Isoform-selective TGF-ß3 inhibition for systemic sclerosis.

BACKGROUND: Transforming growth factor ß (TGF-ß) is implicated as a key mediator of pathological fibrosis, but its pleiotropic activity in a range of homeostatic functions presents challenges to its safe and effective therapeutic targeting. There are three isoforms of TGF-ß, TGF-ß1, TGF-ß2, and TGF-ß3, which bind to a common receptor complex composed of TGF-ßR1 and TGF-ßR2 to induce similar intracellular signals in vitro. We have recently shown that the cellular expression patterns and activation thresholds of TGF-ß2 and TGF-ß3 are distinct from those of TGF-ß1 and that selective short-term TGF-ß2 and TGF-ß3 inhibition can attenuate fibrosis in vivo without promoting excessive inflammation. Isoform-selective inhibition of TGF-ß may therefore provide a therapeutic opportunity for patients with chronic fibrotic disorders. METHODS: Transcriptomic profiling of skin biopsies from patients with systemic sclerosis (SSc) from multiple clinical trials was performed to evaluate the role of TGF-ß3 in this disease. Antibody humanization, biochemical characterization, crystallization, and pre-clinical experiments were performed to further characterize an anti-TGF-ß3 antibody. FINDINGS: In the skin of patients with SSc, TGF-ß3 expression is uniquely correlated with biomarkers of TGF-ß signaling and disease severity. Crystallographic studies establish a structural basis for selective TGF-ß3 inhibition with a potent and selective monoclonal antibody that attenuates fibrosis effectively in vivo at clinically translatable exposures. Toxicology studies suggest that, as opposed to pan-TGF-ß inhibitors, this anti-TGF-ß3 antibody has a favorable safety profile for chronic administration. CONCLUSION: We establish a rationale for targeting TGF-ß3 in SSc with a favorable therapeutic index. FUNDING: This study was funded by Genentech, Inc.

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling.

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

Inhibition of MRTF activation as a clinically achievable anti-fibrotic mechanism for pirfenidone.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease characterised by aberrant fibroblast/myofibroblast accumulation and excessive collagen matrix deposition in the alveolar areas of lungs. As the first approved IPF medication, pirfenidone (PFD) significantly decelerates lung function decline while its underlying anti-fibrotic mechanism remains elusive. METHODS: We performed transcriptomic and immunofluorescence analyses of primary human IPF tissues. RESULTS: We showed that myocardin-related transcription factor (MRTF) signalling is activated in myofibroblasts accumulated in IPF lungs. Furthermore, we showed that PFD inhibits MRTF activation in primary human lung fibroblasts at clinically achievable concentrations (half-maximal inhibitory concentration 50-150 µM, maximal inhibition >90%, maximal concentration of PFD in patients <100 µM). Mechanistically, PFD appears to exert its inhibitory effects by promoting the interaction between MRTF and actin indirectly. Finally, PFD-treated IPF lungs exhibit significantly less MRTF activation in fibroblast foci areas than naïve IPF lungs. CONCLUSIONS: Our results suggest MRTF signalling as a direct target for PFD and implicate that some of the anti-fibrotic effects of PFD may be due to MRTF inhibition in lung fibroblasts.

A WISP1 antibody inhibits MRTF signaling to prevent the progression of established liver fibrosis.

Fibrosis is the major risk factor associated with morbidity and mortality in patients with non-alcoholic steatohepatitis (NASH)-driven chronic liver disease. Although numerous efforts have been made to identify the mediators of the initiation of liver fibrosis, the molecular underpinnings of fibrosis progression remain poorly understood, and therapies to arrest liver fibrosis progression are elusive. Here, we identify a pathway involving WNT1-inducible signaling pathway protein 1 (WISP1) and myocardin-related transcription factor (MRTF) as a central mechanism driving liver fibrosis progression through the integrin-dependent transcriptional reprogramming of myofibroblast cytoskeleton and motility. In mice, WISP1 deficiency protects against fibrosis progression, but not fibrosis onset. Moreover, the therapeutic administration of a novel antibody blocking WISP1 halted the progression of existing liver fibrosis in NASH models. These findings implicate the WISP1-MRTF axis as a crucial determinant of liver fibrosis progression and support targeting this pathway by antibody-based therapy for the treatment of NASH fibrosis.

Oncostatin M expression induced by bacterial triggers drives airway inflammatory and mucus secretion in severe asthma.

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae. These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm-deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.

In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice.

Partial reprogramming by expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) for short periods of time restores a youthful epigenetic signature to aging cells and extends the life span of a premature aging mouse model. However, the effects of longer-term partial reprogramming in physiologically aging wild-type mice are unknown. Here, we performed various long-term partial reprogramming regimens, including different onset timings, during physiological aging. Long-term partial reprogramming lead to rejuvenating effects in different tissues, such as the kidney and skin, and at the organismal level; duration of the treatment determined the extent of the beneficial effects. The rejuvenating effects were associated with a reversion of the epigenetic clock and metabolic and transcriptomic changes, including reduced expression of genes involved in the inflammation, senescence and stress response pathways. Overall, our observations indicate that partial reprogramming protocols can be designed to be safe and effective in preventing age-related physiological changes. We further conclude that longer-term partial reprogramming regimens are more effective in delaying aging phenotypes than short-term reprogramming.

TGFß2 and TGFß3 isoforms drive fibrotic disease pathogenesis.

Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.

A TRPA1 inhibitor suppresses neurogenic inflammation and airway contraction for asthma treatment.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

In silico tools for accurate HLA and KIR inference from clinical sequencing data empower immunogenetics on individual-patient and population scales.

Immunogenetic variation in humans is important in research, clinical diagnosis and increasingly a target for therapeutic intervention. Two highly polymorphic loci play critical roles, namely the human leukocyte antigen (HLA) system, which is the human version of the major histocompatibility complex (MHC), and the Killer-cell immunoglobulin-like receptors (KIR) that are relevant for responses of natural killer (NK) and some subsets of T cells. Their accurate classification has typically required the use of dedicated biological specimens and a combination of in vitro and in silico efforts. Increased availability of next generation sequencing data has led to the development of ancillary computational solutions. Here, we report an evaluation of recently published algorithms to computationally infer complex immunogenetic variation in the form of HLA alleles and KIR haplotypes from whole-genome or whole-exome sequencing data. For both HLA allele and KIR gene typing, we identified tools that yielded >97% overall accuracy for four-digit HLA types, and >99% overall accuracy for KIR gene presence, suggesting the readiness of in silico solutions for use in clinical and high-throughput research settings.

Osteopontin Links Myeloid Activation and Disease Progression in Systemic Sclerosis.

Progressive lung fibrosis is a major cause of mortality in systemic sclerosis (SSc) patients, but the underlying mechanisms remain unclear. We demonstrate that immune complexes (ICs) activate human monocytes to promote lung fibroblast migration partly via osteopontin (OPN) secretion, which is amplified by autocrine monocyte colony stimulating factor (MCSF) and interleukin-6 (IL-6) activity. Bulk and single-cell RNA sequencing demonstrate that elevated OPN expression in SSc lung tissue is enriched in macrophages, partially overlapping with CCL18 expression. Serum OPN is elevated in SSc patients with interstitial lung disease (ILD) and prognosticates future lung function deterioration in SSc cohorts. Serum OPN levels decrease following tocilizumab (monoclonal anti-IL-6 receptor) treatment, confirming the connection between IL-6 and OPN in SSc patients. Collectively, these data suggest a plausible link between autoantibodies and lung fibrosis progression, where circulating OPN serves as a systemic proxy for IC-driven profibrotic macrophage activity, highlighting its potential as a promising biomarker in SSc ILD.

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.

sumrep: A Summary Statistic Framework for Immune Receptor Repertoire Comparison and Model Validation.

The adaptive immune system generates an incredible diversity of antigen receptors for B and T cells to keep dangerous pathogens at bay. The DNA sequences coding for these receptors arise by a complex recombination process followed by a series of productivity-based filters, as well as affinity maturation for B cells, giving considerable diversity to the circulating pool of receptor sequences. Although these datasets hold considerable promise for medical and public health applications, the complex structure of the resulting adaptive immune receptor repertoire sequencing (AIRR-seq) datasets makes analysis difficult. In this paper we introduce sumrep, an R package that efficiently performs a wide variety of repertoire summaries and comparisons, and show how sumrep can be used to perform model validation. We find that summaries vary in their ability to differentiate between datasets, although many are able to distinguish between covariates such as donor, timepoint, and cell type for BCR and TCR repertoires. We show that deletion and insertion lengths resulting from V(D)J recombination tend to be more discriminative characterizations of a repertoire than summaries that describe the amino acid composition of the CDR3 region. We also find that state-of-the-art generative models excel at recapitulating gene usage and recombination statistics in a given experimental repertoire, but struggle to capture many physiochemical properties of real repertoires.

Repertoire-wide phylogenetic models of B cell molecular evolution reveal evolutionary signatures of aging and vaccination.

In order to produce effective antibodies, B cells undergo rapid somatic hypermutation (SHM) and selection for binding affinity to antigen via a process called affinity maturation. The similarities between this process and evolution by natural selection have led many groups to use phylogenetic methods to characterize the development of immunological memory, vaccination, and other processes that depend on affinity maturation. However, these applications are limited by the fact that most phylogenetic models are designed to be applied to individual lineages comprising genetically diverse sequences, while B cell repertoires often consist of hundreds to thousands of separate low-diversity lineages. Further, several features of affinity maturation violate important assumptions in standard phylogenetic models. Here, we introduce a hierarchical phylogenetic framework that integrates information from all lineages in a repertoire to more precisely estimate model parameters while simultaneously incorporating the unique features of SHM. We demonstrate the power of this repertoire-wide approach by characterizing previously undescribed phenomena in affinity maturation. First, we find evidence consistent with age-related changes in SHM hot-spot targeting. Second, we identify a consistent relationship between increased tree length and signs of increased negative selection, apparent in the repertoires of recently vaccinated subjects and those without any known recent infections or vaccinations. This suggests that B cell lineages shift toward negative selection over time as a general feature of affinity maturation. Our study provides a framework for undertaking repertoire-wide phylogenetic testing of SHM hypotheses and provides a means of characterizing dynamics of mutation and selection during affinity maturation.

Early B cell tolerance defects in neuromyelitis optica favour anti-AQP4 autoantibody production.

Neuromyelitis optica spectrum disorders (NMOSD) constitute rare autoimmune disorders of the CNS that are primarily characterized by severe inflammation of the spinal cord and optic nerve. Approximately 75% of NMOSD patients harbour circulating pathogenic autoantibodies targeting the aquaporin-4 water channel (AQP4). The source of these autoantibodies remains unclear, but parallels between NMOSD and other autoantibody-mediated diseases posit compromised B cell tolerance checkpoints as common underlying and contributing factors. Using a well established assay, we assessed tolerance fidelity by creating recombinant antibodies from B cell populations directly downstream of each checkpoint and testing them for polyreactivity and autoreactivity. We examined a total of 863 recombinant antibodies. Those derived from three anti-AQP4-IgG seropositive NMOSD patients (n = 130) were compared to 733 antibodies from 15 healthy donors. We found significantly higher frequencies of poly- and autoreactive new emigrant/transitional and mature naïve B cells in NMOSD patients compared to healthy donors (P-values < 0.003), thereby identifying defects in both central and peripheral B cell tolerance checkpoints in these patients. We next explored whether pathogenic NMOSD anti-AQP4 autoantibodies can originate from the pool of poly- and autoreactive clones that populate the naïve B cell compartment of NMOSD patients. Six human anti-AQP4 autoantibodies that acquired somatic mutations were reverted back to their unmutated germline precursors, which were tested for both binding to AQP4 and poly- or autoreactivity. While the affinity of mature autoantibodies against AQP4 ranged from modest to strong (Kd 15.2-559 nM), none of the germline revertants displayed any detectable binding to AQP4, revealing that somatic hypermutation is required for the generation of anti-AQP4 autoantibodies. However, two (33.3%) germline autoantibody revertants were polyreactive and four (66.7%) were autoreactive, suggesting that pathogenic anti-AQP4 autoantibodies can originate from the pool of autoreactive naïve B cells, which develops as a consequence of impaired early B cell tolerance checkpoints in NMOSD patients.

Identification of Subject-Specific Immunoglobulin Alleles From Expressed Repertoire Sequencing Data.

The adaptive immune receptor repertoire (AIRR) contains information on an individuals' immune past, present and potential in the form of the evolving sequences that encode the B cell receptor (BCR) repertoire. AIRR sequencing (AIRR-seq) studies rely on databases of known BCR germline variable (V), diversity (D), and joining (J) genes to detect somatic mutations in AIRR-seq data via comparison to the best-aligning database alleles. However, it has been shown that these databases are far from complete, leading to systematic misidentification of mutated positions in subsets of sample sequences. We previously presented TIgGER, a computational method to identify subject-specific V gene genotypes, including the presence of novel V gene alleles, directly from AIRR-seq data. However, the original algorithm was unable to detect alleles that differed by more than 5 single nucleotide polymorphisms (SNPs) from a database allele. Here we present and apply an improved version of the TIgGER algorithm which can detect alleles that differ by any number of SNPs from the nearest database allele, and can construct subject-specific genotypes with minimal prior information. TIgGER predictions are validated both computationally (using a leave-one-out strategy) and experimentally (using genomic sequencing), resulting in the addition of three new immunoglobulin heavy chain V (IGHV) gene alleles to the IMGT repertoire. Finally, we develop a Bayesian strategy to provide a confidence estimate associated with genotype calls. All together, these methods allow for much higher accuracy in germline allele assignment, an essential step in AIRR-seq studies.

AIRR Community Standardized Representations for Annotated Immune Repertoires.

Increased interest in the immune system's involvement in pathophysiological phenomena coupled with decreased DNA sequencing costs have led to an explosion of antibody and T cell receptor sequencing data collectively termed "adaptive immune receptor repertoire sequencing" (AIRR-seq or Rep-Seq). The AIRR Community has been actively working to standardize protocols, metadata, formats, APIs, and other guidelines to promote open and reproducible studies of the immune repertoire. In this paper, we describe the work of the AIRR Community's Data Representation Working Group to develop standardized data representations for storing and sharing annotated antibody and T cell receptor data. Our file format emphasizes ease-of-use, accessibility, scalability to large data sets, and a commitment to open and transparent science. It is composed of a tab-delimited format with a specific schema. Several popular repertoire analysis tools and data repositories already utilize this AIRR-seq data format. We hope that others will follow suit in the interest of promoting interoperable standards.

Mutant Cellular AP-1 Proteins Promote Expression of a Subset of Epstein-Barr Virus Late Genes in the Absence of Lytic Viral DNA Replication.

Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator.IMPORTANCE Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.

Reproducibility and Reuse of Adaptive Immune Receptor Repertoire Data.

High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell receptor repertoires has increased dramatically since the technique was introduced in 2009 (1-3). This experimental approach explores the maturation of the adaptive immune system and its response to antigens, pathogens, and disease conditions in exquisite detail. It holds significant promise for diagnostic and therapy-guiding applications. New technology often spreads rapidly, sometimes more rapidly than the understanding of how to make the products of that technology reliable, reproducible, or usable by others. As complex technologies have developed, scientific communities have come together to adopt common standards, protocols, and policies for generating and sharing data sets, such as the MIAME protocols developed for microarray experiments. The Adaptive Immune Receptor Repertoire (AIRR) Community formed in 2015 to address similar issues for HTS data of immune repertoires. The purpose of this perspective is to provide an overview of the AIRR Community's founding principles and present the progress that the AIRR Community has made in developing standards of practice and data sharing protocols. Finally, and most important, we invite all interested parties to join this effort to facilitate sharing and use of these powerful data sets (join@airr-community.org).

Interleukin-10 from CD4+ follicular regulatory T cells promotes the germinal center response.

CD4+ follicular regulatory T (Tfr) cells suppress B cell responses through modulation of follicular helper T (Tfh) cells and germinal center (GC) development. We found that Tfr cells can also promote the GC response through provision of interleukin-10 (IL-10) after acute infection with lymphocytic choriomeningitis virus (LCMV). Sensing of IL-10 by B cells was necessary for optimal development of the GC response. GC B cells formed in the absence of Treg cell-derived IL-10 displayed an altered dark zone state and decreased expression of the transcription factor Forkhead box protein 1 (FOXO1). IL-10 promoted nuclear translocation of FOXO1 in activated B cells. These data indicate that Tfr cells play a multifaceted role in the fine-tuning of the GC response and identify IL-10 as an important mediator by which Tfr cells support the GC reaction.

The Repertoire Dissimilarity Index as a method to compare lymphocyte receptor repertoires.

BACKGROUND: The B and T cells of the human adaptive immune system leverage a highly diverse repertoire of antigen-specific receptors to protect the human body from pathogens. The sequencing and analysis of immune repertoires is emerging as an important tool to understand immune responses, whether beneficial or harmful (in the case of autoimmunity). However, methods for studying these repertoires, and for directly comparing different immune repertoires, are lacking. RESULTS: In this paper, we present a non-parametric method for directly comparing sequencing repertoires, with the goal of rigorously quantifying differences in V, D, and J gene segment utilization. This method, referred to as the Repertoire Dissimilarity Index (RDI), uses a bootstrapped subsampling approach to account for variance in sequencing depth, and, coupled with a data simulation approach, allows for direct quantification of the average variation between repertoires. We use the RDI method to recapitulate known differences in the formation of the CD4+ and CD8+ T cell repertoires, and further show that antigen-driven activation of naïve CD8+ T cells is more selective than in the CD4+ repertoire, resulting in a more specialized CD8+ memory repertoire. CONCLUSIONS: We prove that the RDI method is an accurate and versatile method for comparisons of immune repertoires. The RDI method has been implemented as an R package, and is available for download through Bitbucket.