Astrocytic stress response is induced by exposure to astrocyte-binding antibodies expressed by plasmablasts from pediatric patients with acute transverse myelitis.

BACKGROUND: Pediatric acute transverse myelitis (ATM) accounts for 20-30% of children presenting with a first acquired demyelinating syndrome (ADS) and may be the first clinical presentation of a relapsing ADS such as multiple sclerosis (MS). B cells have been strongly implicated in the pathogenesis of adult MS. However, little is known about B cells in pediatric MS, and even less so in pediatric ATM. Our lab previously showed that plasmablasts (PB), the earliest B cell subtype producing antibody, are expanded in adult ATM, and that these PBs produce self-reactive antibodies that target neurons. The goal of this study was to examine PB frequency and phenotype, immunoglobulin selection, and B cell receptor reactivity in pediatric patients presenting with ATM to gain insight to B cell involvement in disease. METHODS: We compared the PB frequency and phenotype of 5 pediatric ATM patients and 10 pediatric healthy controls (HC) and compared them to previously reported adult ATM patients using cytometric data. We purified bulk IgG from the plasma samples and cloned 20 recombinant human antibodies (rhAbs) from individual PBs isolated from the blood. Plasma-derived IgG and rhAb autoreactivity was measured by mean fluorescence intensity (MFI) in neurons and astrocytes of murine brain or spinal cord and primary human astrocytes. We determined the potential impact of these rhAbs on astrocyte health by measuring stress and apoptotic response. RESULTS: We found that pediatric ATM patients had a reduced frequency of peripheral blood PB. Serum IgG autoreactivity to neurons in EAE spinal cord was similar in the pediatric ATM patients and HC. However, serum IgG autoreactivity to astrocytes in EAE spinal cord was reduced in pediatric ATM patients compared to pediatric HC. Astrocyte-binding strength of rhAbs cloned from PBs was dependent on somatic hypermutation accumulation in the pediatric ATM cohort, but not HC. A similar observation in predilection for astrocyte binding over neuron binding of individual antibodies cloned from PBs was made in EAE brain tissue. Finally, exposure of human primary astrocytes to these astrocyte-binding antibodies increased astrocytic stress but did not lead to apoptosis. CONCLUSIONS: Discordance in humoral immune responses to astrocytes may distinguish pediatric ATM from HC.

VH2+ Antigen-Experienced B Cells in the Cerebrospinal Fluid Are Expanded and Enriched in Pediatric Anti-NMDA Receptor Encephalitis.

Pediatric and adult autoimmune encephalitis (AE) are often associated with Abs to the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor (NMDAR). Very little is known regarding the cerebrospinal fluid humoral immune profile and Ab genetics associated with pediatric anti-NMDAR-AE. Using a combination of cellular, molecular, and immunogenetics tools, we collected cerebrospinal fluid from pediatric subjects and generated 1) flow cytometry data to calculate the frequency of B cell subtypes in the cerebrospinal fluid of pediatric subjects with anti-NMDAR-AE and controls, 2) a panel of recombinant human Abs from a pediatric case of anti-NMDAR-AE that was refractory to treatment, and 3) a detailed analysis of the Ab genes that bound the NR1 subunit of the NMDAR. Ag-experienced B cells including memory cells, plasmablasts, and Ab-secreting cells were expanded in the pediatric anti-NMDAR-AE cohort, but not in the controls. These Ag-experienced B cells in the cerebrospinal fluid of a pediatric case of NMDAR-AE that was refractory to treatment had expanded use of variable H chain family 2 (VH2) genes with high somatic hypermutation that all bound to the NR1 subunit of the NMDAR. A CDR3 motif was identified in this refractory case that likely drove early stage activation and expansion of naive B cells to Ab-secreting cells, facilitating autoimmunity associated with pediatric anti-NMDAR-AE through the production of Abs that bind NR1. These features of humoral immune responses in the cerebrospinal fluid of pediatric anti-NMDAR-AE patients may be relevant for clinical diagnosis and treatment.

Neuronal binding by antibodies can be influenced by low pH stress during the isolation procedure.

Low pH stress and its influence on antibody binding is a common consideration among chemists, but is only recently emerging as a consideration in Immunological studies. Antibody characterizations in Multiple Sclerosis (MS), an autoimmune disease of the Central Nervous System (CNS) has revealed that antibodies in the cerebrospinal fluid (CSF) of patients with Multiple Sclerosis bind to myelin-related and non-myelin antigen targets. Many laboratories have used molecular biology techniques to generate recombinant human antibodies (rhAbs) expressed by individual B cells from healthy donors and patients with systemic autoimmune disease to identify antigen targets. This approach has been adapted within the Neuroimmunology research community to investigate antigen targets of individual B cells in the CSF of MS patients. Our laboratory determines which antibodies to clone based on their immunogenetics and this method enriches for cloning of rhAbs that bind to neurons. However, newer technologies to assist in purification of these rhAbs from culture supernatants use an acidic elution buffer which may enhance low pH stress on the antibody structure. Our laboratory routinely uses a basic elution buffer to purify rhAbs from culture supernatants to avoid low pH stress to the antibody structure. Our goal was to investigate whether acidic elution of our rhAbs using Next Generation Chromatography would impact the rhAbs' ability to bind neurons. The limited data presented here for two neuron-binding rhAbs tested indicated that acidic elution buffers used during rhAb purification impacted the ability of rhAbs with low CDR3 charge to maintain binding to neuronal targets. Reproducibility in a larger panel of rhAbs and factors underlying these observations remain untested.

AIRR community curation and standardised representation for immunoglobulin and T cell receptor germline sets.

Analysis of an individual's immunoglobulin or T cell receptor gene repertoire can provide important insights into immune function. High-quality analysis of adaptive immune receptor repertoire sequencing data depends upon accurate and relatively complete germline sets, but current sets are known to be incomplete. Established processes for the review and systematic naming of receptor germline genes and alleles require specific evidence and data types, but the discovery landscape is rapidly changing. To exploit the potential of emerging data, and to provide the field with improved state-of-the-art germline sets, an intermediate approach is needed that will allow the rapid publication of consolidated sets derived from these emerging sources. These sets must use a consistent naming scheme and allow refinement and consolidation into genes as new information emerges. Name changes should be minimised, but, where changes occur, the naming history of a sequence must be traceable. Here we outline the current issues and opportunities for the curation of germline IG/TR genes and present a forward-looking data model for building out more robust germline sets that can dovetail with current established processes. We describe interoperability standards for germline sets, and an approach to transparency based on principles of findability, accessibility, interoperability, and reusability.

Dynamic kernel matching for non-conforming data: A case study of T cell receptor datasets.

Most statistical classifiers are designed to find patterns in data where numbers fit into rows and columns, like in a spreadsheet, but many kinds of data do not conform to this structure. To uncover patterns in non-conforming data, we describe an approach for modifying established statistical classifiers to handle non-conforming data, which we call dynamic kernel matching (DKM). As examples of non-conforming data, we consider (i) a dataset of T-cell receptor (TCR) sequences labelled by disease antigen and (ii) a dataset of sequenced TCR repertoires labelled by patient cytomegalovirus (CMV) serostatus, anticipating that both datasets contain signatures for diagnosing disease. We successfully fit statistical classifiers augmented with DKM to both datasets and report the performance on holdout data using standard metrics and metrics allowing for indeterminant diagnoses. Finally, we identify the patterns used by our statistical classifiers to generate predictions and show that these patterns agree with observations from experimental studies.

Immune Repertoire Analysis on High-Performance Computing Using VDJServer V1: A Method by the AIRR Community.

AIRR-seq data sets are usually large and require specialized analysis methods and software tools. A typical Illumina MiSeq sequencing run generates 20-30 million 2 × 300 bp paired-end sequence reads, which roughly corresponds to 15 GB of sequence data to be processed. Other platforms like NextSeq, which is useful in projects where the full V gene is not needed, create about 400 million 2 × 150 bp paired-end reads. Because of the size of the data sets, the analysis can be computationally expensive, particularly the early analysis steps like preprocessing and gene annotation that process the majority of the sequence data. A standard desktop PC may take 3-5 days of constant processing for a single MiSeq run, so dedicated high-performance computational resources may be required.VDJServer provides free access to high-performance computing (HPC) at the Texas Advanced Computing Center (TACC) through a graphical user interface (Christley et al. Front Immunol 9:976, 2018). VDJServer is a cloud-based analysis portal for immune repertoire sequence data that provides access to a suite of tools for a complete analysis workflow, including modules for preprocessing and quality control of sequence reads, V(D)J gene assignment, repertoire characterization, and repertoire comparison. Furthermore, VDJServer has parallelized execution for tools such as IgBLAST, so more compute resources are utilized as the size of the input data grows. Analysis that takes days on a desktop PC might take only a few hours on VDJServer. VDJServer is a free, publicly available, and open-source licensed resource. Here, we describe the workflow for performing immune repertoire analysis on VDJServer's high-performance computing.

Data Sharing and Reuse: A Method by the AIRR Community.

High-throughput sequencing of adaptive immune receptor repertoires (AIRR, i.e., IG and TR ) has revolutionized the ability to study the adaptive immune response via large-scale experiments. Since 2009, AIRR sequencing (AIRR-seq) has been widely applied to survey the immune state of individuals (see "The AIRR Community Guide to Repertoire Analysis" chapter for details). One of the goals of the AIRR Community is to make the resulting AIRR-seq data FAIR (Findable, Accessible, Interoperable, and Reusable) (Wilkinson et al. Sci Data 3:1-9, 2016), with a primary goal of making it easy for the research community to reuse AIRR-seq data (Breden et al. Front Immunol 8:1418, 2017; Scott and Breden. Curr Opin Syst Biol 24:71-77, 2020). The basis for this is the MiAIRR data standard (Rubelt et al. Nat Immunol 18:1274-1278, 2017). For long-term preservation, it is recommended that researchers store their sequence read data in an INSDC repository. At the same time, the AIRR Community has established the AIRR Data Commons (Christley et al. Front Big Data 3:22, 2020), a distributed set of AIRR-compliant repositories that store the critically important annotated AIRR-seq data based on the MiAIRR standard, making the data findable, interoperable, and, because the data are annotated, more valuable in its reuse. Here, we build on the other AIRR Community chapters and illustrate how these principles and standards can be incorporated into AIRR-seq data analysis workflows. We discuss the importance of careful curation of metadata to ensure reproducibility and facilitate data sharing and reuse, and we illustrate how data can be shared via the AIRR Data Commons.

CSF-Derived CD4+ T-Cell Diversity Is Reduced in Patients With Alzheimer Clinical Syndrome.

BACKGROUND AND OBJECTIVES: Patients with Alzheimer dementia display evidence of amyloid-related neurodegeneration. Our focus was to determine whether such patients also display evidence of a disease-targeting adaptive immune response mediated by CD4+ T cells. To test this hypothesis, we evaluated the CSF immune profiles of patients with Alzheimer clinical syndrome (ACS), who display clinically defined dementia. METHODS: Innate and adaptive immune profiles of patients with ACS were measured using multicolor flow cytometry. CSF-derived CD4+ and CD8+ T-cell receptor repertoire genetics were measured using next-generation sequencing. Brain-specific autoantibody signatures of CSF-derived antibody pools were measured using array technology or ELISA. CSF from similar-age healthy controls (HCs) was used as a comparator cohort. RESULTS: Innate cells were expanded in the CSF of patients with ACS in comparison to HCs, and innate cell expansion increased with age in the patients with ACS, but not HCs. Despite innate cell expansion in the CSF, the frequency of total CD4+ T cells reduced with age in the patients with ACS. T-cell receptor repertoire genetics indicated that T-cell clonal expansion is enhanced, and diversity is reduced in the patients with ACS compared with similar-age HCs. DISCUSSION: Examination of CSF indicates that CD4+ T cell-mediated adaptive immune responses are altered in patients with ACS. Understanding the underlying mechanisms affecting adaptive immunity will help move us toward the goal of slowing cognitive decline.

Outcome and Immune Correlates of a Phase II Trial of High-Dose Interleukin-2 and Stereotactic Ablative Radiotherapy for Metastatic Renal Cell Carcinoma.

PURPOSE: This phase II clinical trial evaluated whether the addition of stereotactic ablative radiotherapy (SAbR), which may promote tumor antigen presentation, improves the overall response rate (ORR) to high-dose IL2 (HD IL2) in metastatic renal cell carcinoma (mRCC). PATIENTS AND METHODS: Patients with pathologic evidence of clear cell renal cell carcinoma (RCC) and radiographic evidence of metastasis were enrolled in this single-arm trial and were treated with SAbR, followed by HD IL2. ORR was assessed based on nonirradiated metastases. Secondary endpoints included overall survival (OS), progression-free survival (PFS), toxicity, and treatment-related tumor-specific immune response. Correlative studies involved whole-exome and transcriptome sequencing, T-cell receptor sequencing, cytokine analysis, and mass cytometry on patient samples. RESULTS: Thirty ethnically diverse mRCC patients were enrolled. A median of two metastases were treated with SAbR. Among 25 patients evaluable by RECIST v1.1, ORR was 16% with 8% complete responses. Median OS was 37 months. Treatment-related adverse events (AE) included 22 grade ≥3 events that were not dissimilar from HD IL2 alone. There were no grade 5 AEs. A correlation was observed between SAbR to lung metastases and improved PFS (P = 0.0165). Clinical benefit correlated with frameshift mutational load, mast cell tumor infiltration, decreased circulating tumor-associated T-cell clones, and T-cell clonal expansion. Higher regulatory/CD8+ T-cell ratios at baseline in the tumor and periphery correlated with no clinical benefit. CONCLUSIONS: Adding SAbR did not improve the response rate to HD IL2 in patients with mRCC in this study. Tissue analyses suggest a possible correlation between frameshift mutation load as well as tumor immune infiltrates and clinical outcomes.

Reconstituting T cell receptor selection in-silico.

Each T cell receptor (TCR) gene is created without regard for which substances (antigens) the receptor can recognize. T cell selection culls developing T cells when their TCRs (i) fail to recognize major histocompatibility complexes (MHCs) that act as antigen presenting platforms or (ii) recognize with high affinity self-antigens derived from healthy cells and tissue. While T cell selection has been thoroughly studied, little is known about which TCRs are retained or removed by this process. Therefore, we develop an approach using TCR gene sequencing and machine learning to identify patterns in TCR protein sequences influencing the outcome of T cell receptor selection. We verify the trained models classify TCRs from developing T cells as being before selection and TCRs from mature T cells as being after selection. Our approach may provide future avenues for studying the relationship between T cell selection and conditions like autoimmune diseases.

T Cell Receptor Repertoires Acquired via Routine Pap Testing May Help Refine Cervical Cancer and Precancer Risk Estimates.

Cervical cancer is the fourth most common cancer and fourth leading cause of cancer death among women worldwide. In low Human Development Index settings, it ranks second. Screening and surveillance involve the cytology-based Papanicolaou (Pap) test and testing for high-risk human papillomavirus (hrHPV). The Pap test has low sensitivity to detect precursor lesions, while a single hrHPV test cannot distinguish a persistent infection from one that the immune system will naturally clear. Furthermore, among women who are hrHPV-positive and progress to high-grade cervical lesions, testing cannot identify the ~20% who would progress to cancer if not treated. Thus, reliable detection and treatment of cancers and precancers requires routine screening followed by frequent surveillance among those with past abnormal or positive results. The consequence is overtreatment, with its associated risks and complications, in screened populations and an increased risk of cancer in under-screened populations. Methods to improve cervical cancer risk assessment, particularly assays to predict regression of precursor lesions or clearance of hrHPV infection, would benefit both populations. Here we show that women who have lower risk results on follow-up testing relative to index testing have evidence of enhanced T cell clonal expansion in the index cervical cytology sample compared to women who persist with higher risk results from index to follow-up. We further show that a machine learning classifier based on the index sample T cells predicts this transition to lower risk with 95% accuracy (19/20) by leave-one-out cross-validation. Using T cell receptor deep sequencing and machine learning, we identified a biophysicochemical motif in the complementarity-determining region 3 of T cell receptor ß chains whose presence predicts this transition. While these results must still be tested on an independent cohort in a prospective study, they suggest that this approach could improve cervical cancer screening by helping distinguish women likely to spontaneously regress from those at elevated risk of progression to cancer. The advancement of such a strategy could reduce surveillance frequency and overtreatment in screened populations and improve the delivery of screening to under-screened populations.

TCRMatch: Predicting T-Cell Receptor Specificity Based on Sequence Similarity to Previously Characterized Receptors.

The adaptive immune system in vertebrates has evolved to recognize non-self antigens, such as proteins expressed by infectious agents and mutated cancer cells. T cells play an important role in antigen recognition by expressing a diverse repertoire of antigen-specific receptors, which bind epitopes to mount targeted immune responses. Recent advances in high-throughput sequencing have enabled the routine generation of T-cell receptor (TCR) repertoire data. Identifying the specific epitopes targeted by different TCRs in these data would be valuable. To accomplish that, we took advantage of the ever-increasing number of TCRs with known epitope specificity curated in the Immune Epitope Database (IEDB) since 2004. We compared seven metrics of sequence similarity to determine their power to predict if two TCRs have the same epitope specificity. We found that a comprehensive k-mer matching approach produced the best results, which we have implemented into TCRMatch, an openly accessible tool (http://tools.iedb.org/tcrmatch/) that takes TCR ß-chain CDR3 sequences as an input, identifies TCRs with a match in the IEDB, and reports the specificity of each match. We anticipate that this tool will provide new insights into T cell responses captured in receptor repertoire and single cell sequencing experiments and will facilitate the development of new strategies for monitoring and treatment of infectious, allergic, and autoimmune diseases, as well as cancer.

The immuneML ecosystem for machine learning analysis of adaptive immune receptor repertoires.

Adaptive immune receptor repertoires (AIRR) are key targets for biomedical research as they record past and ongoing adaptive immune responses. The capacity of machine learning (ML) to identify complex discriminative sequence patterns renders it an ideal approach for AIRR-based diagnostic and therapeutic discovery. To date, widespread adoption of AIRR ML has been inhibited by a lack of reproducibility, transparency, and interoperability. immuneML (immuneml.uio.no) addresses these concerns by implementing each step of the AIRR ML process in an extensible, open-source software ecosystem that is based on fully specified and shareable workflows. To facilitate widespread user adoption, immuneML is available as a command-line tool and through an intuitive Galaxy web interface, and extensive documentation of workflows is provided. We demonstrate the broad applicability of immuneML by (i) reproducing a large-scale study on immune state prediction, (ii) developing, integrating, and applying a novel deep learning method for antigen specificity prediction, and (iii) showcasing streamlined interpretability-focused benchmarking of AIRR ML.

Mapping the immunogenic landscape of near-native HIV-1 envelope trimers in non-human primates.

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several human-derived broadly neutralizing antibodies.

Fecal microbiota transplant rescues mice from human pathogen mediated sepsis by restoring systemic immunity.

Death due to sepsis remains a persistent threat to critically ill patients confined to the intensive care unit and is characterized by colonization with multi-drug-resistant healthcare-associated pathogens. Here we report that sepsis in mice caused by a defined four-member pathogen community isolated from a patient with lethal sepsis is associated with the systemic suppression of key elements of the host transcriptome required for pathogen clearance and decreased butyrate expression. More specifically, these pathogens directly suppress interferon regulatory factor 3. Fecal microbiota transplant (FMT) reverses the course of otherwise lethal sepsis by enhancing pathogen clearance via the restoration of host immunity in an interferon regulatory factor 3-dependent manner. This protective effect is linked to the expansion of butyrate-producing Bacteroidetes. Taken together these results suggest that fecal microbiota transplantation may be a treatment option in sepsis associated with immunosuppression.

Comparative genetics of Enterococcus faecalis intestinal tissue isolates before and after surgery in a rat model of colon anastomosis.

We have recently demonstrated that collagenolytic Enterococcus faecalis plays a key and causative role in the pathogenesis of anastomotic leak, an uncommon but potentially lethal complication characterized by disruption of the intestinal wound following segmental removal of the colon (resection) and its reconnection (anastomosis). Here we hypothesized that comparative genetic analysis of E. faecalis isolates present at the anastomotic wound site before and after surgery would shed insight into the mechanisms by which collagenolytic strains are selected for and predominate at sites of anastomotic disruption. Whole genome optical mapping of four pairs of isolates from rat colonic tissue obtained following surgical resection (herein named "pre-op" isolates) and then 6 days later from the anastomotic site (herein named "post-op" isolates) demonstrated that the isolates with higher collagenolytic activity formed a distinct cluster. In order to perform analysis at a deeper level, a single pair of E. faecalis isolates (16A pre-op and 16A post-op) was selected for whole genome sequencing and assembled using a hybrid assembly algorithm. Comparative genomics demonstrated absence of multiple gene clusters, notably a pathogenicity island in the post-op isolate. No differences were found in the fsr-gelE-sprE genes (EF1817-1822) responsible for regulation and production of collagenolytic activity. Analysis of unique genes among the 16A pre-op and post-op isolates revealed the predominance of transporter systems-related genes in the pre-op isolate and phage-related and hydrolytic enzyme-encoding genes in the post-op isolate. Despite genetic differences observed between pre-op and post-op isolates, the precise genetic determinants responsible for their differential expression of collagenolytic activity remains unknown.

Biophysicochemical motifs in T cell receptor sequences as a potential biomarker for high-grade serous ovarian carcinoma.

We previously showed, in a pilot study with publicly available data, that T cell receptor (TCR) repertoires from tumor infiltrating lymphocytes (TILs) could be distinguished from adjacent healthy tissue repertoires by the presence of TCRs bearing specific, biophysicochemical motifs in their antigen binding regions. We hypothesized that such motifs might allow development of a novel approach to cancer detection. The motifs were cancer specific and achieved high classification accuracy: we found distinct motifs for breast versus colorectal cancer-associated repertoires, and the colorectal cancer motif achieved 93% accuracy, while the breast cancer motif achieved 94% accuracy. In the current study, we sought to determine whether such motifs exist for ovarian cancer, a cancer type for which detection methods are urgently needed. We made two significant advances over the prior work. First, the prior study used patient-matched TILs and healthy repertoires, collecting healthy tissue adjacent to the tumors. The current study collected TILs from patients with high-grade serous ovarian carcinoma (HGSOC) and healthy ovary repertoires from cancer-free women undergoing hysterectomy/salpingo-oophorectomy for benign disease. Thus, the classification task is distinguishing women with cancer from women without cancer. Second, in the prior study, classification accuracy was measured by patient-hold-out cross-validation on the training data. In the current study, classification accuracy was additionally assessed on an independent cohort not used during model development to establish the generalizability of the motif to unseen data. Classification accuracy was 95% by patient-hold-out cross-validation on the training set and 80% when the model was applied to the blinded test set. The results on the blinded test set demonstrate a biophysicochemical TCR motif found overwhelmingly in women with HGSOC but rarely in women with healthy ovaries, strengthening the proposal that cancer detection approaches might benefit from incorporation of TCR motif-based biomarkers. Furthermore, these results call for studies on large cohorts to establish higher classification accuracies, as well as for studies in other cancer types.

The ADC API: A Web API for the Programmatic Query of the AIRR Data Commons.

The Adaptive Immune Receptor Repertoire (AIRR) Community is a research-driven group that is establishing a clear set of community-accepted data and metadata standards; standards-based reference implementation tools; and policies and practices for infrastructure to support the deposit, curation, storage, and use of high-throughput sequencing data from B-cell and T-cell receptor repertoires (AIRR-seq data). The AIRR Data Commons is a distributed system of data repositories that utilizes a common data model, a common query language, and common interoperability formats for storage, query, and downloading of AIRR-seq data. Here is described the principal technical standards for the AIRR Data Commons consisting of the AIRR Data Model for repertoires and rearrangements, the AIRR Data Commons (ADC) API for programmatic query of data repositories, a reference implementation for ADC API services, and tools for querying and validating data repositories that support the ADC API. AIRR-seq data repositories can become part of the AIRR Data Commons by implementing the data model and API. The AIRR Data Commons allows AIRR-seq data to be reused for novel analyses and empowers researchers to discover new biological insights about the adaptive immune system.

Inferred Allelic Variants of Immunoglobulin Receptor Genes: A System for Their Evaluation, Documentation, and Naming.

Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT®, the international ImMunoGeneTics information system® (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT®. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC-focusing, to begin with, on human IGHV genes-with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species.

Biophysicochemical Motifs in T-cell Receptor Sequences Distinguish Repertoires from Tumor-Infiltrating Lymphocyte and Adjacent Healthy Tissue.

Immune repertoire deep sequencing allows comprehensive characterization of antigen receptor-encoding genes in a lymphocyte population. We hypothesized that this method could enable a novel approach to diagnose disease by identifying antigen receptor sequence patterns associated with clinical phenotypes. In this study, we developed statistical classifiers of T-cell receptor (TCR) repertoires that distinguish tumor tissue from patient-matched healthy tissue of the same organ. The basis of both classifiers was a biophysicochemical motif in the complementarity determining region 3 (CDR3) of TCRß chains. To develop each classifier, we extracted 4-mers from every TCRß CDR3 and represented each 4-mer using biophysicochemical features of its amino acid sequence combined with quantification of 4-mer (or receptor) abundance. This representation was scored using a logistic regression model. Unlike typical logistic regression, the classifier is fitted and validated under the requirement that at least 1 positively labeled 4-mer appears in every tumor repertoire and no positively labeled 4-mers appear in healthy tissue repertoires. We applied our method to publicly available data in which tumor and adjacent healthy tissue were collected from each patient. Using a patient-holdout cross-validation, our method achieved classification accuracy of 93% and 94% for colorectal and breast cancer, respectively. The parameter values for each classifier revealed distinct biophysicochemical properties for tumor-associated 4-mers within each cancer type. We propose that such motifs might be used to develop novel immune-based cancer screening assays. SIGNIFICANCE: This study presents a novel computational approach to identify T-cell repertoire differences between normal and tumor tissue.See related commentary by Zoete and Coukos, p. 1299.