SEARCH · bioRxiv Science

Results for “Immunology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources

bioRxiv2017

Lipid mediator class-switching downstream of PGE2 determines the outcome of inflammation resolution in vivo.

Neutrophils are the first immune cells recruited to a site of injury or infection, where they perform many functions. Having completed their role, neutrophils must be removed from the inflammatory site - either by apoptosis and efferocytosis or by reverse migration away from the wound - for restoration of normal tissue homeostasis. Disruption of these tightly controlled physiological processes of neutrophil removal can lead to a range of inflammatory diseases. We used an in vivo zebrafish model to understand the role of lipid mediator production in neutrophil removal. Following tailfin amputation in the absence of macrophages, neutrophillic inflammation does not resolve. This is due to loss of macrophage-dependent production of eicosanoid prostaglandin E2, which drives neutrophil removal via promotion of reverse migration. Knockdown of endogenous prostaglandin E synthase gene reveals PGE2 as essential for neutrophil inflammation resolution. Furthermore, PGE2 is able to signal through EP4 receptors to enhance Alox15 production, causing a switch towards anti-inflammatory eicosanoid signalling, specifically Lipoxin A4. Our data confirm regulation of neutrophil migration by PGE2 and LXA4 in an in vivo model of inflammation resolution. This pathway may contain therapeutic targets for driving inflammation resolution in chronic inflammatory disease.

immunology↗

bioRxiv2017

Evolutionary Origin of the Mammalian Hematopoietic System Found in a Colonial Chordate

Hematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are hematopoietic stem cells (HSCs), which are multipotent, self-renewing and generate the entire repertoire of blood and immune cells throughout life. Here we studied the hematopoietic system of Botryllus schlosseri, a colonial tunicate that has vasculature, circulating blood cells, and interesting characteristics of stem cell biology and immunity. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other. Using flow-cytometry, whole-transcriptome sequencing of defined cell populations, and diverse functional assays, we identified HSCs, progenitors, immune-effector cells, the HSC niche, and demonstrated that self-recognition inhibits cytotoxic reaction. Our study implies that the HSC and myeloid lineages emerged in a common ancestor of tunicates and vertebrates and suggests that hematopoietic bone marrow and the B. schlosseri endostyle niche evolved from the same origin.

immunology↗

bioRxiv2017

Integrin alpha 4/beta 1 (CD49d/CD29) is a component of the murine IgG3 receptor

Antibodies exert several of their effector functions by binding to cell surface receptors. For murine IgG3 (mIgG3) the identity of its receptors (and the very existence of a receptor) is still under debate, as not all mIgG3 functions can be explained by interaction with Fc{gamma}-receptor I (Fc{gamma}RI). This implies the existence of an alternate receptor, whose identity we sought to pinpoint. We found that blockage of the alpha4/beta1 integrin (Itga4/Itgb1) selectively hampered binding of mIgG3 to macrophages and mIgG3-mediated phagocytosis. Manganese, an integrin activator, increased mIgG3 binding to macrophages. Blockage of Fc{gamma}RI or Itgb1 inhibited binding of different mIgG3 antibodies to variable extents. Our results indicate an integrin component in the mIgG3 receptor. Given the more ancient origin of integrins in comparison with Fc{gamma}R, this observation could have far ranging implications for our understanding of the evolution of antibody-mediated immunity, as well as in immunity to microorganisms, pathogenesis of autoimmune diseases and antibody engineering.

immunology↗

bioRxiv2017

Id proteins suppress E2A-driven innate-like T cell development prior to TCR selection

Id proteins have been shown to promote the differentiation of conventional {beta} and {gamma}{delta}T cells, and to suppress the expansion of invariant Natural Killer T (iNKT) cells and innate-like {gamma}{delta}NKT within their respective cell lineages. However, it remains to be determined whether Id proteins regulate lineage specification in developing T cells that give rise to these distinct cell fates. Here we report that in the absence of Id2 and Id3 proteins, E2A prematurely activates genes critical for the iNKT cell lineage prior to TCR expression. Enhanced iNKT development in Id3-deficient mice lacking {gamma}{delta} NKT cells suggests that Id3 regulates the lineage competition between these populations. RNA-Seq analysis establishes E2A as the transcriptional regulator of both iNKT and {gamma}{delta}NKT development. In the absence of pre-TCR signaling, Id2/Id3 deletion gives rise to a large population of iNKT cells and a unique innate-like DP population, despite the block in conventional {beta} T cell development. The transcriptional profile of these unique DP cells reflects enrichment of innate-like signature genes, including PLZF (Zbtb16) and Granzyme A (Gzma). Results from these genetic models and genome-wide analyses suggest that Id proteins suppress E2A-driven innate-like T cell programs prior to TCR selection to enforce predominance of conventional T cells.

immunology↗

bioRxiv2017

Terminal uridylyltransferases target RNA viruses as part of the innate immune system in animals

RNA viruses are a major threat to animals and plants. RNA interference (RNAi) and the interferon response provide innate antiviral defense against RNA viruses. Here we performed a large-scale screen using C. elegans and its natural pathogen, the Orsay virus (OrV), and identified cde-1 as important for antiviral defense. CDE-1 is a homologue of the mammalian TUT4/7 terminal uridylyltransferases; its catalytic activity is required for its antiviral function. CDE-1 uridylates the 3' end of the OrV RNA genome and promotes its degradation, independently of the RNAi pathway. Likewise, TUT4/7 uridylate influenza A virus (IAV) mRNAs in mammalian cells. Deletion of TUT4/7 leads to increased IAV mRNA and protein levels. We have defined 3' terminal uridylation of viral RNAs as a conserved antiviral defense mechanism.

immunology↗

bioRxiv2017

Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility

Ca2+ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca2+ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca2+ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca2+ transients: short-duration \"sparkles\" and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance.

immunology↗

bioRxiv2017

Identification of Tumor-Reactive B Cells and Systemic IgG in Breast Cancer Based on Clonal Frequency in the Sentinel Lymph Node

A better understanding of antitumor immune responses is key to advancing the field of cancer immunotherapy. Endogenous immunity in cancer patients, such as circulating anticancer antibodies or tumor-reactive B cells, has been historically yet incompletely described. Here, we demonstrate that tumor-draining (sentinel) lymph node (SN) is a rich source for tumor-reactive B cells that give rise to systemic IgG anticancer antibodies circulating in the bloodstream of breast cancer patients. Using a synergistic combination of high-throughput B-cell sequencing and quantitative immunoproteomics, we describe the prospective identification of tumor-reactive SN B cells (based on clonal frequency) and also demonstrate an unequivocal link between affinity-matured expanded B-cell clones in the SN and antitumor IgG in the blood. This technology could facilitate the discovery of antitumor antibody therapeutics and conceivably identify novel tumor antigens. Lastly, these findings highlight the unique and specialized niche the SN can fill in the advancement of cancer immunotherapy.\n\nSIGNIFICANCEUsing high-throughput molecular cloning and antibody proteomics to study coordinated antitumor immunity in breast cancer patients, we simultaneously demonstrate that the sentinel lymph node is a localized source of expanded antitumor B cells undergoing affinity maturation and that their secreted antibodies are abundant as systemic IgG circulating in blood.

immunology↗

bioRxiv2017

Application of the immunoregulatory receptor LILRB1 as a novel crystallisation chaperone for class I peptide-MHC complexes

X-ray crystallographic studies of class I peptide-MHC molecules (pMHC) continue to provide important insights into immune recognition, however their success depends on generation of diffraction-quality crystals, which remains a significant challenge. While protein engineering techniques such as surface-entropy reduction and lysine methylation have proven utility in facilitating and/or improving protein crystallisation, they risk affecting the conformation and biochemistry of the class I MHC antigen binding groove. An attractive alternative is the use of noncovalent crystallisation chaperones, however these have not been developed for pMHC. Here we describe a method for promoting class I pMHC crystallisation, by exploiting its natural ligand interaction with the immunoregulatory receptor LILRB1 as a novel crystallisation chaperone. First, focussing on a model HIV-1-derived HLA-A2-restricted peptide, we determined a 2.4[A] HLA-A2/LILRB1 structure, which validated that co-crystallisation with LILRB1 does not alter conformation of the antigenic peptide. We then demonstrated that addition of LILRB1 enhanced the crystallisation of multiple pMHC complexes, and identified a generic condition for initial co-crystallisation. LILRB1 chaperone-based pMHC crystallisation enabled structure determination for class I pMHC previously intransigent to crystallisation, including both conventional and post-translationally-modified peptides, of diverse lengths. LILRB1 chaperone-mediated crystallisation should expedite molecular insights into the immunobiology of diverse immune-related diseases and immunotherapeutic strategies, particularly involving class I pMHC complexes that are challenging to crystallise. Moreover, since the LILRB1 recognition interface involves predominantly non-polymorphic regions of the MHC molecule, the approach we outline could prove applicable to a diverse range of class I pMHC.

immunology↗

bioRxiv2017

The C-terminal extension landscape of naturally presented HLA-I ligands

HLA-I molecules play a central role in antigen presentation. They typically bind 9- to 12-mer peptides and their canonical binding mode involves anchor residues at the second and last positions of their ligands. To investigate potential non-canonical binding modes we collected in-depth and accurate HLA peptidomics datasets covering 54 HLA-I alleles and developed novel algorithms to analyze these data. Our results reveal frequent (442 unique peptides) and statistically significant C-terminal extensions for at least eight alleles, including the common HLA-A03:01, HLA-A31:01 and HLA-A68:01. High resolution crystal structure of HLA-A68:01 with such a ligand uncovers structural changes taking place to accommodate C-terminal extensions and helps unraveling sequence and structural properties predictive of the presence of these extensions. Scanning viral proteomes with the new C-terminal extension motifs identifies many putative epitopes and we demonstrate direct recognition by human CD8+ T cells of a C-terminally extended epitope from cytomegalovirus.

immunology↗

bioRxiv2017

Single-cell sequencing reveals αβ chain pairing shapes the T cell repertoire

A diverse T cell repertoire is a critical component of the adaptive immune system, providing protection against invading pathogens and neoplastic changes, relying on the recognition of foreign antigens and neoantigen peptides by T cell receptors (TCRs). However, the statistical properties and function of the T cell pool in an individual, under normal physiological conditions, are poorly understood. In this study, we report a comprehensive, quantitative characterization of the T cell repertoire from over 1.9 million cells, yielding over 200,000 high quality paired {beta} sequences in 5 healthy human subjects. The dataset was obtained by leveraging recent biotechnology developments in deep RNA sequencing of lymphocytes via single-cell barcoding in emulsion. We report non-random associations and non-monogamous pairing between the and {beta} chains, lowering the theoretical diversity of the T cell repertoire, and increasing the frequency of public clones shared among individuals. T cell clone size distributions closely followed a power law, with markedly longer tails for CD8+ cytotoxic T cells than CD4+ helper T cells. Furthermore, clonality estimates based on paired chains from single T cells were lower than that from single chain data. Taken together, these results highlight the importance of sequencing {beta} pairs to accurately quantify lymphocyte receptor diversity.

immunology↗

bioRxiv2017

A Polymorphic Residue That Attenuates Interferon Lambda 4 Activity in Hominid Lineages

As antimicrobial signalling molecules, type III or lambda interferons (IFN{lambda}s) are critical for defence against infection by diverse pathogens. Counter-intuitively, expression of one member of the family, IFN{lambda}4, is associated with decreased clearance of hepatitis C virus (HCV) in the human population; by contrast, a natural in-frame nucleotide insertion that abrogates IFN{lambda}4 production improves viral clearance. To further understand how genetic variation between and within species affects IFN{lambda}4 function, we screened a panel of extant coding variants of human IFN{lambda}4 and identified three variants that substantially affect antiviral activity (P70S, L79F and K154E). The most notable variant was K154E, which enhanced in vitro activity in a range of antiviral and interferon stimulated gene (ISG) assays. This more active E154 variant of IFN{lambda}4 was found only in African Congo rainforest Pygmy hunter-gatherers. Remarkably, E154 was highly conserved as the ancestral residue in mammalian IFN{lambda}4s yet K154 is the dominant variant throughout evolution of the hominid genus Homo. Compared to chimpanzee IFN{lambda}4, the human orthologue had reduced activity due to amino acid substitution of glutamic acid with lysine at position 154. Meta-analysis of published gene expression data from humans and chimpanzees showed that this difference in activity between K154 and E154 in IFN{lambda}4 is consistent with differences in antiviral gene expression in vivo during HCV infection. Mechanistically, our data suggest that human-specific K154 likely affects IFN{lambda}4 activity by reducing secretion and potency. We postulate that evolution of an IFN{lambda}4 with attenuated activity in humans (K154) likely contributes to distinct host-specific responses to and outcomes of infection, such as HCV.

immunology↗

bioRxiv2017

B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage

B-1a cells are long-lived, self-renewing innate like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells they have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver (FL) versus bone marrow (BM) environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa (Igk) recombination at the early pro-B cell stage. As a result, B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain (SLC). This alternate pathway of development enables the production of B cells with self reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing models of B-1a cell development and explain how these cells acquire their unique properties.

immunology↗

bioRxiv2017

PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt

Phosphatidylinositol-3 kinases (PI3Ks) modulate numerous cellular functions, including growth, proliferation and survival. Dysregulation of the PI3K pathway can lead to autoimmune disease and cancer. PIK3IP1 (or Transmembrane Inhibitor of PI3K - TrIP) is a novel transmembrane regulator of PI3K. TrIP contains an extracellular kringle domain and an intracellular \"p85-like\" domain with homology to the inter-SH2 domain of the regulatory subunit of PI3K. Although TrIP has been shown to bind to the p110 catalytic subunit of PI3K in fibroblasts, the mechanism by which TrIP functions is poorly understood. We show that both the kringle and \"p85-like\" domains are necessary for TrIP inhibition of PI3K. We also demonstrate that TrIP protein is down-modulated from the surface of T cells to allow T cell activation. In addition, we present evidence that the kringle domain may modulate TrIP function by binding an as-yet-unidentified ligand. Using an inducible knockout mouse model that we developed, we show that TrIP-deficient T cells exhibit more robust T cell activation, show a preference for Th1 polarization and can mediate clearance of Listeria monocytogenes infection faster than WT mice. Thus, TrIP is an important negative regulator of T cell activation and may represent a novel target for immune modulation therapies.

immunology↗

bioRxiv2017

A modular transcriptional signature identifies phenotypic heterogeneity of human tuberculosis infection

Whole blood transcriptional signatures distinguishing active tuberculosis patients from asymptomatic latently infected individuals exist. Consensus has not been achieved regarding the optimal reduced gene sets as diagnostic biomarkers that also achieve discrimination from other diseases. Here we show a blood transcriptional signature of active tuberculosis using RNA-Seq, confirming microarray results, that discriminates active tuberculosis from latently infected and healthy individuals, validating this signature in an independent cohort. Using an advanced modular approach, we utilise information from the entire transcriptome, which includes over-abundance of type I interferon-inducible genes and under-abundance of IFNG and TBX21, to develop a signature that discriminates active tuberculosis patients from latently infected individuals, or those with acute viral and bacterial infections. We suggest methods targeting gene selection across multiple discriminant modules can improve development of diagnostic biomarkers with improved performance. Finally, utilising the modular approach we demonstrate dynamic heterogeneity in a longitudinal study of recent tuberculosis contacts.

immunology↗

bioRxiv2017

T helper cells modulate intestinal stem cell renewal and differentiation

In the small intestine, a cellular niche of diverse accessory cell types supports the rapid generation of mature epithelial cell types through self-renewal, proliferation, and differentiation of intestinal stem cells (ISCs). However, not much is known about interactions between immune cells and ISCs, and it is unclear if and how immune cell dynamics affect eventual ISC fate or the balance between self-renewal and differentiation. Here, we used single-cell RNA-seq (scRNA-Seq) of intestinal epithelial cells (IECs) to identify new mechanisms for ISC-immune cell interactions. Surprisingly, MHC class II (MHCII) is enriched in two distinct subsets of Lgr5+ crypt base columnar ISCs, which are also distinguished by higher proliferation rates. Using co-culture of T cells with intestinal organoids, cytokine stimulations, and in vivo mouse models, we confirm that CD4+ T helper (Th) cells communicate with ISCs and affect their differentiation, in a manner specific to the Th subtypes and their signature cytokines and dependent on MHCII expression by ISCs. Specific inducible knockout of MHCII in intestinal epithelial cells in mice in vivo results in expansion of the ISC pool. Mice lacking T cells have expanded ISC pools, whereas specific depletion of Treg cells in vivo results in substantial reduction of ISC numbers. Our findings show that interactions between Th cells and ISCs mediated via MHCII expressed in intestinal epithelial stem cells help orchestrate tissue-wide responses to external signals.

immunology↗

bioRxiv2017

Single cell transcriptomics of regulatory T cells reveals trajectories of tissue adaptation

Non-lymphoid tissues (NLTs) harbour a pool of adaptive immune cells, the development and phenotype of which remains largely unexplored. Here, we used single-cell RNA-seq to characterise CD4+ regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, the respective draining lymph nodes and spleen. From this data, we modelled a continuous lymphoid-to-NLT trajectory for Treg, and reconstructed the mechanisms of cell migration and NLT adaption. This revealed a shared transcriptional programme of NLT priming in both skin and colon-associated lymph nodes, followed by tissue-specific adaptation. Predicted migration kinetics were validated using a melanoma-induction model, emphasizing the relevance of key regulators and receptors, including Batf, Rora, Ccr8, Samsn1. Finally, we profiled human blood and NLT Treg and Tmem cells, identifying cross-mammalian conserved tissue signatures. In summary, we have identified molecular signals mediating NLT Treg recruitment and tissue adaptation through the combined use of computational prediction and in vivo validation.

immunology↗

bioRxiv2017

Comprehensive profiling of TCRβ repertoire in a non-model species (the bank vole) using high-throughput sequencing

In recent years, immune repertoire profiling with high-throughput sequencing (HTS) has advanced our understanding of adaptive immunity. However, fast progress in the field applied mostly to human and mouse research, with only few studies devoted to other model vertebrates. We present the first in-depth characterization of the TCR{beta} repertoire in a non-model mammal with limited genomic resources available - the bank vole (Myodes glareolus). We used 5'RACE and Illumina HTS to describe V and J segments and to qualitatively characterize preferential V-J segment usage and CDR3 length distribution. Finally, a molecular protocol integrating unique molecular identifiers was used for quantitative analysis of CDR3 repertoire with stringent error correction. We found 37 V and 11 J genes that were orthologous to mice genes. A conservative, lower bound estimation of the TCR{beta} repertoire was 1.7-2.3x105 clonotypes, and the degree of sharing of the observed repertoire between any two individuals was 3.6% of nucleotide sequences and 14.3% of amino acid sequences. Our work adds a crucial element to the immunogenetic resources available for the bank vole, an important species in ecological and evolutionary research. The workflow that we developed can be applied for immune repertoire sequencing of non-model species, including endangered vertebrates.

immunology↗

bioRxiv2017

One-step generation of monoclonal B cell receptor mice capable of class switch recombination and somatic hypermutation

We developed a method for rapid generation of B cell receptor (BCR) monoclonal mice expressing pre-rearranged Igh and Igk chains monoallelically from the Igh locus by CRISPR/Cas9 injection into fertilized oocytes. B cells from these mice undergo somatic hypermutation (SHM), class switch recombination (CSR), and affinity-based selection in germinal centers. This method combines the practicality of BCR transgenes with the ability to study Ig SHM, CSR and affinity maturation.

immunology↗