Repeated mRNA vaccination sequentially boosts SARS-CoV-2-specific CD8+ T cells in persons with previous COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hybrid immunity is more protective than vaccination or previous infection alone. To investigate the kinetics of spike-reactive T (TS) cells from SARS-CoV-2 infection through messenger RNA vaccination in persons with hybrid immunity, we identified the T cell receptor (TCR) sequences of thousands of index TS cells and tracked their frequency in bulk TCRß repertoires sampled longitudinally from the peripheral blood of persons who had recovered from coronavirus disease 2019 (COVID-19). Vaccinations led to large expansions in memory TS cell clonotypes, most of which were CD8+ T cells, while also eliciting diverse TS cell clonotypes not observed before vaccination. TCR sequence similarity clustering identified public CD8+ and CD4+ TCR motifs associated with spike (S) specificity. Synthesis of longitudinal bulk ex vivo single-chain TCRß repertoires and paired-chain TCRÉ‘ß sequences from droplet sequencing of TS cells provides a roadmap for the rapid assessment of T cell responses to vaccines and emerging pathogens.

Fundamental immune-oncogenicity trade-offs define driver mutation fitness.

Missense driver mutations in cancer are concentrated in a few hotspots1. Various mechanisms have been proposed to explain this skew, including biased mutational processes2, phenotypic differences3-6 and immunoediting of neoantigens7,8; however, to our knowledge, no existing model weighs the relative contribution of these features to tumour evolution. We propose a unified theoretical 'free fitness' framework that parsimoniously integrates multimodal genomic, epigenetic, transcriptomic and proteomic data into a biophysical model of the rate-limiting processes underlying the fitness advantage conferred on cancer cells by driver gene mutations. Focusing on TP53, the most mutated gene in cancer1, we present an inference of mutant p53 concentration and demonstrate that TP53 hotspot mutations optimally solve an evolutionary trade-off between oncogenic potential and neoantigen immunogenicity. Our model anticipates patient survival in The Cancer Genome Atlas and patients with lung cancer treated with immunotherapy as well as the age of tumour onset in germline carriers of TP53 variants. The predicted differential immunogenicity between hotspot mutations was validated experimentally in patients with cancer and in a unique large dataset of healthy individuals. Our data indicate that immune selective pressure on TP53 mutations has a smaller role in non-cancerous lesions than in tumours, suggesting that targeted immunotherapy may offer an early prophylactic opportunity for the former. Determining the relative contribution of immunogenicity and oncogenic function to the selective advantage of hotspot mutations thus has important implications for both precision immunotherapies and our understanding of tumour evolution.

Germline genetic regulation of the colorectal tumor immune microenvironment.

OBJECTIVE: To evaluate the contribution of germline genetics to regulating the briskness and diversity of T cell responses in CRC, we conducted a genome-wide association study to examine the associations between germline genetic variation and quantitative measures of T cell landscapes in 2,876 colorectal tumors from participants in the Molecular Epidemiology of Colorectal Cancer Study (MECC). METHODS: Germline DNA samples were genotyped and imputed using genome-wide arrays. Tumor DNA samples were extracted from paraffin blocks, and T cell receptor clonality and abundance were quantified by immunoSEQ (Adaptive Biotechnologies, Seattle, WA). Tumor infiltrating lymphocytes per high powered field (TILs/hpf) were scored by a gastrointestinal pathologist. Regression models were used to evaluate the associations between each variant and the three T-cell features, adjusting for sex, age, genotyping platform, and global ancestry. Three independent datasets were used for replication. RESULTS: We identified a SNP (rs4918567) near RBM20 associated with clonality at a genome-wide significant threshold of 5 × 10- 8, with a consistent direction of association in both discovery and replication datasets. Expression quantitative trait (eQTL) analyses and in silico functional annotation for these loci provided insights into potential functional roles, including a statistically significant eQTL between the T allele at rs4918567 and higher expression of ADRA2A (P = 0.012) in healthy colon mucosa. CONCLUSIONS: Our study suggests that germline genetic variation is associated with the quantity and diversity of adaptive immune responses in CRC. Further studies are warranted to replicate these findings in additional samples and to investigate functional genomic mechanisms.

Cytomegalovirus Exposure in the Elderly Does Not Reduce CD8 T Cell Repertoire Diversity.

With age, the immune system becomes less effective, causing increased susceptibility to infection. Chronic CMV infection further impairs immune function and is associated with increased mortality in the elderly. CMV exposure elicits massive CD8+ T cell clonal expansions and diminishes the cytotoxic T cell response to subsequent infections, leading to the hypothesis that to maintain homeostasis, T cell clones are expelled from the repertoire, reducing T cell repertoire diversity and diminishing the ability to combat new infections. However, in humans, the impact of CMV infection on the structure and diversity of the underlying T cell repertoire remains uncharacterized. Using TCR ß-chain immunosequencing, we observed that the proportion of the peripheral blood T cell repertoire composed of the most numerous 0.1% of clones is larger in the CMV seropositive and gradually increases with age. We found that the T cell repertoire in the elderly grows to accommodate CMV-driven clonal expansions while preserving its underlying diversity and clonal structure. Our observations suggest that the maintenance of large CMV-reactive T cell clones throughout life does not compromise the underlying repertoire. Alternatively, we propose that the diminished immunity in elderly individuals with CMV is due to alterations in cellular function rather than a reduction in CD8+ T cell repertoire diversity.

Lymphocytic infiltration in stage II microsatellite stable colorectal tumors: A retrospective prognosis biomarker analysis.

BACKGROUND: Identifying stage II patients with colorectal cancer (CRC) at higher risk of progression is a clinical priority in order to optimize the advantages of adjuvant chemotherapy while avoiding unnecessary toxicity. Recently, the intensity and the quality of the host immune response in the tumor microenvironment have been reported to have an important role in tumorigenesis and an inverse association with tumor progression. This association is well established in microsatellite instable CRC. In this work, we aim to assess the usefulness of measures of T-cell infiltration as prognostic biomarkers in 640 stage II, CRC tumors, 582 of them confirmed microsatellite stable. METHODS AND FINDINGS: We measured both the quantity and clonality index of T cells by means of T-cell receptor (TCR) immunosequencing in a discovery dataset (95 patients with colon cancer diagnosed at stage II and microsatellite stable, median age 67, 30% women) and replicated the results in 3 additional series of stage II patients from 2 countries. Series 1 and 2 were recruited in Barcelona, Spain and included 112 fresh frozen (FF, median age 69, 44% women) and 163 formalin-fixed paraffin-embedded (FFPE, median age 67, 39% women) samples, respectively. Series 3 included 270 FFPE samples from patients recruited in Haifa, Northern Israel, as part of a large case-control study of CRC (median age 73, 46% women). Median follow-up time was 81.1 months. Cox regression models were fitted to evaluate the prognostic value of T-cell abundance and Simpson clonality of TCR variants adjusting by sex, age, tumor location, and stage (IIA and IIB). In the discovery dataset, higher TCR abundance was associated with better prognosis (hazard ratio [HR] for ≥Q1 = 0.25, 95% CI 0.10-0.63, P = 0.003). A functional analysis of gene expression on these tumors revealed enrichment in pathways related to immune response. Higher values of clonality index (lower diversity) were not associated with worse disease-free survival, though the HR for ≥Q3 was 2.32 (95% CI 0.90-5.97, P = 0.08). These results were replicated in an independent FF dataset (TCR abundance: HR = 0.30, 95% CI 0.12-0.72, P = 0.007; clonality: HR = 3.32, 95% CI 1.38-7.94, P = 0.007). Also, the association with prognosis was tested in 2 independent FFPE datasets. The same association was observed with TCR abundance (HR = 0.41, 95% CI 0.18-0.93, P = 0.03 and HR = 0.56, 95% CI 0.31-1, P = 0.042, respectively, for each FFPE dataset). However, the clonality index was associated with prognosis only in the FFPE dataset from Israel (HR = 2.45, 95% CI 1.39-4.32, P = 0.002). Finally, a combined analysis combining all microsatellite stable (MSS) samples demonstrated a clear prognosis value both for TCR abundance (HR = 0.39, 95% CI 0.26-0.57, P = 1.3e-06) and the clonality index (HR = 2.13, 95% CI 1.44-3.15, P = 0.0002). These associations were also observed when variables were considered continuous in the models (HR per log2 of TCR abundance = 0.85, 95% CI 0.78-0.93, P = 0.0002; HR per log2 or clonality index = 1.16, 95% CI 1.03-1.31, P = 0.016). LIMITATIONS: This is a retrospective study, and samples had been preserved with different methods. Validation series lack complete information about microsatellite instability (MSI) status and pathology assessment. The Molecular Epidemiology of Colorectal Cancer (MECC) study had information about overall survival instead of progression-free survival. CONCLUSION: Results from this study demonstrate that tumor lymphocytes, assessed by TCR repertoire quantification based on a sequencing method, are an independent prognostic factor in microsatellite stable stage II CRC.

Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma.

BACKGROUND: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines. METHODS: Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels. RESULTS: LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low. CONCLUSIONS: These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

A Diverse Lipid Antigen-Specific TCR Repertoire Is Clonally Expanded during Active Tuberculosis.

Human T cells that recognize lipid Ags presented by highly conserved CD1 proteins often express semi-invariant TCRs, but the true diversity of lipid Ag-specific TCRs remains unknown. We use CD1b tetramers and high-throughput immunosequencing to analyze thousands of TCRs from ex vivo-sorted or in vitro-expanded T cells specific for the mycobacterial lipid Ag, glucose monomycolate. Our results reveal a surprisingly diverse repertoire resulting from editing of germline-encoded gene rearrangements analogous to MHC-restricted TCRs. We used a distance-based metric (TCRDist) to show how this diverse TCR repertoire builds upon previously reported conserved motifs by including subject-specific TCRs. In a South African cohort, we show that TCRDist can identify clonal expansion of diverse glucose monomycolate-specific TCRs and accurately distinguish patients with active tuberculosis from control subjects. These data suggest that similar mechanisms govern the selection and expansion of peptide and lipid Ag-specific T cells despite the nonpolymorphic nature of CD1.

Longitudinal immunosequencing in healthy people reveals persistent T cell receptors rich in highly public receptors.

BACKGROUND: The adaptive immune system maintains a diversity of T cells capable of recognizing a broad array of antigens. Each T cell's specificity for antigens is determined by its T cell receptors (TCRs), which together across all T cells form a repertoire of millions of unique receptors in each individual. Although many studies have examined how TCR repertoires change in response to disease or drugs, few have explored the temporal dynamics of the TCR repertoire in healthy individuals. RESULTS: Here we report immunosequencing of TCR ß chains (TCRß) from the blood of three healthy individuals at eight time points over one year. TCRß repertoires of all peripheral-blood T cells and sorted memory T cells clustered clearly by individual, systematically demonstrating that TCRß repertoires are specific to individuals across time. This individuality was absent from TCRßs from naive T cells, suggesting that the differences resulted from an individual's antigen exposure history, not genetic background. Many characteristics of the TCRß repertoire (e.g., diversity, clonality) were stable across time, although we found evidence of T cell expansion dynamics even within healthy individuals. We further identified a subset of "persistent" TCRßs present across all time points. These receptors were rich in clonal and highly public receptors and may play a key role in immune system maintenance. CONCLUSIONS: Our results highlight the importance of longitudinal sampling of the immune system, providing a much-needed baseline for TCRß dynamics in healthy individuals. Such a baseline will improve interpretation of changes in the TCRß repertoire during disease or treatment.

Clonal expansion of CD8 T cells in the systemic circulation precedes development of ipilimumab-induced toxicities.

Immune checkpoint therapies, such as ipilimumab, induce dramatic antitumor responses in a subset of patients with advanced malignancies, but they may also induce inflammatory responses and toxicities termed immune-related adverse events (irAEs). These irAEs are often low grade and manageable, but severe irAEs may lead to prolonged hospitalizations or fatalities. Early intervention is necessary to minimize morbidities that occur with severe irAEs. However, correlative biomarkers are currently lacking. In a phase II clinical trial that treated 27 patients with metastatic prostate cancer, we aimed to test the safety and efficacy of androgen deprivation therapy plus ipilimumab. In this study, we observed grade 3 toxicities in >40% of treated patients, which led to early closure of the study. Because ipilimumab enhances T-cell responses, we hypothesized that increased clonal T-cell responses in the systemic circulation may contribute to irAEs. Sequencing of the T-cell receptor ß-chains in purified T cells revealed clonal expansion of CD8 T cells, which occurred in blood samples collected before the onset of grade 2-3 irAEs. These initial results suggested that expansion of ≥55 CD8 T-cell clones preceded the development of severe irAEs. We further evaluated available blood samples from a second trial and determined that patients who experienced grade 2-3 irAEs also had expansion of ≥55 CD8 T-cell clones in blood samples collected before the onset of irAEs. We propose that CD8 T-cell clonal expansion may be a correlative biomarker to enable close monitoring and early intervention for patients receiving ipilimumab.

Targeted single molecule mutation detection with massively parallel sequencing.

Next-generation sequencing (NGS) technologies have transformed genomic research and have the potential to revolutionize clinical medicine. However, the background error rates of sequencing instruments and limitations in targeted read coverage have precluded the detection of rare DNA sequence variants by NGS. Here we describe a method, termed CypherSeq, which combines double-stranded barcoding error correction and rolling circle amplification (RCA)-based target enrichment to vastly improve NGS-based rare variant detection. The CypherSeq methodology involves the ligation of sample DNA into circular vectors, which contain double-stranded barcodes for computational error correction and adapters for library preparation and sequencing. CypherSeq is capable of detecting rare mutations genome-wide as well as those within specific target genes via RCA-based enrichment. We demonstrate that CypherSeq is capable of correcting errors incurred during library preparation and sequencing to reproducibly detect mutations down to a frequency of 2.4 × 10(-7) per base pair, and report the frequency and spectra of spontaneous and ethyl methanesulfonate-induced mutations across the Saccharomyces cerevisiae genome.

Contribution of systemic and somatic factors to clinical response and resistance to PD-L1 blockade in urothelial cancer: An exploratory multi-omic analysis.

BACKGROUND: Inhibition of programmed death-ligand 1 (PD-L1) with atezolizumab can induce durable clinical benefit (DCB) in patients with metastatic urothelial cancers, including complete remissions in patients with chemotherapy refractory disease. Although mutation load and PD-L1 immune cell (IC) staining have been associated with response, they lack sufficient sensitivity and specificity for clinical use. Thus, there is a need to evaluate the peripheral blood immune environment and to conduct detailed analyses of mutation load, predicted neoantigens, and immune cellular infiltration in tumors to enhance our understanding of the biologic underpinnings of response and resistance. METHODS AND FINDINGS: The goals of this study were to (1) evaluate the association of mutation load and predicted neoantigen load with therapeutic benefit and (2) determine whether intratumoral and peripheral blood T cell receptor (TCR) clonality inform clinical outcomes in urothelial carcinoma treated with atezolizumab. We hypothesized that an elevated mutation load in combination with T cell clonal dominance among intratumoral lymphocytes prior to treatment or among peripheral T cells after treatment would be associated with effective tumor control upon treatment with anti-PD-L1 therapy. We performed whole exome sequencing (WES), RNA sequencing (RNA-seq), and T cell receptor sequencing (TCR-seq) of pretreatment tumor samples as well as TCR-seq of matched, serially collected peripheral blood, collected before and after treatment with atezolizumab. These parameters were assessed for correlation with DCB (defined as progression-free survival [PFS] >6 months), PFS, and overall survival (OS), both alone and in the context of clinical and intratumoral parameters known to be predictive of survival in this disease state. Patients with DCB displayed a higher proportion of tumor-infiltrating T lymphocytes (TIL) (n = 24, Mann-Whitney p = 0.047). Pretreatment peripheral blood TCR clonality below the median was associated with improved PFS (n = 29, log-rank p = 0.048) and OS (n = 29, log-rank p = 0.011). Patients with DCB also demonstrated more substantial expansion of tumor-associated TCR clones in the peripheral blood 3 weeks after starting treatment (n = 22, Mann-Whitney p = 0.022). The combination of high pretreatment peripheral blood TCR clonality with elevated PD-L1 IC staining in tumor tissue was strongly associated with poor clinical outcomes (n = 10, hazard ratio (HR) (mean) = 89.88, HR (median) = 23.41, 95% CI [2.43, 506.94], p(HR > 1) = 0.0014). Marked variations in mutation loads were seen with different somatic variant calling methodologies, which, in turn, impacted associations with clinical outcomes. Missense mutation load, predicted neoantigen load, and expressed neoantigen load did not demonstrate significant association with DCB (n = 25, Mann-Whitney p = 0.22, n = 25, Mann-Whitney p = 0.55, and n = 25, Mann-Whitney p = 0.29, respectively). Instead, we found evidence of time-varying effects of somatic mutation load on PFS in this cohort (n = 25, p = 0.044). A limitation of our study is its small sample size (n = 29), a subset of the patients treated on IMvigor 210 (NCT02108652). Given the number of exploratory analyses performed, we intend for these results to be hypothesis-generating. CONCLUSIONS: These results demonstrate the complex nature of immune response to checkpoint blockade and the compelling need for greater interrogation and data integration of both host and tumor factors. Incorporating these variables in prospective studies will facilitate identification and treatment of resistant patients.

CMV reactivation drives posttransplant T-cell reconstitution and results in defects in the underlying TCRß repertoire.

Although cytomegalovirus (CMV) reactivation has long been implicated in posttransplant immune dysfunction, the molecular mechanisms that drive this phenomenon remain undetermined. To address this, we combined multiparameter flow cytometric analysis and T-cell subpopulation sorting with high-throughput sequencing of the T-cell repertoire, to produce a thorough evaluation of the impact of CMV reactivation on T-cell reconstitution after unrelated-donor hematopoietic stem cell transplant. We observed that CMV reactivation drove a >50-fold specific expansion of Granzyme B(high)/CD28(low)/CD57(high)/CD8(+) effector memory T cells (Tem) and resulted in a linked contraction of all naive T cells, including CD31(+)/CD4(+) putative thymic emigrants. T-cell receptor ß (TCRß) deep sequencing revealed a striking contraction of CD8(+) Tem diversity due to CMV-specific clonal expansions in reactivating patients. In addition to querying the topography of the expanding CMV-specific T-cell clones, deep sequencing allowed us, for the first time, to exhaustively evaluate the underlying TCR repertoire. Our results reveal new evidence for significant defects in the underlying CD8 Tem TCR repertoire in patients who reactivate CMV, providing the first molecular evidence that, in addition to driving expansion of virus-specific cells, CMV reactivation has a detrimental impact on the integrity and heterogeneity of the rest of the T-cell repertoire. This trial was registered at www.clinicaltrials.gov as #NCT01012492.

PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3' UTRs and coding sequences.

The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3' untranslated regions (3' UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3' UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA-mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA-mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats.

Dynamics of the cytotoxic T cell response to a model of acute viral infection.

UNLABELLED: A detailed characterization of the dynamics and breadth of the immune response to an acute viral infection, as well as the determinants of recruitment to immunological memory, can greatly contribute to our basic understanding of the mechanics of the human immune system and can ultimately guide the design of effective vaccines. In addition to neutralizing antibodies, T cells have been shown to be critical for the effective resolution of acute viral infections. We report the first in-depth analysis of the dynamics of the CD8(+) T cell repertoire at the level of individual T cell clonal lineages upon vaccination of human volunteers with a single dose of YF-17D. This live attenuated yellow fever virus vaccine yields sterile, long-term immunity and has been previously used as a model to understand the immune response to a controlled acute viral infection. We identified and enumerated unique CD8(+) T cell clones specifically induced by this vaccine through a combined experimental and statistical approach that included high-throughput sequencing of the CDR3 variable region of the T cell receptor ß-chain and an algorithm that detected significantly expanded T cell clones. This allowed us to establish that (i) on average, ∼ 2,000 CD8(+) T cell clones were induced by YF-17D, (ii) 5 to 6% of the responding clones were recruited to long-term memory 3 months postvaccination, (iii) the most highly expanded effector clones were preferentially recruited to the memory compartment, and (iv) a fraction of the YF-17D-induced clones could be identified from peripheral blood lymphocytes solely by measuring clonal expansion. IMPORTANCE: The exhaustive investigation of pathogen-induced effector T cells is essential to accurately quantify the dynamics of the human immune response. The yellow fever vaccine (YFV) has been broadly used as a model to understand how a controlled, self-resolving acute viral infection induces an effective and long-term protective immune response. Here, we extend this previous work by reporting the identity of activated effector T cell clones that expand in response to the YFV 2 weeks postvaccination (as defined by their unique T cell receptor gene sequence) and by tracking clones that enter the memory compartment 3 months postvaccination. This is the first study to use high-throughput sequencing of immune cells to characterize the breadth of the antiviral effector cell response and to determine the contribution of unique virus-induced clones to the long-lived memory T cell repertoire. Thus, this study establishes a benchmark against which future vaccines can be compared to predict their efficacy.

High-throughput sequencing of T-cell receptors reveals a homogeneous repertoire of tumour-infiltrating lymphocytes in ovarian cancer.

The cellular adaptive immune system mounts a response to many solid tumours mediated by tumour-infiltrating T lymphocytes (TILs). Basic measurements of these TILs, including total count, show promise as prognostic markers for a variety of cancers, including ovarian and colorectal. In addition, recent therapeutic advances are thought to exploit this immune response to effectively fight melanoma, with promising studies showing efficacy in additional cancers. However, many of the basic properties of TILs are poorly understood, including specificity, clonality, and spatial heterogeneity of the T-cell response. We utilize deep sequencing of rearranged T-cell receptor beta (TCRB) genes to characterize the basic properties of TILs in ovarian carcinoma. Due to somatic rearrangement during T-cell development, the TCR beta chain sequence serves as a molecular tag for each T-cell clone. Using these sequence tags, we assess similarities and differences between infiltrating T cells in discretely sampled sections of large tumours and compare to T cells from peripheral blood. Within the limits of sensitivity of our assay, the TIL repertoires show strong similarity throughout each tumour and are distinct from the circulating T-cell repertoire. We conclude that the cellular adaptive immune response within ovarian carcinomas is spatially homogeneous and distinct from the T-cell compartment of peripheral blood.

Ultra-deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas.

The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α- and ß-chains (TCRb). Our aim was to assess whether ultra-deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples can provide insights into the clonality and heterogeneity of intratumoural T cells in ccRCCs, a tumour type that can display extensive genetic intratumour heterogeneity (ITH). For this purpose, DNA was extracted from two to four tumour regions from each of four primary ccRCCs and was analysed by ultra-deep TCR sequencing. In parallel, tumour infiltration by CD4, CD8 and Foxp3 regulatory T cells was evaluated by immunohistochemistry and correlated with TCR-sequencing data. A polyclonal T cell repertoire with 367-16 289 (median 2394) unique TCRb sequences was identified per tumour region. The frequencies of the 100 most abundant T cell clones/tumour were poorly correlated between most regions (Pearson correlation coefficient, -0.218 to 0.465). 3-93% of these T cell clones were not detectable across all regions. Thus, the clonal composition of T cell populations can be heterogeneous across different regions of the same ccRCC. T cell ITH was higher in tumours pretreated with an mTOR inhibitor, which could suggest that therapy can influence adaptive tumour immunity. These data show that ultra-deep TCR-sequencing technology can be applied directly to DNA extracted from unfractionated tumour samples, allowing novel insights into the clonality of T cell populations in cancers. These were polyclonal and displayed ITH in ccRCC. TCRb sequencing may shed light on mechanisms of cancer immunity and the efficacy of immunotherapy approaches.

Shaping of human germline IgH repertoires revealed by deep sequencing.

To understand better how selection processes balance the benefits of Ig repertoire diversity with the risks of autoreactivity and nonfunctionality of highly variable IgH CDR3s, we collected millions of rearranged germline IgH CDR3 sequences by deep sequencing of DNA from mature human naive B cells purified from four individuals and analyzed the data with computational methods. Long HCDR3 regions, often components of HIV-neutralizing Abs, appear to derive not only from incorporation of long D genes and insertion of large N regions but also by usage of multiple D gene segments in tandem. However, comparison of productive and out-of-frame IgH rearrangements revealed a selection bias against long HCDR3 loops, suggesting these may be disproportionately either poorly functional or autoreactive. Our data suggest that developmental selection removes HCDR3 loops containing patches of hydrophobicity, which are commonly found in some auto-antibodies, and at least 69% of the initial productive IgH rearrangements are removed from the repertoire during B cell development. Additionally, we have demonstrated the potential utility of this new technology for vaccine development with the identification in all four individuals of related candidate germline IgH precursors of the HIV-neutralizing Ab 4E10.

Multimodal, broadly neutralizing antibodies against SARS-CoV-2 identified by high-throughput native pairing of BCRs from bulk B cells.

TruAB Discovery is an approach that integrates cellular immunology, high-throughput immunosequencing, bioinformatics, and computational biology in order to discover naturally occurring human antibodies for prophylactic or therapeutic use. We adapted our previously described pairSEQ technology to pair B cell receptor heavy and light chains of SARS-CoV-2 spike protein-binding antibodies derived from enriched antigen-specific memory B cells and bulk antibody-secreting cells. We identified approximately 60,000 productive, in-frame, paired antibody sequences, from which 2,093 antibodies were selected for functional evaluation based on abundance, isotype and patterns of somatic hypermutation. The exceptionally diverse antibodies included RBD-binders with broad neutralizing activity against SARS-CoV-2 variants, and S2-binders with broad specificity against betacoronaviruses and the ability to block membrane fusion. A subset of these RBD- and S2-binding antibodies demonstrated robust protection against challenge in hamster and mouse models. This high-throughput approach can accelerate discovery of diverse, multifunctional antibodies against any target of interest.

Perturbations of the T-cell immune repertoire in kidney transplant rejection.

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 post-transplantation, 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 post-transplant predictors of rejection risk.