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Results for “Immunology”

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bioRxiv2018

Hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing

Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains uncovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cells (IECs). Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion, gut inflammation, and enterocyte pyroptosis in orally-infected mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.\n\nSignificanceSensing of lipopolysaccharide (LPS) in the cytosol triggers non-canonical inflammasome-mediated innate responses. Recent work revealed that bacterial outer membrane vesicles (OMVs) enables LPS to access the cytosol for extracellular bacteria. However, since intracellular OMVs are generally constrained in endosomes, how OMV-derived LPS gain access to the cytosol remains unknown. Here, we reported that hemolysin largely bound with OMVs and entered cells through dynamin-dependent endocytosis. Intracellular hemolysin significantly impaired OMVs-constrained vacuole integrity and increased OMV-derived LPS exposure to the cytosolic sensor, which promoted non-canonical inflammasome activation and restricted bacterial gut infections. This work reveals the role of hemolysin in promoting non-canonical inflammasome activation and alerting host immune recognition, which provides insights into the more sophisticated biological functions of hemolysin upon infection.

immunology↗

bioRxiv2018

Opposing roles of endosomal innate immunity proteins IFITM3 and TLR7 in human metapneumovirus infection

Human metapneumovirus (hMPV) utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosome membrane. Whether cellular factors restrict or enhance either entry pathway is largely unknown. We found that the interferon-induced transmembrane protein 3 (IFITM3) inhibits hMPV infection to an extent similar to endocytosis-inhibiting drugs, and an IFITM3 variant that accumulates at the plasma membrane in addition to its endosome localization provided increased virus restriction. Mechanistically, IFITM3 blocks hMPV F protein-mediated membrane fusion, and inhibition of infection was reversed by the membrane destabilizing drug amphotericin B. Conversely, we unexpectedly found that infection by some hMPV strains is enhanced by Toll-like receptor 7 (TLR7), an endosomal protein, suggesting that cellular entry via endocytosis may be particularly advantageous for hMPV despite eventual restriction of this pathway upon induction of IFITM3. Overall, our results identify IFITM3 and TLR7 as endosomal factors differentially regulating hMPV infection.

immunology↗

bioRxiv2018

IL-33 regulates gene expression in intestinal epithelial cells independently of its nuclear localization

IL-33 is a cytokine found in the extracellular space (mature IL-33) or in the cell nucleus (full-length IL-33). Nuclear accumulation of IL-33 has been reported in intestinal epithelial cells (IEC) during intestinal inflammation and cancer, but a functional role for this nuclear form remains unclear. To study the role of nuclear IL-33 in IEC, we generated transgenic mice expressing full-length IL-33 in the intestinal epithelium (Vfl33 mice). Expression of full-length IL-33 in the epithelium resulted in accumulation of IL-33 protein in the nucleus and secretion of IL-33. Over-expression of full-length IL-33 by IEC did not promote gut inflammation, but induced expression of genes in the IEC and lamina propria lymphocytes (LPL) that correlated negatively with genes expressed in inflammatory bowel diseases (IBD). Because the IL-33 receptor ST2 is expressed by IEC, there was the potential that both the mature and full-length forms could mediate this effect. To specifically interrogate the transcriptional role of nuclear IL-33, we intercrossed the Vfl33 mice with ST2-deficient mice. ST2 deficiency completely abrogated the transcriptional effects elicited by IL-33 expression, suggesting that the transcriptional effects of IL-33 on IEC are mediated by its mature, not its nuclear form.\n\nHighlightsO_LIExpression of full-length IL-33 in the epithelium resulted in accumulation of IL-33 protein in the nucleus and secretion of IL-33.\nC_LIO_LIFull-length IL-33 induced differential gene expression in IEC and LPL that was negatively associated with intestinal inflammatory diseases\nC_LIO_LIIL-33 regulated gene expression in IEC via its extracellular (mature) form not via its nuclearform.\nC_LI

immunology↗

bioRxiv2018

NK cells inhibit Plasmodium falciparum growth in red blood cells via antibody dependent cellular cytotoxicity

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite-host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.

immunology↗

bioRxiv2018

Antibody-based vaccine for TB: pre-clinical validation in horse foals challenged with the TB-related pathogen Rhodococcus equi

Immune correlates for protection against Mycobacterium tuberculosis (Mtb) infection and other intracellular pathogens are largely undetermined. Whether there is a role for antibody-mediated immunity is controversial. Rhodococcus equi is an intracellular pathogen causing severe pneumonia in young horse foals, eliciting a disease with many similarities to TB including intracellular residence, formation of granulomas and induction of severe respiratory distress. No purified vaccine antigens exist for R. equi or Mtb infections. Both express the microbial surface polysaccharide antigen poly-N-acetyl glucosamine (PNAG). Vaccination of pregnant mares with a synthetic PNAG oligosaccharide conjugated to tetanus toxoid elicited antibody that transferred to foals via colostrum and provided nearly complete protection against R. equi pneumonia in a randomized, controlled, blinded challenge trial. Infusion of PNAG-hyperimmune plasma protected 100% of foals against R. equi pneumonia. Vaccination induced opsonic antibodies that killed extracellular and intracellular R. equi and other intracellular pathogens. Killing of intracellular organisms was dependent on antibody recognition of surface expression of PNAG on infected macrophages, complement deposition and PMN-assisted lysis of infected macrophages. Protection also correlated with PBMC release of interferon-{gamma} in response to PNAG. Antibody-mediated opsonic killing and interferon-{gamma} release in response to PNAG may protect against disease caused by intracellular bacterial pathogens.

immunology↗

bioRxiv2018

T-cell receptor αβ chain pairing is associated with CD4+ and CD8+ lineage specification

While a highly diverse T-cell receptor (TCR) repertoire is the hallmark of a healthy adaptive immune system, relatively little is understood about how the CD4+ and CD8+ TCR repertoires differ from one another. We here utilize high-throughput single T-cell sequencing to obtain approximately 100,000 TCR {beta} chain pairs from human subjects, stratified into CD4+ and CD8+ lineages. We reveal that substantial information about T-cell lineage is encoded by V{beta} gene pairs and, to a lesser extent, by several other TCR features such as CDR3 length and charge. We further find that the strength of association between the {beta} chain and T-cell lineage is surprisingly weak, similar in strength to that of the chain. Using machine learning classifiers to predict T-cell lineage from TCR features, we demon-strate that {beta} chain pairs are significantly more informative than individual chains alone. These findings provide unprecedented insight into the CD4+ and CD8+ TCR repertoires and highlight the importance of {beta} chain pairing in TCR function and specificity.

immunology↗

bioRxiv2018

The expression of class II major histocompatibility molecules on breast tumors delays T cell exhaustion, expands the T cell repertoire and slows tumor growth

The expression of major histocompatibility complex II (MHCII) on tumor cells correlates with survival and responsiveness to immunotherapy. However, the mechanisms underlying these observations are poorly defined. Using a murine breast tumor line, we tested how MHCII expression affected anti-tumor immunity. We found that MHCII-expressing tumors grew more slowly than controls and recruited more functional CD4+ and CD8+ T cells. Additionally, MHCII-expressing tumors contained more TCR clonotypes expanded to a larger degree than control tumors. Functional CD8+ T cells in tumors depended on CD4+ T cells. However, both CD4+ and CD8+ T cells eventually became exhausted, even in MHCII-expressing tumors. PD1 blockade had no impact on tumor growth, potentially because tumor cells poorly expressed PD-L1. These results suggest tumor cell expression of MHCII facilitates the local activation of CD4+ T cells and indirectly helps the activation and expansion of CD8+ T cells, but by itself, cannot prevent T cell exhaustion.\n\nPrecisThe expression of MHCII on tumor cells augments CD4 and CD8 T cell responses, expands the TCR repertoire and delays exhaustion. Hence, strategies to induce MHCII expression may be a powerful adjuvant to immunotherapeutic regimens of solid tumors.

immunology↗

bioRxiv2018

Toward a model-free feedback control synthesis for treating acute inflammation -- A mathematical perspective

An effective and patient-specific feedback control synthesis for inflammation resolution is still an ongoing research area. A strategy consisting of manipulating a pro and anti-inflammatory mediator is considered here as used in some promising model-based control studies. These earlier studies, unfortunately, suffer from the difficultly of calibration due to the heterogeneity of individual patient responses even under similar initial conditions. We exploit a new model-free control approach and its corresponding \"intelligent\" controllers for this biomedical problem. A crucial feature of the proposed control problem is as follows: the two most important outputs which must be driven to their respective desired states are sensorless. This difficulty is overcome by assigning suitable reference trajectories to the other two outputs that do have sensors. A mathematical model, via a system of ordinary differential equations, is nevertheless employed as a \"virtual\" patient for in silico testing. We display several simulation results with respect to the most varied situations, which highlight the effectiveness of our viewpoint.

immunology↗

bioRxiv2018

Characterisation of subpopulations of chicken mononuclear phagocytes that express TIM4 and the macrophage colony-stimulating factor receptor (CSF1R)

The phosphatidylserine receptor, TIM4, encoded by TIMD4, mediates the phagocytic uptake of apoptotic cells. We applied anti-chicken TIM4 monoclonal antibodies, in combination with CSF1R reporter transgenes to dissect the function of TIM4 in chick (Gallus gallus). During development in ovo, TIM4 was present on the large majority of macrophages but expression became more heterogeneous post-hatch. Blood monocytes expressed KUL01, class II MHC and CSF1R-mApple uniformly. Around 50% of monocytes were positive for surface TIM4. They also expressed many other monocyte-specific transcripts at a higher level than TIM4- monocytes. In liver, highly-phagocytic TIM4hi cells shared many transcripts with mammalian Kupffer cells and were associated with uptake of apoptotic cells. Although they expressed CSF1R mRNA, Kupffer cells did not express the CSF1R-mApple transgene, suggesting that additional CSF1R transcriptional regulatory elements are required by these cells. By contrast, CSF1R-mApple was detected in liver TIM4lo and TIM4- cells which were not phagocytic and were more abundant than Kupffer cells. These cells expressed CSF1R, alongside high levels of FLT3, MHCII, XCR1 and other markers associated with conventional dendritic cells (cDC) in mice. In bursa, TIM4 was present on the cell surface of two populations. Like Kupffer cells, bursal TIM4hi phagocytes co-expressed many receptors involved in apoptotic cell recognition. TIM4lo cells appear to be a sub-population of bursal B cells. In overview, TIM4 is associated with phagocytes that eliminate apoptotic cells in the chick. In the liver, TIM4 and CSF1R reporters distinguished Kupffer cells from an abundant population of DC-like cells.

immunology↗

bioRxiv2018

Epigenomic and functional dynamics of human bone marrow myeloid differentiation to mature blood neutrophils

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic and functional overview of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of epigenetic and transcriptome dynamics reveals an enforced regulation of differentiation, through cellular functions such as: release of proteases, respiratory burst, cell cycle regulation and apoptosis. We observe an early establishment of the cytotoxic capability, whilst the signaling components that activate antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the epigenetic landscape orchestrate the daily production of large number of neutrophils required for innate host defense and provide a comprehensive overview of the epigenomes of differentiating human neutrophils.\n\nKey pointsO_LIDynamic acetylation enforces human neutrophil progenitor differentiation.\nC_LI\n\nO_LINeutrophils cytotoxic capability is established early at the (pro)myelocyte stage.\nC_LI\n\nO_LICoordinated signaling component expression prevents unwanted toxic effects to the bone marrow niche.\nC_LI

immunology↗

bioRxiv2018

Genetic variation/evolution and differential host responses resulting from in-patient adaptation of Mycobacterium avium

Mycobacterium avium (Mav) complex (MAC) are characterized as non-tuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact on the infection outcome. The current study aimed to characterize the genomic changes within Mav isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed field gel electrophoresis and whole genome sequencing was performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Patients (4, 9 and 13) who contributed more than two isolates were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single nucleotide polymorphisms (SNPs), suggesting that Mav accumulates mutations at high rates during persistent infections. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency towards increased persistence and down-regulation of inflammatory cytokines by host-adapted Mav strains. The study revealed rapid genetic evolution of Mav in chronically infected patients accompanied with change in virulence properties of the sequential mycobacterial isolates.\n\nIMPORTANCEMAC are a group of opportunistic pathogens, consisting of Mav and M. intracellulare species. Mav is found ubiquitously in the environment. In Mav infected individuals, Mav has been known to persist for long periods of time, and anti-mycobacterial drugs are unable to effectively clear the infection. The continued presence of the bacteria, could be attributed to either a single persistent strain or reinfection with the same or different strain. We examined sequential isolates collected over time from Mav infected individuals and observed that most patients carried the same strain overtime and were not re infected. We observed high rates of mutation within the serial isolates, accompanied with changes in virulence properties. In the light of increase in incidence of MAC related infections, this study highlights the possibility that host adapted Mav undergo genetic modifications to cope with the host environment and thereby persisting longer.

immunology↗

bioRxiv2018

High prevalence of S. pyogenes Cas9-specific T cell sensitization within the adult human population - A balanced effector/regulatory T cell response

Summary ParagraphThe field of gene therapy has been galvanized by the discovery of the highly efficient and adaptable site-specific nuclease system CRISPR/Cas9 from bacteria.1,2 Immunity against therapeutic gene vectors or gene-modifying cargo nullifies the effect of a possible curative treatment and may pose significant safety issues.3-5 Immunocompetent mice treated with CRISPR/Cas9-encoding vectors exhibit humoral and cellular immune responses against the Cas9 protein, that impact the efficacy of treatment and can cause tissue damage.5,6 Most applications aim to temporarily express the Cas9 nuclease in or deliver the protein directly into the target cell. Thus, a putative humoral antibody response may be negligible.5 However, intracellular protein degradation processes lead to peptide presentation of Cas9 fragments on the cellular surface of gene-edited cells that may be recognized by T cells. While a primary T cell response could be prevented or delayed, a pre-existing memory would have major impact. Here, we show the presence of a ubiquitous memory/effector T cell response directed towards the most popular Cas9 homolog from Streptococcus pyogenes (SpCas9) within healthy human subjects. We have characterized SpCas9-reactive memory/effector T cells (TEFF) within the CD4/CD8 compartments for multi-effector potency and lineage determination. Intriguingly, SpCas9-specific regulatory T cells (TREG) profoundly contribute to the pre-existing SpCas9-directed T cell immunity. The frequency of SpCas9-reactive TREG cells inversely correlates with the magnitude of the respective TEFF response. SpCas9-specific TREG may be harnessed to ensure the success of SpCas9-mediated gene therapy by combating undesired TEFF response in vivo. Furthermore, the equilibrium of Cas9-specific TEFF and TREG cells may have greater importance in Streptococcus pyogenes-associated diseases. Our results shed light on the T cell mediated immunity towards the much-praised gene scissor SpCas9 and offer a possible solution to overcome the problem of pre-existing immunity.

immunology↗

bioRxiv2018

Peptide-binding groove contraction linked to the lack of T-cell response: Using complex structure and energy to identify neoantigens

Using personalized peptide vaccines (PPVs) to target tumor-specific non-self antigens (neoantigens) is a promising approach to cancer treatment. However, the development of PPVs is hindered by the challenge of identifying tumor-specific neoantigens, in part because current in silico methods for identifying such neoantigens have limited effectiveness. Here we report the results of molecular dynamics simulations of 12 oligopeptides bound with a human leukocyte antigen (HLA), revealing a previously unrecognized association between the inability of an oligopeptide to elicit a T-cell response and the contraction of the peptide-binding groove upon binding of the oligopeptide to the HLA. Our conformational analysis showed that this association was due to incompatibility at the interface between the contracted groove and its {beta}-T-cell antigen receptor (TCR). This structural demonstration that having the capability to bind HLA does not guarantee immunogenicity prompted us to develop an atom-based method [Formula] to predict immunogenicity through using the structure and energy of a peptide*HLA complex to assess the propensity of the complex for forming a ternary complex with its TCR. In predicting the immunogenicities of the 12 oligopeptides, [Formula] achieved a 100% success rate compared with success rates of 25-50% for 11 publicly available residue-based methods including NetMHC-4.0. While further validation and refinements of [Formula] are required, our results suggest a need to develop in silico methods that assess peptide characteristics beyond their capability to form stable binary complexes with HLAs to help remove hurdles in using the patient tumor DNA information to develop PPVs for personalized cancer immunotherapy.

immunology↗

bioRxiv2018

An inter-chromosomal transcription hub activates the unfolded protein response in plasma cells

Previous studies have indicated that the transcription signature of antibody-secreting cells is closely associated with the induction of the unfolded protein response pathway (UPR). Here we have used genome-wide and single cell analyses to examine the folding patterns of plasma cell genomes. We found that plasma cells adopt a cartwheel configuration and undergo large-scale changes in chromatin folding at genomic regions associated with a plasma cell specific transcription signature. During plasma cell differentiation, Blimp1 assembles into an inter-chromosomal transcription hub with genes associated with the UPR, biosynthesis of the endoplasmic reticulum (ER) as well as a cluster of genes linked with Alzheimers disease. We suggest that the assembly of the Blimp1-UPR-ER transcription hub permits the coordinate activation of a wide spectrum of genes that collectively establish plasma cell identity.

immunology↗

bioRxiv2018

CCL3 promotes germinal center B cells sampling by follicular regulatory T cells and ensures optimal humoral response

Previous studies and our findings suggest upregulated expression of proinflammatory chemokines CCL3/4 in germinal center (GC) centrocytes. However, the role of CCL3/4 for centrocyte interactions with follicular T cells and regulation of humoral immunity is poorly understood. We found that CCL3 promotes chemotaxis of Tfr cells ex vivo. In vivo CCL3 is not required for Tfr cells recruitment of into the GC light zone. However, B cells-intrinsic production of CCL3 promotes their direct interactions and negative regulation by follicular regulatory T cells (Tfr) within GCs.

immunology↗

bioRxiv2018

Transient μm scale protein accumulation at the center of the T cell antigen presenting cell interface drives efficient IL-2 secretion

Supramolecular signaling assemblies are of interest for their unique signaling properties. A {micro}m scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5{micro}m3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates.

immunology↗

bioRxiv2018

Maternal tamoxifen treatment expands the macrophage population of early mouse embryos.

Several different transgenic tamoxifen-inducible cre reporter lines have been used to analyse the contribution of embryonic precursors to the development of the mononuclear phagocyte system in mice. Here we show that tamoxifen treatment of the mother at 8.5dpc with doses commonly-used in lineage trace studies produces a 4-5-fold expansion of the embryonic leukocyte populations by 10.5dpc, detected in whole mounts of embryos using a Csf1r reporter gene or separately by expression of Csf1r, Itgam (CD11b), Adgre1 (F4/80) or Ptprc (CD45) mRNA. These findings indicate that tamoxifen cannot be considered a neutral agonist in macrophage lineage trace studies.\n\nSummary sentenceTreatment of pregnant mice with tamoxifen in early gestation produces a large expansion of the embryonic macrophage population.

immunology↗

bioRxiv2018

powerTCR: a model-based approach to comparative analysis of the clone size distribution of the T cell receptor repertoire

Sequencing of the T cell receptor repertoire is a powerful tool for deeper study of immune response, but the unique structure of this type of data makes its meaningful quantification challenging. We introduce a new method, the Gamma-GPD spliced threshold model, to address this difficulty. This biologically interpretable model captures the distribution of the TCR repertoire, demonstrates stability across varying sequencing depths, and permits comparative analysis across any number of sampled individuals. We apply our method to several datasets and obtain insights regarding the differentiating features in the T cell receptor repertoire among sampled individuals across conditions. We have implemented our method in the open-source R package powerTCR.\n\nAuthor summaryA more detailed understanding of the immune response can unlock critical information concerning diagnosis and treatment of disease. Here, in particular, we study T cells through T cell receptor sequencing, as T cells play a vital role in immune response. One important feature of T cell receptor sequencing data is the frequencies of each receptor in a given sample. These frequencies harbor global information about the landscape of the immune response. We introduce a flexible method that extracts this information by modeling the distribution of these frequencies, and show that it can be used to quantify differences in samples from individuals of different biological conditions.

immunology↗