Revealing Clonal Responses of Tumor-Reactive T-Cells Through T Cell Receptor Repertoire Analysis.

CD8+ T cells are the key effector cells that contribute to the antitumor immune response. They comprise various T-cell clones with diverse antigen-specific T-cell receptors (TCRs). Thus, elucidating the overall antitumor responses of diverse T-cell clones is an emerging challenge in tumor immunology. With the recent advancement in next-generation DNA sequencers, comprehensive analysis of the collection of TCR genes (TCR repertoire analysis) is feasible and has been used to investigate the clonal responses of antitumor T cells. However, the immunopathological significance of TCR repertoire indices is still undefined. In this review, we introduce two approaches that facilitate an immunological interpretation of the TCR repertoire data: inter-organ clone tracking analysis and single-cell TCR sequencing. These approaches for TCR repertoire analysis will provide a more accurate understanding of the response of tumor-specific T cells in the tumor microenvironment.

huARdb: human Antigen Receptor database for interactive clonotype-transcriptome analysis at the single-cell level.

T-cell receptors (TCRs) and B-cell receptors (BCRs) are critical in recognizing antigens and activating the adaptive immune response. Stochastic V(D)J recombination generates massive TCR/BCR repertoire diversity. Single-cell immune profiling with transcriptome analysis allows the high-throughput study of individual TCR/BCR clonotypes and functions under both normal and pathological settings. However, a comprehensive database linking these data is not yet readily available. Here, we present the human Antigen Receptor database (huARdb), a large-scale human single-cell immune profiling database that contains 444 794 high confidence T or B cells (hcT/B cells) with full-length TCR/BCR sequence and transcriptomes from 215 datasets. All datasets were processed in a uniform workflow, including sequence alignment, cell subtype prediction, unsupervised cell clustering, and clonotype definition. We also developed a multi-functional and user-friendly web interface that provides interactive visualization modules for biologists to analyze the transcriptome and TCR/BCR features at the single-cell level. HuARdb is freely available at https://huarc.net/database with functions for data querying, browsing, downloading, and depositing. In conclusion, huARdb is a comprehensive and multi-perspective atlas for human antigen receptors.

Pan-cancer multi-omics analyses reveal crosstalk between the Hippo and immune signaling pathways in the tumor microenvironment.

The Hippo signaling pathway is critical for carcinogenesis. However, the roles of the Hippo signaling pathway in the tumor immune microenvironment have been rarely investigated. This study systematically analyzed the relationship between the Hippo signaling pathway and immune cell infiltration across 32 cancer types. Both bioinformatics analyses and biological experiments revealed that the downstream effector of Hippo signaling YAP1 might inhibit CD8+ T cell infiltration by upregulating the expression of the transcription factor CREB1 in uterine corpus endometrial carcinoma. In addition, esophageal carcinoma (ESCA) patients were classified into three subtypes based on the Hippo-immune gene panel. The subtypes of ESCA had distinct characteristics in immune cell infiltration, immune pathways, and prognosis. Thus, this study also reveals a new classification of the immune subtypes with prognostic characteristics in ESCA.

GIANA allows computationally-efficient TCR clustering and multi-disease repertoire classification by isometric transformation.

Similarity in T-cell receptor (TCR) sequences implies shared antigen specificity between receptors, and could be used to discover novel therapeutic targets. However, existing methods that cluster T-cell receptor sequences by similarity are computationally inefficient, making them impractical to use on the ever-expanding datasets of the immune repertoire. Here, we developed GIANA (Geometric Isometry-based TCR AligNment Algorithm) a computationally efficient tool for this task that provides the same level of clustering specificity as TCRdist at 600 times its speed, and without sacrificing accuracy. GIANA also allows the rapid query of large reference cohorts within minutes. Using GIANA to cluster large-scale TCR datasets provides candidate disease-specific receptors, and provides a new solution to repertoire classification. Querying unseen TCR-seq samples against an existing reference differentiates samples from patients across various cohorts associated with cancer, infectious and autoimmune disease. Our results demonstrate how GIANA could be used as the basis for a TCR-based non-invasive multi-disease diagnostic platform.

Autoencoder based local T cell repertoire density can be used to classify samples and T cell receptors.

Recent advances in T cell repertoire (TCR) sequencing allow for the characterization of repertoire properties, as well as the frequency and sharing of specific TCR. However, there is no efficient measure for the local density of a given TCR. TCRs are often described either through their Complementary Determining region 3 (CDR3) sequences, or theirV/J usage, or their clone size. We here show that the local repertoire density can be estimated using a combined representation of these components through distance conserving autoencoders and Kernel Density Estimates (KDE). We present ELATE-an Encoder-based LocAl Tcr dEnsity and show that the resulting density of a sample can be used as a novel measure to study repertoire properties. The cross-density between two samples can be used as a similarity matrix to fully characterize samples from the same host. Finally, the same projection in combination with machine learning algorithms can be used to predict TCR-peptide binding through the local density of known TCRs binding a specific target.

T cell receptor repertoires within liver allografts are different to those in the peripheral blood.

BACKGROUND & AIMS: T cells are the main mediators of allogeneic immune responses. Specific T cell clones can be tracked by their unique T cell receptor (TCR), but specificity and function remain elusive and have not been investigated in human liver biopsies thus far. METHODS: TCR repertoire analysis of CD4+, CD8+, and regulatory T cells of the peripheral blood and liver graft was performed in 7 liver transplant recipients with either stable course (non-rejector, NR), subclinical cellular rejection (SCR), or acute cellular rejection (ACR) during an observation period from pre-transplant to 6 years post-transplant. Furthermore, donor-reactive T cells, identified by their expression of CD154 and glycoprotein A repetitions predominant (GARP) after allogeneic activation, were tracked longitudinally in peripheral blood and within the liver allograft. RESULTS: Although overall clonality of the TCR repertoire did not increase in peripheral blood after liver transplantation, clonality of donor-reactive CD4+ and regulatory T cells increased and these clones accumulated within the liver graft. Surprisingly, the TCR repertoires between the liver graft and the periphery were distinct and showed only limited overlap. Notably, during ACR, TCR repertoires aligned suggesting either graft-specific homing or release of activated T cells from the graft. CONCLUSIONS: This is the first study comparing TCR repertoires between liver grafts and blood in patients with NR, SCR, and ACR. Moreover, we attribute specificity and function to a subgroup of intragraft T cell populations. Given the limited overlap between peripheral blood and intragraft repertoires, future studies investigating function and specificities of T cells after liver transplantation should focus on the intragraft immune response. LAY SUMMARY: In solid organ transplantation, T cells are key mediators of the recipient's immune response directed at the transplanted organ. In our study, we characterised the T cell repertoire in a cohort of 7 liver transplant recipients. We demonstrate that donor-specific T cells expand clonally and accumulate in the transplanted liver. Moreover, we show that the composition of T cells in peripheral blood differs from the T cells in the liver allograft, only aligning in the context of acute cellular rejection but not in normal graft or subclinical cellular rejection. This indicates that the intragraft immune response is not mirrored in the peripheral blood. Our findings clarify the importance of protocol liver biopsies in identifying intragraft immune responses for future investigations of allo-directed immune responses.

Adaptive immune responses to SARS-CoV-2 infection in severe versus mild individuals.

The global Coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has affected more than eight million people. There is an urgent need to investigate how the adaptive immunity is established in COVID-19 patients. In this study, we profiled adaptive immune cells of PBMCs from recovered COVID-19 patients with varying disease severity using single-cell RNA and TCR/BCR V(D)J sequencing. The sequencing data revealed SARS-CoV-2-specific shuffling of adaptive immune repertories and COVID-19-induced remodeling of peripheral lymphocytes. Characterization of variations in the peripheral T and B cells from the COVID-19 patients revealed a positive correlation of humoral immune response and T-cell immune memory with disease severity. Sequencing and functional data revealed SARS-CoV-2-specific T-cell immune memory in the convalescent COVID-19 patients. Furthermore, we also identified novel antigens that are responsive in the convalescent patients. Altogether, our study reveals adaptive immune repertories underlying pathogenesis and recovery in severe versus mild COVID-19 patients, providing valuable information for potential vaccine and therapeutic development against SARS-CoV-2 infection.

TCR Repertoire Characterization for T Cells Expanded in Response to hRSV Infection in Mice Immunized with a Recombinant BCG Vaccine.

T cells play an essential role in the immune response against the human respiratory syncytial virus (hRSV). It has been described that both CD4+ and CD8+ T cells can contribute to the clearance of the virus during an infection. However, for some individuals, such an immune response can lead to an exacerbated and detrimental inflammatory response with high recruitment of neutrophils to the lungs. The receptor of most T cells is a heterodimer consisting of α and ß chains (αßTCR) that upon antigen engagement induces the activation of these cells. The αßTCR molecule displays a broad sequence diversity that defines the T cell repertoire of an individual. In our laboratory, a recombinant Bacille Calmette-Guérin (BCG) vaccine expressing the nucleoprotein (N) of hRSV (rBCG-N-hRSV) was developed. Such a vaccine induces T cells with a Th1 polarized phenotype that promote the clearance of hRSV infection without causing inflammatory lung damage. Importantly, as part of this work, the T cell receptor (TCR) repertoire of T cells expanded after hRSV infection in naïve and rBCG-N-hRSV-immunized mice was characterized. A more diverse TCR repertoire was observed in the lungs from rBCG-N-hRSV-immunized as compared to unimmunized hRSV-infected mice, suggesting that vaccination with the recombinant rBCG-N-hRSV vaccine triggers the expansion of T cell populations that recognize more viral epitopes. Furthermore, differential expansion of certain TCRVß chains was found for hRSV infection (TCRVß+8.3 and TCRVß+5.1,5.2) as compared to rBCG-N-hRSV vaccination (TCRVß+11 and TCRVß+12). Our findings contribute to better understanding the T cell response during hRSV infection, as well as the functioning of a vaccine that induces a protective T cell immunity against this virus.

Multiple sevoflurane exposures don't disturb the T-cell receptor repertoire in infant rhesus monkeys' thymus.

AIMS: Multiple surgical procedures and anesthesia increase the risk of the development in children. However, the influence of such exposures on the developing childhood immunity organs is rarely reported. MATERIALS AND METHODS: High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) from rhesus monkeys' thymus was performed to investigate whether anesthetics could induce de novo antigen recognition via TCR or TCR development impairments. KEY FINDINGS: No significant difference between sevoflurane and control groups regarding VJ gene combinations and diversity of V and J gene was seen, nor was there an obvious change in similar average number of Complementarity Determining Region 3 (CDR3) aa clonotypes. Our analysis of Rank abundance, Gini coefficient, Simpson index, Normalized Shannon Diversity Entropy (NSDE), Morisita-Horn Similarity Index (MHSI) and Bhattacharyya Distance (BD) indicated there is no difference in TCR diversity and similarity. SIGNIFICANCE: These results suggest early events in thymic T cell development and repertoire generation are not abnormality after multiple sevoflurane exposure during childhood. The stabilization of the immune repertoires suggested the safety of sevoflurane in host immune response in children.

Topology and expressed repertoire of the Felis catus T cell receptor loci.

BACKGROUND: The domestic cat (Felis catus) is an important companion animal and is used as a large animal model for human disease. However, the comprehensive study of adaptive immunity in this species is hampered by the lack of data on lymphocyte antigen receptor genes and usage. The objectives of this study were to annotate the feline T cell receptor (TR) loci and to characterize the expressed repertoire in lymphoid organs of normal cats using high-throughput sequencing. RESULTS: The Felis catus TRG locus contains 30 genes: 12 TRGV, 12 TRGJ and 6 TRGC, the TRB locus contains 48 genes: 33 TRBV, 2 TRBD, 11 TRBJ, 2 TRBC, the TRD locus contains 19 genes: 11 TRDV, 2 TRDD, 5 TRDJ, 1 TRDC, and the TRA locus contains 127 genes: 62 TRAV, 64 TRAJ, 1 TRAC. Functional feline V genes form monophyletic clades with their orthologs, and clustering of multimember subgroups frequently occurs in V genes located at the 5' end of TR loci. Recombination signal (RS) sequences of the heptamer and nonamer of functional V and J genes are highly conserved. Analysis of the TRG expressed repertoire showed preferential intra-cassette over inter-cassette rearrangements and dominant usage of the TRGV2-1 and TRGJ1-2 genes. The usage of TRBV genes showed minor bias but TRBJ genes of the second J-C-cluster were more commonly rearranged than TRBJ genes of the first cluster. The TRA/TRD V genes almost exclusively rearranged to J genes within their locus. The TRAV/TRAJ gene usage was relatively balanced while the TRD repertoire was dominated by TRDJ3. CONCLUSIONS: This is the first description of all TR loci in the cat. The genomic organization of feline TR loci was similar to that of previously described jawed vertebrates (gnathostomata) and is compatible with the birth-and-death model of evolution. The large-scale characterization of feline TR genes provides comprehensive baseline data on immune repertoires in healthy cats and will facilitate the development of improved reagents for the diagnosis of lymphoproliferative diseases in cats. In addition, these data might benefit studies using cats as a large animal model for human disease.

A High-Avidity T-cell Receptor Redirects Natural Killer T-cell Specificity and Outcompetes the Endogenous Invariant T-cell Receptor.

T-cell receptor (TCR) gene transfer redirects T cells to target intracellular antigens. However, the potential autoreactivity generated by TCR mispairing and occurrence of graft-versus-host disease in the allogenic setting due to the retention of native TCRs remain major concerns. Natural killer T cells (NKT) have shown promise as a platform for adoptive T-cell therapy in cancer patients. Here, we showed their utility for TCR gene transfer. We successfully engineered and expanded NKTs expressing a functional TCR (TCR NKTs), showing HLA-restricted antitumor activity in xenogeneic mouse models in the absence of graft-versus-mouse reactions. We found that TCR NKTs downregulated the invariant TCR (iTCR), leading to iTCR+TCR+ and iTCR-TCR+ populations. In-depth analyses of these subsets revealed that in iTCR-TCR+ NKTs, the iTCR, although expressed at the mRNA and protein levels, was retained in the cytoplasm. This effect resulted from a competition for binding to CD3 molecules for cell-surface expression by the transgenic TCR. Overall, our results highlight the feasibility and advantages of using NKTs for TCR expression for adoptive cell immunotherapies. NKT-low intrinsic alloreactivity that associated with the observed iTCR displacement by the engineered TCR represents ideal characteristics for "off-the-shelf" products without further TCR gene editing.

TCR Repertoire Diversity of Peripheral PD-1+CD8+ T Cells Predicts Clinical Outcomes after Immunotherapy in Patients with Non-Small Cell Lung Cancer.

T-cell receptor (TCR)-based biomarkers might predict patient response to immune checkpoint blockade (ICB) but need further exploration and validation for that use. We sequenced complementarity-determining region 3 of TCRß chains isolated from PD-1+ CD8+ T cells to investigate its value for predicting the response to anti-programmed cell death 1 (PD-1)/PD-ligand 1 (PD-L1) therapy in patients with non-small cell lung cancer (NSCLC). Two independent patient cohorts (cohort A, n = 25; cohort B, n = 15) were used as discovery and validation sets, respectively. Pre- and post-ICB peripheral blood samples were collected. In cohort A, patients with high PD-1+ CD8+ TCR diversity before ICB treatment showed better response to ICB and progression-free survival (PFS) compared with patients with low diversity [6.4 months vs. 2.5 months, HR, 0.39; 95% confidence interval (CI), 0.17-0.94; P = 0.021]. The results were validated in cohort B. Pre-ICB PD-1+ CD8+ TCR diversity achieved an optimal Youden's index of 0.81 (sensitivity = 0.87 and specificity = 0.94) for differentiating the ICB response in the merged dataset (cohort A plus cohort B). Patients with increased PD-1+ CD8+ TCR clonality after ICB treatment had longer PFS (7.3 months vs. 2.6 months, HR, 0.26; 95% CI, 0.08-0.86; P = 0.002) than those with decreased clonality. Thus, TCR diversity and clonality in peripheral blood PD-1+ CD8+ T cells may serve as noninvasive predictors of patient response to ICB and survival outcomes in NSCLC.

Comprehensive annotation and evolutionary insights into the canine (Canis lupus familiaris) antigen receptor loci.

Dogs are an excellent model for human disease. For example, the treatment of canine lymphoma has been predictive of the human response to that treatment. However, an incomplete picture of canine (Canis lupus familiaris) immunoglobulin (IG) and T cell receptor (TR)-or antigen receptor (AR)-gene loci has restricted their utility. This work advances the annotation of the canine AR loci and looks into breed-specific features of the loci. Bioinformatic analysis of unbiased RNA sequence data was used to complete the annotation of the canine AR genes. This annotation was used to query 107 whole genome sequences from 19 breeds and identified over 5500 alleles across the 550 genes of the seven AR loci: the IG heavy, kappa, and lambda loci; and the TR alpha, beta, gamma, and delta loci. Of note was the discovery that half of the IGK variable (V) genes were located downstream of, and inverted with respect to, the rest of the locus. Analysis of the germline sequences of all the AR V genes identified greater conservation between dog and human than mouse with either. This work brings our understanding of the genetic diversity and expression of AR in dogs to the same completeness as that of mice and men, making it the third species to have all AR loci comprehensively and accurately annotated. The large number of germline sequences serves as a reference for future studies, and has allowed statistically powerful conclusions to be drawn on the pressures that have shaped these loci.

T follicular helper and T follicular regulatory cells have different TCR specificity.

Immunization leads to the formation of germinal centres (GCs) that contain both T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Whether T-cell receptor (TCR) specificity defines the differential functions of Tfh and Tfr cells is unclear. Here we show that antigen-specific T cells after immunization are preferentially recruited to the GC to become Tfh cells, but not Tfr cells. Tfh cells, but not Tfr cells, also proliferate efficiently on restimulation with the same immunizing antigen in vitro. Ex vivo TCR repertoire analysis shows that immunization induces oligoclonal expansion of Tfh cells. By contrast, the Tfr pool has a TCR repertoire that more closely resembles that of regulatory T (Treg) cells. Our data thus indicate that the GC Tfh and Tfr pools are generated from distinct TCR repertoires, with Tfh cells expressing antigen-responsive TCRs to promote antibody responses, and Tfr cells expressing potentially autoreactive TCRs to suppress autoimmunity.

The Role of Natural Killer T Cells in Acute Kidney Injury: Angel or Evil?

Acute kidney injury (AKI) is manifested by inflammation, and an early feature in the pathogenesis is the accumulation of immune cells in the kidney. Natural killer T (NKT) cells, a peculiar T cells subtype, serve as a bridge between innate and adaptive immunity. Due to the difference between type I and type II subsets, NKT cells were supposed to play a dual role in IR-related tissue injury. Furthermore, membrane receptors and clinical immunosuppressive agents remain involved in the modulation of NKT cell function. Therefore, regulation of the amount and viability of NKT cells becomes a potential strategy in amelioration of AKI. This review will highlight the recent insights gained into the role and mechanisms of NKT cells in AKI.

ANARCI: antigen receptor numbering and receptor classification.

MOTIVATION: Antibody amino-acid sequences can be numbered to identify equivalent positions. Such annotations are valuable for antibody sequence comparison, protein structure modelling and engineering. Multiple different numbering schemes exist, they vary in the nomenclature they use to annotate residue positions, their definitions of position equivalence and their popularity within different scientific disciplines. However, currently no publicly available software exists that can apply all the most widely used schemes or for which an executable can be obtained under an open license. RESULTS: ANARCI is a tool to classify and number antibody and T-cell receptor amino-acid variable domain sequences. It can annotate sequences with the five most popular numbering schemes: Kabat, Chothia, Enhanced Chothia, IMGT and AHo. AVAILABILITY AND IMPLEMENTATION: ANARCI is available for download under GPLv3 license at opig.stats.ox.ac.uk/webapps/anarci. A web-interface to the program is available at the same address. CONTACT: deane@stats.ox.ac.uk.

Qualifying high-throughput immune repertoire sequencing.

Diversity of B and T cell receptors, achieved by gene recombination and somatic hypermutation, allows the immune system for recognition and targeted reaction against various threats. Next-generation sequencing for assessment of a cell's gene composition and variation makes deep analysis of one individual's immune spectrum feasible. An easy to apply but detailed analysis and visualization strategy is necessary to process all sequences generated. We performed sequencing utilizing the 454 system for CLL and control samples, utilized the IMGT database and applied the presented analysis tools. With the applied protocol, malignant clones are found and characterized, mutational status compared to germline identity is elaborated in detail showing that the CLL mutation status is not as monoclonal as generally thought. On the other hand, this strategy is not solely applicable to the 454 sequencing system but can easily be transferred to any other next-generation sequencing platform.

Correlation of contact sensitizer potency with T cell frequency and TCR repertoire diversity.

Allergic contact dermatitis is a T cell-mediated skin disease. Many hundreds of organic chemicals and some metal ions are contact sensitizers. They induce an innate inflammatory immune response in the skin that results in the priming of contact sensitizer-specific T cells by dendritic cells in the draining lymph nodes. The factors that determine the strength of this T cell response and thereby define the potency of a contact sensitizer are largely unknown. This chapter highlights different variables such as precursor frequency of antigen-specific T cells, possible bystander activation, and T cell receptor diversity or avidity of the TCR/peptide-MHC interactions, which might impact the quality and strength of T cell responses to contact sensitizers. In addition, different methods available to determine both the frequency of antigen-specific T cells and T cell receptor repertoires are discussed. Identification of the factors determining potency may allow for the development of suitable in vitro assays for potency assessment of contact sensitizers.

Using synthetic templates to design an unbiased multiplex PCR assay.

T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.

The important role of T cells and receptor expression in Sjögren's syndrome.

Sjögren's syndrome (SjS), an autoimmune disease characterized by exocrine gland dysfunction leading to dry mouth and dry eye diseases, is typified by progressive leucocyte infiltrations of the salivary and lacrimal glands. Histologically, these leucocyte infiltrations generally establish periductal aggregates, referred to as lymphocytic foci (LF), which occasionally appear as germinal centre (GC)-like structures. The formation and organization of these LF suggest an important and dynamic role for helper T cells (TH), specifically TH1, TH2 and the recently discovered TH17, in development and onset of clinical SjS, considered a B cell-mediated hypersensitivity type 2 disease. Despite an ever-increasing focus on identifying the underlying aetiology of SjS, defining factors that initiate this autoimmune disease remain a mystery. Thus, determining interactions between infiltrating TH cells and exocrine gland tissue (auto-)antigens represents a fertile research endeavour. This review discusses pathological functions of TH cells in SjS, the current status of TH cell receptor gene rearrangements associated with human and mouse models of SjS and potential future prospects for identifying receptor-autoantigen interactions.