Background T cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. Objective To determine the mechanism(s) of immune suppression mediated by the nanovesicles. Methods T cell tolerance was induced by i.v. injections of hapten conjugated to self antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated for testing in active and adoptive cell transfer models of CS. Results Tolerance was shown due to exosome-like nanovesicles in the supernatant of CD8+ suppressor T cells that were not Treg. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains, or possibly whole antibody, allowing targeted delivery of selected inhibitory miRNA-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin deficient JH-/- or miRNA-150-/- mice that produced non-suppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. Conclusions This is the first example of T cell regulation via systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains.

Asf1 is an evolutionarily conserved chaperone of H3 and H4 histones that functions in replication dependent and independent chromatin assembly. Although Asf1 has been well studied in humans and yeast (members of the Opisthokonta lineage of eukaryotes), questions remain concerning its mechanism of function. To obtain additional insight into the Asf1 function we have initiated a proteomic analysis in the ciliate protozoan T. thermophila, a member of the Alveolata lineage of eukaryotes. Our results suggest that an evolutionarily conserved function of Asf1 is mediating the nuclear transport of newly synthesized histones H3 and H4.

, triggers a transcriptional program that includes the production of type I IFNs. These antiviral cytokines signal in both autocrine and paracrine fashion through the JAK-STAT pathway leading to additional transcription events involving the differential expression of many hundreds of genes. The antiviral state produced by this extensive genetic reprogramming involves a core set of genes as well as pathogenspecific components (1).The DC response to individual pathogens involves multiple signals that must be integrated to initiate an appropriate immune response. Pathogenic viruses attempt to subvert normal immune function through the expression of IFN antagonists (2, 3). For example, IFN regulatory factor (IRF) 3 activation and IFN-b expression are blocked by the NS1 protein of influenza (4). Unraveling the impact of these immune antagonists would be aided by a detailed understanding of the genetic regulatory network that operates during an uninhibited antiviral response. This knowledge is lacking because previous human studies have used viruses that interfere with the immune response (4-6). One fundamental unresolved question is to what extent the antiviral response is a single interconnected transcriptional cascade (convergent architecture) or a combination of transcriptional events operating independently in reaction to the multiple signals that arise following viral insult (parallel architecture). Newcastle disease virus (NDV) infection of human DCs provides an ideal system to define the uninhibited regulatory network (7,8). NDV is an avian virus that is able to stimulate innate immunity and DC maturation, but lacks the ability to evade the human interferon response (9). By focusing on NDV, we can accurately depict the baseline network of transcription factor (TF) interactions that underlie a broad range of immune responses. Through comparative studies, this network will enable detailed analysis of other infections and greatly improve our understanding of the control mechanisms in antiviral immunity and the myriad ways through which pathogenic viruses subvert normal immune function.Systems biology methods combined with high-throughput experimental technologies are providing new insights into virus-host interactions (10, 11). Genome-wide transcriptional profiling has suggested that the DC antiviral response is characterized by temporal waves of gene activation, which may be controlled by different combinations of transcriptional regulators (1). Potential regulators can be implicated using direct approaches, such as differential expression of the TF mRNA (1) -regulatory motifs (12, 13). These methods typically provide a static view of the network. Other computational methods have been proposed to identify TFs driving time-dependent changes in expression, but these do not explicitly account for the regulation of the TF itself (14, 15). The most common approaches are based on the hypothesis that genes sharing a similar temporal profile are regulated by common TFs (16). In mammals, a variety of posttranscriptional ...

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.

Highlights d Tetrahymena Mediator contains at least 10 conserved subunits d ChIP-seq suggests a role for Mediator in global transcription regulation d MED31 localizes to the crescent stage of meiotic prophase in MIC during development d MED31 KD results in ectopic expression of developmental genes during mitotic growth

IgD 2 CD27 2 double negative (DN) B cells with proinflammatory characteristics are abnormally elevated in a proportion of multiple sclerosis (MS) patients. In this study, the origin and selection characteristics of DN B cells were studied in MS patients and healthy controls (HC). Expression of developmental markers on peripheral blood DN, IgD 2 CD27 + class-switched memory (CSM) and IgD + CD27 2 naive B cells of HC (n = 48) and MS patients (n = 96) was determined by flow cytometry. High-throughput adaptive immune receptor repertoire sequencing was performed on peripheral blood DN and CSM B cells of HC and MS patients (n = 3 each). DN B cells from HC and MS patients showed similar phenotypic and Ig repertoire characteristics. Phenotypic analysis indicated a mature state of DN B cells by low CD5, CD10, and CD38 expression. However, the frequency of CD95 + and IgA + cells was lower in DN versus CSM B cells. DN B cells are Ag experienced, as shown by somatic hypermutation of their Ig genes in adaptive immune receptor repertoire sequencing, although they showed a lower mutation load than CSM B cells. Shared clones were found between DN and CSM B cells, although >95% of the clones were unique to each population, and differences in V(D)J usage and CDR3 physicochemical properties were found. Thus, DN B cells arise in HC and MS patients via a common developmental pathway that is probably linked to immune aging. However, DN and CSM B cells develop through unique differentiation pathways, with most DN B cells representing an earlier maturation state.

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.

Summary Antibody repertoires reveal insights into the biology of the adaptive immune system and empower diagnostics and therapeutics. There are currently multiple tools available for the annotation of antibody sequences. All downstream analyses such as choosing lead drug candidates depend on the correct annotation of these sequences; however, a thorough comparison of the performance of these tools has not been investigated. Here, we benchmark the performance of commonly used immunoinformatic tools, i.e. IMGT/HighV-QUEST, IgBLAST and MiXCR, in terms of reproducibility of annotation output, accuracy and speed using simulated and experimental high-throughput sequencing datasets. We analyzed changes in IMGT reference germline database in the last 10 years in order to assess the reproducibility of the annotation output. We found that only 73/183 (40%) V, D and J human genes were shared between the reference germline sets used by the tools. We found that the annotation results differed between tools. In terms of alignment accuracy, MiXCR had the highest average frequency of gene mishits, 0.02 mishit frequency and IgBLAST the lowest, 0.004 mishit frequency. Reproducibility in the output of complementarity determining three regions (CDR3 amino acids) ranged from 4.3% to 77.6% with preprocessed data. In addition, run time of the tools was assessed: MiXCR was the fastest tool for number of sequences processed per unit of time. These results indicate that immunoinformatic analyses greatly depend on the choice of bioinformatics tool. Our results support informed decision-making to immunoinformaticians based on repertoire composition and sequencing platforms. Availability and implementation All tools utilized in the paper are free for academic use. Supplementary information Supplementary data are available at Bioinformatics online.