1200 highly public CD4+ T cell clonotypes reactive to SARS-CoV-2. TCR similarity analysis showed six prominent TCR clusters, for which we predicted both HLA-restriction and cognate SARS-CoV-2 immunodominant epitopes. To validate our predictions we used an independent cohort of TCR repertoires before and after vaccination with ChAdOx1 , a replication-deficient simian adenovirus-vectored vaccine, encoding the SARS-CoV-2 spike protein. We find statistically significant enrichment of the predicted spike-reactive TCRs after vaccination with ChAdOx1 , while the frequency of TCRs specific to other SARS-CoV-2 proteins remains stable. Thus, the CD4-associated TCR repertoire differentiates vaccination from natural infection. In conclusion, our study presents a novel reverse epitope discovery approach that can be used to infer HLA- and antigen-specificity of orphan TCRs in any context, such as viral infections, antitumor immune responses, or autoimmune disease., Highlights: Identification of highly public CD4+ T cell responses to SARS-CoV-2Systematic prediction of exact immunogenic HLA class II epitopes for CD4+ T cell responseMethodological framework for reverse epitope discovery, which can be applied to other disease contexts and may provide essential insights for future studies and clinical applications.

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in-depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK-deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo., (© 2020 Federation of American Societies for Experimental Biology.)

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.

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.)

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.

Retroelements are considered as one of the important sources of genomic variability in modern humans. It is known that transposition activity of retroelements in germline cells generates new insertions in various genomic loci and sometimes results in genetic diseases. Retroelements activity in somatic cells is restricted by different cellular mechanisms; however, there is an evidence for it in some tissue types. Somatic insertions can trigger tumorigenesis or participate in normal functioning such as generation of neurons' plasticity. In spite of the rapid development of high-throughput sequencing methods a confident detection of somatic insertions is still quite a challenging task. That, in part, is due to the absence of adequate bioinformatic tools for the analysis of sequencing data. Here, we propose an advanced computational pipeline for the identification of somatic insertions in datasets generated by selective amplification and high-throughput sequencing of genomic regions flanking insertions of AluYa5. Particular attention is paid for the identification of various artifacts arising in course of library preparation and the parameters for their filtration. Pipeline sensitivity is confirmed by in silico experiments with artificial datasets. Using the proposed pipeline we remove at least 80% of artifacts and preserve 75% of potentially somatic insertions. The approaches used in this work can be applied for the study of other mobile elements insertion variability.

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: There is increasing evidence that the transpositional activity of retroelements (REs) is not limited to germ line cells, but often occurs in tumor and normal somatic cells. Somatic transpositions were found in several human tissues and are especially typical for the brain. Several computational and experimental approaches for detection of somatic retroelement insertions was developed in the past few years. These approaches were successfully applied to detect somatic insertions in clonally expanded tumor cells. At the same time, identification of somatic insertions presented in small proportion of cells, such as neurons, remains a considerable challenge., Results: In this study, we developed a normalization procedure for library enrichment by DNA sequences corresponding to rare somatic RE insertions. Two rounds of normalization increased the number of fragments adjacent to somatic REs in the sequenced sample by more than 26-fold, and the number of identified somatic REs was increased by 8-fold., Conclusions: The developed technique can be used in combination with vast majority of modern RE identification approaches and can dramatically increase their capacity to detect rare somatic RE insertions in different types of cells., Competing Interests: The study was approved by the local ethics committee and conducted in accordance with the Declaration of Helsinki.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.)

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant neoplasm of the lymphocyte precursors that suffered malignant transformation arresting the lymphoid cell differentiation. Clinical studies revealed monoor, more rarely, oligoclonal nature of the disease. A precise identification of malignant clone markers is both the crucial stage of early diagnostics and the essential prognostic factor for therapeutic treatment. Here we present an improved system for unbiased detection of lymphoblastic clones in bone marrow aspirates of T-ALL patients. The system based on multiplex PCR of rearranged T-cell receptor locus (TRB) and straightforward sequencing of the resulted PCR fragments. Testing of the system on genomic DNA from Jurkat cell line and four clinical bone marrow aspirates revealed a set of unique TRB rearrangements that precisely characterize each of tested samples. Therefore, the outcome of the system produces highly informative molecular genetic markers for further monitoring of minimal residual disease in T-ALL patients.

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.