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Background: Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma (CTCL). Comprehensive analysis of MF cells in situ and ex vivo is complicated by the fact that is challenging to distinguish malignant from reactive T cells with certainty. Methods: To overcome this limitation, we performed combined single-cell RNA (scRNAseq) and T-cell receptor TCR sequencing (scTCRseq) of skin lesions of cutaneous MF lesions from 12 patients. A sufficient quantity of living T cells was obtained from 9 patients, but 2 had to be excluded due to unclear diagnoses (coexisting CLL or revision to a fixed toxic drug eruption). Results: From the remaining patients we established single-cell mRNA expression profiles and the corresponding TCR repertoire of 18,630 T cells. TCR clonality unequivocally identified 13,592 malignant T cells. Reactive T cells of all patients clustered together, while malignant cells of each patient formed a unique cluster expressing genes typical of naive/memory, such as CD27, CCR7 and IL7R, or cytotoxic T cells, e.g., GZMA, NKG7 and GNLY. Genes encoding classic CTCL markers were not detected in all clusters, consistent with the fact that mRNA expression does not correlate linearly with protein expression. Nevertheless, we successfully pinpointed distinctive gene signatures differentiating reactive malignant from malignant T cells: keratins (KRT81, KRT86), galectins (LGALS1, LGALS3) and S100 genes (S100A4, S100A6) being overexpressed in malignant cells. Conclusions: Combined scRNAseq and scTCRseq not only allows unambiguous identification of MF cells, but also revealed marked heterogeneity between and within patients with unexpected functional phenotypes. While the correlation between mRNA and protein abundance was limited with respect to established MF markers, we were able to identify a single-cell gene expression signature that distinguishes malignant from reactive T cells. [ABSTRACT FROM AUTHOR]

An individual's T-cell repertoire constantly changes under the influence of external and internal factors. Cells that do not receive a stimulatory signal die, while those that encounter and recognize a pathogen or receive a costimulatory signal divide, resulting in clonal expansions. T-cell clones can be traced by monitoring the presence of their unique T-cell receptor (TCR) sequence, which is assembled de novo through a process known as V(D)J rearrangement. Tracking T cells can provide valuable insights into the survival of cells after hematopoietic stem cell transplantation (HSCT) or cancer treatment response and can indicate the induction of protective immunity by vaccination. In this study, we report a bioinformatic method for quantifying the T-cell repertoire dynamics from TCR sequencing data. We demonstrate its utility by measuring the T-cell repertoire stability in healthy donors, by quantifying the effect of donor lymphocyte infusion (DLI), and by tracking the fate of the different T-cell subsets in HSCT patients and the expansion of pathogen-specific clones in vaccinated individuals. [ABSTRACT FROM AUTHOR]

Adenovirus vaccines, particularly the COVID-19 Ad5-nCoV adenovirus vaccine, have emerged as promising tools in the fight against infectious diseases. In this study, we investigated the structure of the T cell response to the Spike protein of the SARS-CoV-2 virus used in the COVID-19 Ad5-nCoV adenoviral vaccine in a phase 3 clinical trial (NCT04540419). In 69 participants, we collected peripheral blood samples at four time points after vaccination or placebo injection. Sequencing of T cell receptor repertoires from Spike-stimulated T cell cultures at day 14 from 17 vaccinated revealed a more diverse CD4+ T cell repertoire compared to CD8+. Nevertheless, CD8+ clonotypes accounted for more than half of the Spike-specific repertoire. Our longitudinal analysis showed a peak T cell response at day 14, followed by a decline untilmonth 6. Remarkably, multiple T cell clonotypes persisted for at least 6 months after vaccination, as demonstrated by ex vivo stimulation. Examination of CDR3 regions revealed homologous sequences in both CD4+ and CD8+ clonotypes, with major CD8+ clonotypes sharing high similarity with annotated sequences specific for the NYNYLYRLF peptide, suggesting potential immunodominance. In conclusion, our study demonstrates the immunogenicity of the Ad5-nCoV adenoviral vaccine and highlights its ability to induce robust and durable T cell responses. These findings provide valuable insight into the efficacy of the vaccine against COVID-19 and provide critical information for ongoing efforts to control infectious diseases. [ABSTRACT FROM AUTHOR]

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αβ-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes. [ABSTRACT FROM AUTHOR]

Background: Accumulating evidence supports the involvement of adaptive immunity in the development of radiation‐induced brain injury (RIBI). Our previous work has emphasized the cytotoxic function of CD8+ T cells in RIBI. In this study, we aimed to investigate the presence and potential roles of cytotoxic CD4+ T cells (CD4+ CTLs) in RIBI to gain a more comprehensive understanding of adaptive immunity in this context. Main Text: Utilizing single‐cell RNA sequencing (scRNA‐seq), we analyzed 3934 CD4+ T cells from the brain lesions of four RIBI patients and identified six subclusters within this population. A notable subset, the cytotoxic CD4+ T cells (CD4+ CTLs), was marked with high expression of cytotoxicity‐related genes (NKG7, GZMH, GNLY, FGFBP2, and GZMB) and several chemokine and chemokine receptors (CCL5, CX3CR1, and CCL4L2). Through in‐depth pseudotime analysis, which simulates the development of CD4+ T cells, we observed that the CD4+ CTLs exhibited signatures of terminal differentiation. Their functions were enriched in protein serine/threonine kinase activity, GTPase regulator activity, phosphoprotein phosphatase activity, and cysteine‐type endopeptidase activity involved in the apoptotic signaling pathway. Correspondingly, mice subjected to gamma knife irradiation on the brain showed a time‐dependent infiltration of CD4+ T cells, an increase of MHCII+ cells, and the existence of CD4+ CTLs in lesions, along with an elevation of apoptotic‐related proteins. Finally, and most crucially, single‐cell T‐cell receptor sequencing (scTCR‐seq) analysis at the patient level determined a large clonal expansion of CD4+ CTLs in lesion tissues of RIBI. Transcriptional factor‐encoding genes TBX21, RORB, and EOMES showed positive correlations with the cytotoxic functions of CD4+ T cells, suggesting their potential to distinguish RIBI‐related CD4+ CTLs from other subsets. Conclusion: The present study enriches the understanding of the transcriptional landscape of adaptive immune cells in RIBI patients. It provides the first description of a clonally expanded CD4+ CTL subset in RIBI lesions, which may illuminate new mechanisms in the development of RIBI and offer potential biomarkers or therapeutic targets for the disease. [ABSTRACT FROM AUTHOR]

T-cell–based diagnostic tools identify pathogen exposure but lack differentiation between recent and historical exposures in acute infectious diseases. Here, T-cell receptor (TCR) RNA sequencing was performed on HLA-DR+/CD38+CD8+ T-cell subsets of hospitalized coronavirus disease 2019 (COVID-19) patients (n = 30) and healthy controls (n = 30; 10 of whom had previously been exposed to severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]). CDR3α and CDR3β TCR regions were clustered separately before epitope specificity annotation using a database of SARS-CoV-2–associated CDR3α and CDR3β sequences corresponding to >1000 SARS-CoV-2 epitopes. The depth of the SARS-CoV-2–associated CDR3α/β sequences differentiated COVID-19 patients from the healthy controls with a receiver operating characteristic area under the curve of 0.84 ± 0.10. Hence, annotating TCR sequences of activated CD8+ T cells can be used to diagnose an acute viral infection and discriminate it from historical exposure. In essence, this work presents a new paradigm for applying the T-cell repertoire to accomplish TCR-based diagnostics. [ABSTRACT FROM AUTHOR]

Immunoglobulin A nephropathy (IgAN) is an autoimmune disease characterized by abnormal IgA deposition in glomerulus. Current diagnosis of IgAN still depends on renal biopsy, an invasive method that might increase the risk of clinical outcomes. Therefore, we aimed to explore the characteristics of T cell repertoire in IgAN from peripheral blood samples for identifying innovative diagnostic biomarkers. Herein, we included 8 IgAN patients, 25 non-IgAN patients, and 10 healthy controls in the study. A high-throughput immune repertoire sequencing was conducted to investigate the T-cell receptor beta-chain (TCRβ) repertoire of peripheral blood. Characteristics of TCRβ repertoire were assessed for these three distinct groups. A reduced TCRβ repertoire diversity was observed in IgAN patients compared to non-IgAN and healthy individuals. A skewed distribution toward shorter TCRβ complementarity determining region (CDR3) length was found in non-IgAN relative to IgAN patients. In addition, the differences in usages of five TRBV genes (TRBV5-4, TRBV6-4, TRBV12-1, TRBV16, and TRBV21-1) were identified between IgAN, non-IgAN, and healthy subjects. Of note, the TRBV6-4 gene, which is associated with mucosal-associated invariant T (MAIT) cells, exhibited higher usage in IgAN patients, suggesting potential importance of MAIT cells in IgAN. In short, our findings supported TCR repertoire characteristics as potential biomarkers for IgAN diagnosis. [ABSTRACT FROM AUTHOR]

Objectives: Infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes coronavirus disease 2019 (COVID‐19). Although an acute SARS‐CoV‐2 infection mainly presents with respiratory illness, neurologic symptoms and sequelae are increasingly recognised in the long‐term treatment of COVID‐19 patients. The pathophysiology and the neuropathogenesis behind neurologic complications of COVID‐19 remain poorly understood, but mounting evidence points to endothelial dysfunction either directly caused by viral infection or indirectly by inflammatory cytokines, followed by a local immune response that may include virus‐specific T cells. However, the type and role of central nervous system‐infiltrating T cells in COVID‐19 are complex and not fully understood. Methods: We analysed distinct anatomical brain regions of patients who had deceased as a result of COVID‐19‐associated pneumonia or complications thereof and performed T cell receptor Vβ repertoire sequencing. Clonotypes were analysed for SARS‐CoV‐2 association using public TCR repertoire data. Results: Our descriptive study demonstrates that SARS‐CoV‐2‐associated T cells are found in almost all brain areas of patients with fatal COVID‐19 courses. The olfactory bulb, medulla and cerebellum were brain regions showing the most SARS‐CoV‐2 specific sequence patterns. Neuropathological workup demonstrated primary CD8+ T‐cell infiltration with a perivascular infiltration pattern. Conclusion: Future research is needed to better define the relationship between T‐cell infiltration and neurological symptoms and its long‐term impact on patients' cognitive and mental health. [ABSTRACT FROM AUTHOR]

Analysis of T‐cell receptor (TCR) repertoires in different stages of hepatocellular carcinoma (HCC) might help to elucidate its pathogenesis and progression. This study aimed to investigate TCR profiles in liver biopsies and peripheral blood mononuclear cells (PBMCs) in different Barcelona Clinic liver cancer (BCLC) stages of HCC. Ten patients in early stage (BCLC_A), 10 patients in middle stage (BCLC_B), and 10 patients in late stage (BCLC_C) cancer were prospectively enrolled. The liver tumor tissues, adjacent tissues, and PBMCs of each patient were collected and examined by TCR β sequencing. Based on the ImMunoGeneTics (IMGT) database, we aligned the V, D, J, and C gene segments and identified the frequency of CDR3 sequences and amino acids sequence. Diversity of TCR in PBMCs was higher than in both tumor tissues and adjacent tissues, regardless of BCLC stage and postoperative recurrence. TCR clonality was increased in T cells from peripheral blood in advanced HCC, compared with the early and middle stages. No statistical differences were observed between different BCLC stages, either in tumors or adjacent tissues. TCR clonality revealed no significant difference between recurrent tumor and non‐recurrent tumor, therefore PBMCs was better to be representative of TCR characteristics in different stages of HCC compared to tumor tissues. Clonal expansion of T cells was associated with low risk of recurrence in HCC patients. [ABSTRACT FROM AUTHOR]

Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others. [ABSTRACT FROM AUTHOR]

The T-cell receptor (TCR) repertoire is highly diverse among the population and plays an essential role in initiating multiple immune processes. TCR sequencing (TCR-seq) has been developed to profile the T cell repertoire. Similar to other high-throughput experiments, contamination can happen during several steps of TCR-seq, including sample collection, preparation and sequencing. Such contamination creates artifacts in the data, leading to inaccurate or even biased results. Most existing methods assume 'clean' TCR-seq data as the starting point with no ability to handle data contamination. Here, we develop a novel statistical model to systematically detect and remove contamination in TCR-seq data. We summarize the observed contamination into two sources, pairwise and cross-cohort. For both sources, we provide visualizations and summary statistics to help users assess the severity of the contamination. Incorporating prior information from 14 existing TCR-seq datasets with minimum contamination, we develop a straightforward Bayesian model to statistically identify contaminated samples. We further provide strategies for removing the impacted sequences to allow for downstream analysis, thus avoiding any need to repeat experiments. Our proposed model shows robustness in contamination detection compared with a few off-the-shelf detection methods in simulation studies. We illustrate the use of our proposed method on two TCR-seq datasets generated locally. [ABSTRACT FROM AUTHOR]

The ability to identify and track T-cell receptor (TCR) sequences from patient samples is becoming central to the field of cancer research and immunotherapy. Tracking genetically engineered T cells expressing TCRs that target specific tumor antigens is important to determine the persistence of these cells and quantify tumor responses. The available high-throughput method to profile TCR repertoires is generally referred to as TCR sequencing (TCR-Seq). However, the available TCR-Seq data are limited compared with RNA sequencing (RNA-Seq). In this paper, we have benchmarked the ability of RNA-Seq-based methods to profile TCR repertoires by examining 19 bulk RNA-Seq samples across 4 cancer cohorts including both T-cell-rich and T-cell-poor tissue types. We have performed a comprehensive evaluation of the existing RNA-Seq-based repertoire profiling methods using targeted TCR-Seq as the gold standard. We also highlighted scenarios under which the RNA-Seq approach is suitable and can provide comparable accuracy to the TCR-Seq approach. Our results show that RNA-Seq-based methods are able to effectively capture the clonotypes and estimate the diversity of TCR repertoires, as well as provide relative frequencies of clonotypes in T-cell-rich tissues and low-diversity repertoires. However, RNA-Seq-based TCR profiling methods have limited power in T-cell-poor tissues, especially in highly diverse repertoires of T-cell-poor tissues. The results of our benchmarking provide an additional appealing argument to incorporate RNA-Seq into the immune repertoire screening of cancer patients as it offers broader knowledge into the transcriptomic changes that exceed the limited information provided by TCR-Seq. [ABSTRACT FROM AUTHOR]

This document is an editorial published in the journal Frontiers in Immunology. It discusses the role of adaptive immunity in atherosclerosis, a condition that contributes to acute cardiovascular events. The editorial highlights the importance of chronic inflammation in atherosclerosis and the potential for new intervention strategies. It also explores the use of new immunological techniques to study the role of the adaptive immune system in the development, progression, and regression of atherosclerosis. The document includes summaries of several research papers that investigate different aspects of adaptive immunity in atherosclerosis, including T cell and B cell responses. Overall, the editorial provides an overview of the latest findings in this field and serves as a valuable resource for researchers interested in studying adaptive immunity in atherosclerosis. [Extracted from the article]

T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions. [ABSTRACT FROM AUTHOR]

In this cross-sectional and longitudinal analysis of mapping the T-cell repertoire in kidney transplant recipients, we have investigated and validated T-cell clonality, immune repertoire chronology at rejection, and contemporaneous allograft biopsy quantitative tissue injury, to better understand the pathobiology of acute T-cell fraction, T-cell repertoire and antibody-mediated kidney transplant rejection. To follow the dynamic evolution of T-cell repertoire changes before and after engraftment and during biopsy-confirmed acute rejection, we sequenced 323 peripheral blood samples from 200 unique kidney transplant recipients, with (n=100) and without (n=100) biopsy-confirmed acute rejection. We report that patients who develop acute allograft rejection, have lower (p=0.01) T-cell fraction even before transplantation, followed by its rise after transplantation and at the time of acute rejection accompanied by high TCR repertoire turnover (p=0.004). Acute rejection episodes occurring after the first 6 months posttransplantation, and those with a component of antibody-mediated rejection, had the highest turnover; p=0.0016) of their T-cell repertoire. In conclusion, we validated that detecting repertoire changes in kidney transplantation correlates with post-transplant rejection episodes suggesting that T-cell receptor sequencing may provide recipient pre-transplant and posttransplant predictors of rejection risk. [ABSTRACT FROM AUTHOR]

Background: Vγ9Vδ2+ T cells are a major innate T cell subset in human peripheral blood. Their Vd2+ VDJ-rearrangements are short and simple in the fetal thymus and gradually increase in diversity and CDR3 length along with development. So-called "public" versions of Vd2+ TCRs are shared among individuals of all ages. However, it is unclear whether such frequently occurring "public" Vγ9Vδ2+ T cell clones are derived from the fetal thymus and whether they are fitter to proliferate and persist than infrequent "private" clones. Methods: Shared "public" Vδ2+ TCRs were identified from Vδ2+ TCRrepertoires collected from 89 individuals, including newborns (cord blood), infants, and adults (peripheral blood). Distance matrices of Vδ2+ CDR3 were generated by TCRdist3 and then embedded into a UMAP for visualizing the heterogeneity of Vδ2+ TCRs. Results: Vδ2+ CDR3 distance matrix embedded by UMAP revealed that the heterogeneity of Vδ2+ TCRs is primarily determined by the J-usage and CDR3aa length, while age or publicity-specific motifs were not found. The most prevalent public Vδ2+ TCRs showed germline-like rearrangement with low N-insertions. Age-related features were also identified. Public Vδ2+ TRDJ1 TCRs from cord blood showed higher N-insertions and longer CDR3 lengths. Synonymous codons resulting from VDJ rearrangement also contribute to the generation of public Vδ2+ TCRs. Each public TCR was always produced by multiple different transcripts, even with different D gene usage, and the publicity of Vδ2+ TCRs was positively associated with expansion status. Conclusion: To conclude, the heterogeneity of Vδ2+ TCRs is mainly determined by TRDJ-usage and the length of CDR3aa sequences. Public Vδ2+ TCRs result from germline-like rearrangement and synonymous codons, associated with a higher expansion status. [ABSTRACT FROM AUTHOR]

The immune system is a dynamic feature of each individual and a footprint of our unique internal and external exposures. Indeed, the type and level of exposure to physical and biological agents shape the development and behavior of this complex and diffuse system. Many pathological conditions depend on how our immune system responds or does not respond to a pathogen or a disease or on how the regulation of immunity is altered by the disease itself. T-cells are important players in adaptive immunity and, together with B-cells, define specificity and monitor the internal and external signals that our organism perceives through its specific receptors, TCRs and BCRs, respectively. Today, high-throughput sequencing (HTS) applied to the TCR repertoire has opened a window of opportunity to disclose T-cell repertoire development and behavior down to the clonal level. Although TCR repertoire sequencing is easily accessible today, it is important to deeply understand the available technologies for choosing the best fit for the specific experimental needs and questions. Here, we provide an updated overview of TCR repertoire sequencing strategies, providers and applications to infectious diseases and cancer to guide researchers' choice through the multitude of available options. The possibility of extending the TCR repertoire to HLA characterization will be of pivotal importance in the near future to understand how specific HLA genes shape T-cell responses in different pathological contexts and will add a level of comprehension that was unthinkable just a few years ago. [ABSTRACT FROM AUTHOR]

This article is an editorial published in Frontiers in Pharmacology titled "Synthetic peptide vaccine platforms targeting tumor-specific antigens: advances and challenges." The authors discuss the discovery and therapeutic aspects of peptide vaccine platforms, specifically in relation to identifying potential antigen targets on tumors and strategies to enhance their immunogenicity. The article highlights advancements in computational techniques for identifying immunogenic peptide antigens, as well as the use of toll-like receptor agonists to improve the immunogenicity of vaccines. The authors emphasize the importance of confirming peptide binding characteristics through in-vivo or in-vitro determination. The article concludes by discussing the potential advantages of synthetic peptide vaccine platforms and the increasing tools available for designing peptide vaccines for oncology applications. [Extracted from the article]

Glioblastoma is the most common primary malignant brain tumor and one of the most aggressive tumors across all cancer types with remarkable resistance to any treatment. While immunotherapy has shown a robust clinical benefit in systemic cancers, its benefit is still under investigation in brain cancers. The broader use of immunotherapy in clinical trials for glioblastoma has highlighted the challenges of traditional methods of monitoring progression via imaging. Development of new guidelines, advanced imaging techniques, and immune profiling have emerged to counter premature diagnoses of progressive disease. However, these approaches do not provide a timely diagnosis and are costly and time consuming. Surgery is currently the standard of care for diagnosis of pseudoprogression in cases where MRI is equivocal. However, it is invasive, risky, and disruptive to patient's lives and their oncological treatment. With its increased vascularity, glioblastoma is continually shedding tumor components into the vasculature including tumor cells, genetic material, and extracellular vesicles. These elements can be isolated from routine blood draws and provide a real-time non-invasive indicator of tumor progression. Liquid biopsy therefore presents as an attractive alternative to current methods to guide treatment. While the initial evaluation of liquid biopsy for brain tumors via identification of mutations in the plasma was disappointing, novel technologies and use of alternatives to plasma cell-free DNA analytes provide promise for an effective liquid biopsy approach in brain tumors. This review aims to summarize developments in the use of liquid biopsy to monitor glioblastoma, especially in the context of immunotherapy. [ABSTRACT FROM AUTHOR]

Long-term antiretroviral therapy (ART) in people living with HIV (PLHIV) is associated with sustained increases in CD4+ T-cell count, but its effect on the peripheral blood T-cell repertoire has not been comprehensively evaluated. In this study, we performed serial profiling of the composition and diversity of the T-cell receptor β-chain (TRB) repertoire in 30 adults with HIV infection before and after the initiation of ART to define its long-term impact on the TRB repertoire. Serially acquired blood samples from 30 adults with HIV infection collected over a mean of 6 years (range, 1-12) years, with 1-4 samples collected before and 2-8 samples collected after the initiation of ART, were available for analysis. TRB repertoires were characterized via high-throughput sequencing of the TRB variable region performed on genomic DNA extracted from unsorted peripheral blood mononuclear cells. Additional laboratory and clinical metadata including serial measurements of HIV viral load and CD4 + T-cell count were available for all individuals in the cohort. A previously published control group of 189 TRB repertoires from peripheral blood samples of adult bone marrow transplant donors was evaluated for comparison. ART initiation in PLHIV was associated with a sustained reduction in viral load and a significant increase in TRB repertoire diversity. However, repertoire diversity in PLHIV remained significantly lower than in the control group even after long-term ART. The composition of TRB repertoires of PLHIV after ART also remained perturbed compared to the control cohort, as evidenced by large persistent private clonal expansions, reduced efficiency in the generation of TRB CDR3 amino acid sequences, and a narrower range of CDR3 lengths. Network analysis revealed an antigen-experienced structure in the TRB repertoire of PLHIV both before and after ART initiation that was quite distinct from the structure of control repertoires, with a slight shift toward a more naïve structure observed after ART initiation. Though we observe significant improvement in TRB repertoire diversity with durable viral suppression in PLHIV on long-term ART, the composition and structure of these repertoires remain significantly perturbed compared to the control cohort of adult bone marrow transplant donors. [ABSTRACT FROM AUTHOR]

T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell's transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, 'RM-scTCR-Seq', which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR's with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses. [ABSTRACT FROM AUTHOR]

The potential protective or pathogenic role of the adaptive immune response to SARS‐CoV‐2 infection has been vigorously debated. While COVID‐19 patients consistently generate a T lymphocyte response to SARS‐CoV‐2 antigens, evidence of significant immune dysregulation in these patients continues to accumulate. In this study, next generation sequencing of the T cell receptor beta chain (TRB) repertoire was conducted in hospitalized COVID‐19 patients to determine if immunogenetic differences of the TRB repertoire contribute to disease course severity. Clustering of highly similar TRB CDR3 amino acid sequences across COVID‐19 patients yielded 781 shared TRB sequences. The TRB sequences were then filtered for known associations with common diseases such as EBV and CMV. The remaining sequences were cross‐referenced to a publicly accessible dataset that mapped COVID‐19 specific TCRs to the SARS‐CoV‐2 genome. We identified 158 SARS‐CoV‐2 specific TRB sequences belonging to 134 clusters in our COVID‐19 patients. Next, we investigated 113 SARS‐CoV‐2 specific clusters binding only one peptide target in relation to disease course. Distinct skewing of SARS‐CoV‐2 specific TRB sequences toward the nonstructural proteins (NSPs) encoded within ORF1a/b of the SARS‐CoV‐2 genome was observed in clusters associated with critical disease course when compared to COVID‐19 clusters associated with a severe disease course. These data imply that T‐lymphocyte reactivity towards peptides from NSPs of SARS‐CoV‐2 may not constitute an effective adaptive immune response and thus may negatively affect disease severity. [ABSTRACT FROM AUTHOR]

Common approaches for monitoring T cell responses are limited in their multiplexity and sensitivity. In contrast, deep sequencing of the T Cell Receptor (TCR) repertoire provides a global view that is limited only in terms of theoretical sensitivity due to the depth of available sampling; however, the assignment of antigen specificities within TCR repertoires has become a bottleneck. This study combines antigen-driven expansion, deep TCR sequencing, and a novel analysis framework to show that homologous 'Clusters of Expanded TCRs (CETs)' can be confidently identified without cell isolation, and assigned to antigen against a background of non-specific clones. We show that clonotypes within each CET respond to the same epitope, and that protein antigens stimulate multiple CETs reactive to constituent peptides. Finally, we demonstrate the personalized assignment of antigen-specificity to rare clones within fully-diverse uncultured repertoires. The method presented here may be used to monitor T cell responses to vaccination and immunotherapy with high fidelity. [ABSTRACT FROM AUTHOR]

T cell receptors (TCRs) are unique markers that define antigen specificity for a given T cell. With the evolution of sequencing and computational analysis technologies, TCRs are now prime candidates for the development of next-generation non-cell based T cell biomarkers, which provide a surrogate measure to assess the presence of antigen-specific T cells. Type 1 diabetes (T1D), the immune-mediated form of diabetes, is a prototypical organ specific autoimmune disease in which T cells play a pivotal role in targeting pancreatic insulin-producing beta cells. While the disease is now predictable by measuring autoantibodies in the peripheral blood directed to beta cell proteins, there is an urgent need to develop T cell markers that recapitulate T cell activity in the pancreas and can be a measure of disease activity. This review focuses on the potential and challenges of developing TCR biomarkers for T1D. We summarize current knowledge about TCR repertoires and clonotypes specific for T1D and discuss challenges that are unique for autoimmune diabetes. Ultimately, the integration of large TCR datasets produced from individuals with and without T1D along with computational 'big data' analysis will facilitate the development of TCRs as potentially powerful biomarkers in the development of T1D. [ABSTRACT FROM AUTHOR]

Temporal analysis of the T cell receptor (TCR) repertoire has been used to monitor treatment-induced changes in antigen-specific T cells in patients with cancer. However, the lack of experimental models that allow a temporal analysis of the TCR repertoire in the same individual in a homogeneous population limits the understanding of the causal relationship between changes in TCR repertoire and antitumor responses. A bilateral tumor model, where tumor cells were inoculated bilaterally into the backs of mice, could be used for temporal analysis of the TCR repertoire. This study examined the prerequisite for this strategy: the TCR repertoire is conserved between bilateral tumors that grow symmetrically. Bilateral tumors and draining lymph nodes (dLNs) were collected 13 days after tumor inoculation to analyze the TCR repertoire of CD4+ and CD8+ T cells. The tumor-infiltrating T-cell clones were highly similar between the bilateral tumors and expanded to a similar extent. In addition, the differences of TCR repertoire between the bilateral tumors were equivalent to Intra-tumoral heterogeneity on one side. On the other hand, the similarity of the TCR repertoire in the bilateral dLNs was markedly lower than that in the tumor, suggesting that tumor-reactive T cell clones induced independently in each dLN are mixed during recirculation and then proportionally infiltrated the bilateral tumors. These findings provide the basis for future analysis of temporal and treatment-induced changes in tumor-reactive T cell clones using this bilateral tumor model. [ABSTRACT FROM AUTHOR]

This article is a response to a comment on a previous publication about benchmarking T cell receptor repertoire profiling methods for cancer RNA sequencing. The authors address concerns raised by Davydov et al. regarding the comparison of different tools and the inclusion of Phred nucleotide quality filters. They also discuss the issue of clonotype abundance calculation in TRUST4 and the evaluation of immune repertoire extraction tools. The authors explain their methodology and choices, emphasizing the use of default settings, adherence to best practices, and the focus on quantitative measures. They also defend their decision to exclude singletons from the analysis to enhance the reliability of the results. [Extracted from the article]

Transcriptome sequencing has become common in cancer research, resulting in the generation of a substantial volume of RNA sequencing (RNA-Seq) data. The ability to extract immune repertoires from these data is crucial for obtaining information on infiltrating T- and B-lymphocyte clones when dedicated amplicon T-cell/B-cell receptors sequencing (TCR-Seq/BCR-Seq) methods are unavailable. In response to this demand, several dedicated computational methods have been developed, including MiXCR, TRUST and ImRep. In the recent publication in Briefings in Bioinformatics, Peng et al. have conducted an intensive, systematic comparison of the three previously mentioned tools. Although their effort is commendable, we do have a few constructive critiques regarding technical elements of their analysis. [ABSTRACT FROM AUTHOR]

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a public health emergency. The most common symptoms of COVID-19 are fever, cough, and fatigue. While most patients with COVID-19 present with mild illness, some patients develop pneumonia, an important risk factor for mortality, at early stage of viral infection, putting these patients at increased risk of death. So far, little has been known about differences in the T cell repertoires between COVID-19 patients with and without pneumonia during SARS-CoV-2 infection. Herein, we aimed to investigate T cell receptor (TCR) repertoire profiles and patient-specific SARS-CoV-2-associated TCR clusters between COVID-19 patients with mild disease (no sign of pneumonia) and pneumonia. The TCR sequencing was conducted to characterize the peripheral TCR repertoire profile and diversity. The TCR clustering and CDR3 annotation were exploited to further discover groups of patient-specific TCR clonotypes with potential SARS-CoV-2 antigen specificities. Our study indicated a slight decrease in the TCR repertoire diversity and a skewed CDR3 length usage in patients with pneumonia compared to those with mild disease. The SARS-CoV-2-associated TCR clusters enriched in patients with mild disease exhibited significantly higher TCR generation probabilities and most of which were highly shared among patients, compared with those from pneumonia patients. Importantly, using similarity network-based clustering followed by the sequence conservation analysis, we found different patterns of CDR3 sequence motifs between mild disease- and pneumonia-specific SARS-CoV-2-associated public TCR clusters. Our results showed that characteristics of overall TCR repertoire and SARS-CoV-2-associated TCR clusters/clonotypes were divergent between COVID-19 patients with mild disease and patients with pneumonia. These findings provide important insights into the correlation between the TCR repertoire and disease severity in COVID-19 patients. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: Characterization of the localized adaptive immune response in the airway scar of patients with idiopathic subglottic stenosis (iSGS). Study Design: Basic Science. Methods: Utilizing 36 patients with subglottic stenosis (25 idiopathic subglottic stenosis [iSGS], 10 iatrogenic post‐intubation stenosis [iLTS], and one granulomatosis with polyangiitis [GPA]) we applied immunohistochemical and immunologic techniques coupled with RNA sequencing. Results: iSGS, iLTS, and GPA demonstrate a significant immune infiltrate in the subglottic scar consisting of adaptive cell subsets (T cells along with dendritic cells). Interrogation of T cell subtypes showed significantly more CD69+ CD103+ CD8+ tissue resident memory T cells (TRM) in the iSGS airway scar than iLTS specimens (iSGS vs. iLTS; 50% vs. 28%, P =.0065). Additionally, subglottic CD8+ clones possessed T‐cell receptor (TCR) sequences with known antigen specificity for viral and intracellular pathogens. Conclusions: The human subglottis is significantly enriched for CD8+ tissue resident memory T cells in iSGS, which possess TCR sequences proven to recognize viral and intracellular pathogens. These results inform our understanding of iSGS, provide a direction for future discovery, and demonstrate immunologic function in the human proximal airway. Laryngoscope, 131:610–617, 2021 [ABSTRACT FROM AUTHOR]

CD8 T cells play a crucial role in providing protection from viral infections. It has recently been established that a subset of CD8 T cells expressing Tcf1 are responsible for sustaining exhausted T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection. Many of these studies, however, have been performed using T cell receptor (TCR) transgenic mice, in which CD8 T cells express a monoclonal TCR specific for the LCMV glycoprotein. To investigate whether the Tcf1+ and Tcf1- repertoires are naturally composed of similar or different clones in wild-type mice exposed to acute or chronic LCMV infection, we performed TCR repertoire sequencing of virus-specific CD8 T cells, including Tcf1+ and Tcf1- populations. Our analysis revealed that the Tcf1+ TCR repertoire is maintained at an equal or higher degree of clonal diversity despite harboring fewer cells. Additionally, within the same animal, there was extensive clonal overlap between the Tcf1+ and Tcf1- repertoires in both chronic and acute LCMV infection. We could further detect these virus-specific clones in longitudinal blood samples earlier in the infection. With respect to common repertoire parameters (clonal overlap, germline gene usage, and clonal expansion), we found minor differences between the virus-specific TCR repertoire of acute and chronic LCMV infection 40 days post infection. Overall, our results indicate that the Tcf1+ population emerging during chronic LCMV infection is not clonally distinct from the Tcf1- population, supporting the notion that the Tcf1+ pool is indeed a fuel for the more exhausted Tcf1– population within the heterogenous repertoire of LCMV-specific CD8 T cells. [ABSTRACT FROM AUTHOR]

Among Immunotherapeutic approaches for cancer treatment, the adoptive transfer of antigen specific T cells is still a relevant approach, that could have higher efficacy when further combined with immune check-point blockade. A high number of adoptive transfer trials have been performed in metastatic melanoma, due to its high immunogenic potential, either with polyclonal TIL or antigen-specific polyclonal populations. In this setting, the extensive characterization of T cell functions and receptor diversity of infused polyclonal T cells is required, notably for monitoring purposes. We developed a clinical grade procedure for the selection and amplification of polyclonal CD8 T cells, specific for two shared and widely expressed melanoma antigens: Melan-A and MELOE-1. This procedure is currently used in a clinical trial for HLA-A2 metastatic melanoma patients. In this study, we characterized the T-cell diversity (T-cell repertoire) of such T cell populations using a new RNAseq strategy. We first assessed the added-value of TCR receptor sequencing, in terms of sensitivity and specificity, by direct comparison with cytometry analysis of the T cell populations labeled with anti-Vß-specific antibodies. Results from these analyzes also confirmed specific features already reported for Melan-A and MELOE-1 specific T cell repertoires in terms of V-alpha recurrence usage, on a very high number of T cell clonotypes. Furthermore, these analyses also revealed undescribed features, such as the recurrence of a specific motif in the CDR3α region for MELOE-1 specific T cell repertoire. Finally, the analysis of a large number of T cell clonotypes originating from various patients revealed the existence of public CDR3α and ß clonotypes for Melan-A and MELOE-1 specific T cells. In conclusion, this method of high throughput TCR sequencing is a reliable and powerful approach to deeply characterize polyclonal T cell repertoires, and to reveal specific features of a given TCR repertoire, that would be useful for immune follow-up of cancer patients treated by immunotherapeutic approaches. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Comparedwith Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαβ pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9-23 and proinsulin. Consistently, insulin B:9-23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28-/- recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is an aggressive and molecularly diverse breast cancer subtype typified by the presence of p53 mutations (∼80%), elevated immune gene signatures and neoantigen expression, as well as the presence of tumor infiltrating lymphocytes (TILs). As these factors are hypothesized to be strong immunologic prerequisites for the use of immune checkpoint blockade (ICB) antibodies, multiple clinical trials testing single ICBs have advanced to Phase III, with early indications of heterogeneous response rates of <20% to anti-PD1 and anti-PDL1 ICB. While promising, these modest response rates highlight the need for mechanistic studies to understand how different ICBs function, how their combination impacts functionality and efficacy, as well as what immunologic parameters predict efficacy to different ICBs regimens in TNBC. To address these issues, we tested anti-PD1 and anti-CTLA4 in multiple models of TNBC and found that their combination profoundly enhanced the efficacy of either treatment alone. We demonstrate that this efficacy is due to anti-CTLA4-driven expansion of an individually unique T-cell receptor (TCR) repertoire whose functionality is enhanced by both intratumoral Treg suppression and anti-PD1 blockade of tumor expressed PDL1. Notably, the individuality of the TCR repertoire was observed regardless of whether the tumor cells expressed a nonself antigen (ovalbumin) or if tumor-specific transgenic T-cells were transferred prior to sequencing. However, responsiveness was strongly correlated with systemic measures of tumor-specific T-cell and B-cell responses, which along with systemic assessment of TCR expansion, may serve as the most useful predictors for clinical responsiveness in future clinical trials of TNBC utilizing anti-PD1/anti-CTLA4 ICB. [ABSTRACT FROM AUTHOR]

Vaccine-mediated cancer treatment is unlikely to induce long-term survival unless suppressive mechanisms are overcome. Given the success of antibody-mediated immune checkpoint blockade in relieving regulation of endogenous anti-tumor T cell responses in tumor-burdened hosts, we investigated whether checkpoint blockade could improve the efficacy of responses induced with a whole tumor-cell vaccine. We show that administration of a single dose of blocking antibody was sufficient to significantly enhance antitumor activity of the vaccine, inducing complete radiological regression of established intracranial tumors. The antibody or vaccine alone were ineffective in this setting. The antibody had to be administered before, or close to, vaccine administration, suggesting CTLA-4 blockade had an impact on early priming events. The combined treatment resulted in enhanced trapping of leukocytes in the lymphoid tissues, including T cells that had undergone significant proliferation. There were no obvious changes in the stimulatory function of antigen-presenting cells or the number and function of regulatory T cells, suggesting T cells were the targets of the checkpoint blockade. While tumors regressing under combined treatment were highly infiltrated with a variety of leukocytes, tumor eradication was dependent on CD4+T cells. Analysis of the TCR repertoire showed that the addition of anti-CTLA-4 at priming reshaped the repertoire of tumor infiltrating T cells. In particular, the oligoclonal populations became greater in magnitude and more diverse in specificity. Using anti-CTLA-4 in a restricted way to promote the priming phase of an anti-cancer vaccine may offer a useful way of harnessing clinical benefit from this powerful agent. [ABSTRACT FROM PUBLISHER]