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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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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↗

bioRxiv2017

MicroRNA-21 Regulates Metabolic Adaptation of Pathogenic TH17 cells and Controls Autoimmune Inflammation

TH17 cells exhibit great heterogeneity and variable functional states in vivo. However, metabolic reprogramming of TH17 cells in vivo and its regulation during autoimmunity and host defence is unknown. Here we report that TH17 cells derived in vivo show discrete metabolic states. Metabolic states of TH17 cells in vivo were controlled at the epigenetic level, with conserved regulatory region of key metabolic regulators show distinct chromatin accessibility as demonstrated by chromatin landscape profiling. TGF-{beta}1 signaling was further shown to be crucial for remodeling of TH17 cell chromatin states, Ahr and miR-21 were identified as essential metabolic regulators for TH17 cells. Understanding metabolic reprogramming of TH17 cells in vivo may therefore provide more defined therapeutic intervention to TH17 cell mediated autoimmune diseases and insights into TH17 cell mediated host defence.

immunology↗

bioRxiv2017

Reduced cellular diversity and an altered basal progenitor cell state inform epithelial barrier dysfunction in human type 2 immunity

Tissue barrier dysfunction is a poorly defined feature hypothesized to drive chronic human inflammatory disease1,2. The epithelium of the upper respiratory tract represents one such barrier, responsible for separating inhaled agents, such as pathogens and allergens, from the underlying submucosa. Specialized epithelial subsets--including secretory, glandular, and ciliated cells--differentiate from basal progenitors to collectively realize this role3-5. Allergic inflammation in the upper airway barrier can develop from persistent activation of Type 2 immunity (T2I), resulting in the disease spectrum known as chronic rhinosinusitis (CRS), ranging from rhinitis to severe nasal polyps6-8. Whether recently identified epithelial progenitor subsets, and their differentiation trajectory, contribute to the clinical presentation and barrier dysfunction in T2I-mediated disease in humans remains unexplored3,9,10. Profiling twelve primary human samples spanning the range of clinical severity with the Seq-Well platform11 for massively-parallel single-cell RNA-sequencing (scRNA-seq), we report the first single-cell transcriptomes for human respiratory epithelial cell subsets, immune cells, and parenchymal cells (18,036 total cells) from a T2I inflammatory disease, and map key mediators. We find striking differences between non-polyp and polyp tissues within the epithelial compartments of human T2I cellular ecosystems. More specifically, across 10,383 epithelial cells, we identify a global reduction in epithelial diversity in polyps characterized by basal cell hyperplasia, a concomitant decrease in glandular and ciliated cells, and phenotypic shifts in secretory cell function. We validate these findings through flow cytometry, histology, and bulk tissue RNA-seq of an independent cohort. Furthermore, we detect an aberrant basal progenitor differentiation trajectory in polyps, and uncover cell-intrinsic and extrinsic factors that may lock polyp basal cells into an uncommitted state. Overall, our data define severe T2I barrier dysfunction as a reduction in epithelial diversity, characterized by profound functional shifts stemming from basal cell defects, and nominate a cellular mechanism for the persistence and chronicity of severe human respiratory disease.

immunology↗

bioRxiv2017

The CD11a and EPCR marker combination simplifies and improves the purification of mouse hematopoietic stem cells

Hematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish it from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a, and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, EPCR, can be used to effectively identify and purify HSCs. We introduce a new two-color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologists toolkit improves the purity of and simplifies isolation of HSCs.\n\nSignificance StatementThe study of hematopoietic stem cells (HSCs) and their purification for transplantation requires a panel of surface markers that can be used to distinguish HSCs from other cell types. The number of markers necessary to identify HSCs continues to grow, making it increasingly difficult to identify HSCs by flow cytometry. In this study, we identified a combination of two surface markers, CD11a and EPCR, to enrich for HSCs in the mouse bone marrow without the need for additional markers. This simplified panel could aid HSC research by reducing the number of markers necessary to identify and isolate HSCs.

immunology↗

bioRxiv2017

Potent and Selective Antitumor Activity of a T-Cell Engaging Bispecific Antibody Targeting a Membrane-Proximal Epitope of ROR1

T-cell engaging bispecific antibodies present a promising strategy for cancer immunotherapy and numerous bispecific formats have been developed for retargeting cytolytic T cells toward tumor cells. To explore the therapeutic utility of T-cell engaging bispecific antibodies targeting the receptor tyrosine kinase ROR1, which is expressed by tumor cells of various hematologic and solid malignancies, we used a bispecific ROR1 x CD3 scFv-Fc format based on a heterodimeric and aglycosylated Fc domain designed for extended circulatory half-life and diminished systemic T-cell activation. A diverse panel of ROR1-targeting scFv derived from immune and naive rabbit antibody repertoires was compared in this bispecific format for target-dependent T-cell recruitment and activation. A ROR1-targeting scFv with a membrane-proximal epitope, R11, revealed potent and selective antitumor activity in vitro and in vivo and emerged as a prime candidate for further preclinical and clinical studies. To elucidate the precise location and engagement of this membrane-proximal epitope, which is conserved between human and mouse ROR1, the three-dimensional structure of scFv R11 in complex with the kringle domain of ROR1 was determined by X-ray crystallography at 1.6-[A] resolution.

immunology↗

bioRxiv2017

Peptide Terminus Tilting: an Unusual conformational transition of MHC Class I Revealed by Molecular Dynamics Simulations

A conventional picture for major histocompatibility complex class I (MHCI) antigen presentation is that the terminal anchor residues of the antigenic peptide bind to the pockets at the bottom of the MHC cleft, leaving the central peptide residues exposed for T cell antigen receptor (TCR) recognition. However, in the present study, we show that in canonical or accelerated molecular dynamics (MD) simulations, the peptide terminus in some immunodominant peptide-MHCI (pMHCI) complexes can detach from their binding pockets and stretch outside the MHC cleft. These pMHCI complexes include the complex of the H-2Kb and the lymphocytic choriomeningitis virus (LCMV) gp33 peptide, and the complex of the HLA-A*0201 and the influenza A virus M1 peptide. The detached peptide terminus becomes the most prominent spot at the pMHC interface, and so can serves as a novel TCR recognition target. Thus, peptide terminus detaching may be a novel mechanism for MHC antigen presentation.

immunology↗

bioRxiv2017

TGF-β1 enhances mouse mast cell release of IL-6 and IL-13

For immune cells, TGF-{beta}1 can enhance or repress effector functions. Here, we characterize the effects of TGF-{beta}1 on IgE-mediated activation of primary murine mast cells derived from hematopoietic stem cells (BMMC). We also investigated potential interaction between TGF-{beta}1 and stem-cell factor (SCF). Resting IL-6 production was increased with TGF-{beta}1 but significance was lost following BMMC activation via IgE receptor (Fc{varepsilon}RI) crosslinking. SCF also enhanced resting levels of IL-6, but there was no difference from control once Fc{varepsilon}RI was engaged. SCF had no effect on IL-13 production; however, TGF-{beta}1 treatment enhanced release of IL-13 upon Fc{varepsilon}RI activation. Lastly, percent colocalization of SCF receptor (CD117) and Fc{varepsilon}RI were unaffected by TGF-{beta}1 treatment. These data reveal a novel positive effect of soluble TGF-{beta}1 on mast cell activation.

immunology↗

bioRxiv2017

HIV-1 unmasks the plasticity of innate lymphoid cells

HIV-1-infected people who take medications that suppress viremia, preserve CD4+ T cells, and prevent AIDS, have chronic inflammation with increased cardiovascular mortality. To investigate the etiology of this inflammation, the effect of HIV-1 on innate lymphoid cells (ILCs) and NK cells was examined. Homeostatic ILCs in blood and intestine were depleted permanently. NK cells were skewed towards a memory subset. Cytokines that are elevated during HIV-1 infection reproduced both abnormalities ex vivo. Pseudotime analysis of single NK cell transcriptomes revealed a developmental trajectory towards a subset with expression profile, chromatin state, and biological function like memory T lymphocytes. Expression of TCF7, a WNT transcription factor, increased over the course of the trajectory. TCF7 disruption, or WNT inhibition, prevented memory NK cell induction by inflammatory cytokines. These results demonstrate that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt homeostatic ILCs and cause developmental shift towards TCF7+ memory NK cells.\n\nHighlightsO_LIHIV-1 infection depletes homeostatic ILCs in blood and intestine and shifts NK cells towards a memory cell phenotype, irrespective of viremia or CD4 count\nC_LIO_LIInflammatory cytokines recapitulate ILC and NK cell abnormalities ex vivo\nC_LIO_LITCF7 expression correlates with a developmental trajectory that culminates in memory NK cells\nC_LIO_LITCF7/WNT signaling is required for establishment of memory NK cells\nC_LI\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=172 HEIGHT=200 SRC=\"FIGDIR/small/221010v3_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (32K):\norg.highwire.dtl.DTLVardef@ae82a7org.highwire.dtl.DTLVardef@103b2b4org.highwire.dtl.DTLVardef@1c9585aorg.highwire.dtl.DTLVardef@1c7bb2d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

immunology↗

bioRxiv2017

Time intervals in sequence sampling, not data modifications, have a major impact on estimates of HIV escape rates

The ability of HIV to avoid recognition by humoral and cellular immunity (viral escape) is well documented but the strength of the immune response needed to cause such a viral escape remains poorly quantified. Several previous studies observed a more rapid escape of HIV from CD8 T cell responses in the acute phase of infection as compared to the chronic infection. With the help of simple mathematical models the rate of HIV escape was estimated and results were interpreted to suggest that CD8 T cell responses causing escape in acute HIV infection may be more efficient at killing virus-infected cells than responses that cause escape in chronic infection, or alternatively, early escapes occur in epitopes mutations in which there is minimal fitness cost to the virus. These conclusions, however, were challenged on several grounds, including linkage and interference of multiple escape mutations due to a low population size and because of potential issues associated with modifying the data to estimate escape rates. Here we use a parametric resampling method which does not require data modification to show that previous results on the decline of the viral escape rate with time since infection remain unchanged. However, using this method we also show that estimates of the escape rate are highly sensitive to the time interval between measurements with longer intervals biasing estimates of the escape rate downwards. Our results thus suggest that data modifications for early and late escapes were not the primary reason for the observed decline in the escape rate with time since infection. However, longer sampling periods for escapes in chronic infection strongly influence estimates of the escape rate. More frequent sampling of viral sequences in the chronic infection may improve our understanding of factors influencing the rate of HIV escape from CD8 T cell responses.

immunology↗

bioRxiv2017

Single-Cell Immune Map of Breast Carcinoma Reveals Diverse Phenotypic States Driven by the Tumor Microenvironment

Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We created an immune map of breast cancer using single-cell RNA-seq data from 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph node. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous tumor-specific phenotypic expansions driven by environmental cues. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer, with important implications for characterizing tumor-infiltrating immune cells.

immunology↗

bioRxiv2017

Cytomegalovirus infection is a risk factor for TB disease in Infants

Immune activation is associated with increased risk of tuberculosis (TB) disease in infants. We performed a case-control analysis to identify drivers of immune activation and disease risk. Among 49 infants who developed TB disease over the first two years of life, and 129 matched controls who remained healthy, we found the cytomegalovirus (CMV) stimulated IFN-{gamma} response at age 4-6 months to be associated with CD8+ T-cell activation (Spearmans rho, p=6x10-8). A CMV specific IFN-{gamma} response was also associated with increased risk of developing TB disease (Conditional Logistic Regression, p=0.043, OR 2.2, 95% CI 1.02-4.83), and shorter time to TB diagnosis (Log Rank Mantel-Cox p=0.037). CMV positive infants who developed TB disease had lower expression of natural killer cell associated gene signatures and a lower frequency of CD3-CD4-CD8-lymphocytes. We identified transcriptional signatures predictive of risk of TB disease among CMV ELISpot positive (AUROC 0.98, accuracy 92.57%) and negative (AUROC 0.9, accuracy 79.3%) infants; the CMV negative signature validated in an independent infant study (AUROC 0.71, accuracy 63.9%). Understanding and controlling the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants.

immunology↗

bioRxiv2017

Previously Hidden Dynamics at the TCR-pMHC Interface Revealed

A structural characterization of the interaction between T cell receptors (TCR) and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the X-ray data. We found that in a large number of systems ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and discriminating between induced fit and conformational selection models of TCR binding. We show that the currently widespread practise of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from X-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data, but also provides previously missing information concerning the dynamics of existing characterized TCR-pMHC interactions.

immunology↗

bioRxiv2017

Cyclin-Dependent Kinase Activity is Required for Type I Interferon Production

Recognition of nucleic acids results in the production of type I interferons (IFN), which activate the JAK/STAT pathway and promote the expression of IFN-stimulated genes (ISG). In a search for modulators of this pathway, we discovered a previously unknown requirement for cyclin-dependent kinases (CDK) in the production of type I IFN following nucleic acid sensing and virus infection. Inhibition of CDK activity or knockdown of CDK levels leads to a striking block in STAT activation and ISG expression. CDKs are not required for the initial nucleic acid sensing leading to IFN-{beta} mRNA induction, nor for the response to exogenous IFN-/{beta}, but are critical for IFN-{beta} release into culture supernatants, suggesting a post-transcriptional role for CDKs in type I IFN production. In the absence of CDK activity, we demonstrate a translational block specific for IFN-{beta}, in which IFN-{beta} mRNA is removed from the actively translating polysomes, while the distribution of other cellular mRNAs or global translation rates are unaffected. Our findings reveal a critical role for CDKs in the translation of IFN-{beta}.

immunology↗

bioRxiv2017

A single cycle influenza virus coated in H7 hemagglutinin provides heterotypic protection and neutralising antibody responses to both glycoproteins.

A non-replicating form of pseudotyped influenza virus, inactivated by suppression of the hemagglutinin signal sequence (S-FLU), can act as a broadly protective vaccine. S-FLU can infect for a single round only, and induces heterotypic protection predominantly through activation of cross-reactive T cells in the lung. Unlike the licensed live attenuated virus, it cannot reassort a pandemic HA into seasonal influenza. Here we present data on four new forms of S-FLU coated with the H7 hemagglutinins from A/Anhui/1/2013 and A/Shanghai/1/2013, H7N9 viruses that emerged recently in China, and A/Netherlands/219/2003 and A/New York/107/2003. We show that vaccination in the lung induced a strong local CD8 T cell response and protected against heterosubtypic X31 (H3N2) virus and highly virulent PR8 (H1N1), but not influenza B virus. Lung vaccination also induced a strong neutralising antibody response to the encoded neuraminidase. If given at higher dose in the periphery, H7 S-FLU induced a specific neutralising antibody response to H7 hemagglutinin coating the particle. Polyvalent vaccination with mixed H7 S-FLU induced a broadly neutralising antibody response to all four H7 strains. S-FLU is a versatile vaccine candidate that could be rapidly mobilized ahead of a new pandemic threat.

immunology↗

bioRxiv2017

Antigen-induced but not innate memory CD8 T cells express NKG2D and are recruited to the lung parenchyma upon viral infection

The pool of memory-phenotype CD8 T cells is composed of antigen-induced (AI) and cytokine-induced innate (IN) cells. IN have been described as having similar properties to AI memory cells. However, we found that pathogen-induced AI memory cells can be distinguished from naturally-generated IN memory cells by surface expression of NKG2D. Using this marker, we described the increased functionalities of AI and IN memory CD8 T cells compared to naive cells, as shown by comprehensive analysis of cytokine secretion and gene expression. However, AI differed from IN memory CD8 T cells by their capacity to migrate to the lung parenchyma upon inflammation or infection, a process dependent on their expression of ITGA1/CD49a and ITGA4/CD49d integrins.

immunology↗

bioRxiv2017

A Computational Model for the Evaluation of Complement System Regulation under Homeostasis, Disease, and Drug Intervention

HighlightsO_LIComputational model describing dynamics of complement system activation pathways\nC_LIO_LIComplement dysregulation leads to deviation from homeostasis and to inflammatory diseases\nC_LIO_LIModel identifies biomarkers to quantify the effects of complement dysregulation\nC_LIO_LIKnown drugs restore impaired dynamics of complement biomarkers under dysregulation\nC_LIO_LIDisease-specific models are suitable for diagnosis and patient-specific drug treatment\nC_LI\n\nAbstractThe complement system is a part of innate immunity that rapidly removes invading pathogens and impaired host-cells. Activation of the complement system is balanced under homeostasis by regulators that protect healthy host-cells. Impairment of complement regulators tilts the balance, favoring activation and propagation that leads to inflammatory diseases. The most potent regulator of the complement system is Factor H (FH), and its impairment induces improper complement activation that leads to inflammatory diseases, such as atypical hemolytic uremic syndrome and age related macular degeneration. To understand the dynamics involved in the pivotal balance between activation and regulation, we have developed a comprehensive computational model of the alternative and classical pathways of the complement system. The model is composed of 290 ordinary differential equations with 142 kinetic parameters that describe the state of complement system under homeostasis and disorder through FH impairment. We have evaluated the state of the system by generating concentration-time profiles for the biomarkers C3, C3a-desArg, C5, C5a-desArg, Factor B (FB), Ba, Bb, and fC5b-9 that are influenced by complement dysregulation. We show that FH-mediated disorder induces substantial levels of complement activation compared to homeostasis, by generating reduced levels of C3 and FB, and to a lesser extent C5, and elevated levels of C3a-desArg, Ba, Bb, C5a-desArg, and fC5b-9. These trends are consistent with clinically observed biomarkers associated with complement-mediated diseases. Furthermore, we introduced therapy states by modeling known drugs of the complement system, a compstatin variant (C3 inhibitor) and eculizumab (a C5 inhibitor). Compstatin demonstrates strong restorative effects for early-stage biomarkers, such as C3a-desArg, FB, Ba, and Bb, and milder restorative effects for late-stage biomarkers, such as C5a-desArg and fC5b-9, whereas eculizumab has strong restorative effects on late-stage biomarkers, and negligible effects on early-stage biomarkers. These results highlight the need for patient-specific therapies that target early complement activation at the C3 level, or late-stage propagation of the terminal cascade at the C5 level, depending on the specific FH-mediated disease and the manifestations of a patients genetic profile in complement regulatory function.

immunology↗

bioRxiv2017

Zika virus infection preferentially counterbalances human peripheral monocyte and/or NK-cell activity

Zika virus (ZIKV) has re-emerged in the population and caused unprecedented global outbreaks. Here, the transcriptomic consequences of ZIKV infection were studied systematically firstly in human peripheral blood CD14+ monocytes and monocyte-derived macrophages with high density RNA-sequencing. Analyses of the ZIKV genome revealed that the virus underwent genetic diversification and differential mRNA abundance was found in host cells during infection. Notably, there was a significant change in the cellular response with crosstalk between monocytes and natural killer (NK) cells as one of the highly identified pathway. Immune-phenotyping of peripheral blood from ZIKV-infected patients further confirmed the activation of NK cells during acute infection. ZIKV infection in peripheral blood cells isolated from healthy donors led to the induction of IFN{gamma} and CD107a -- two key markers of NK-cell function. Depletion of CD14+ monocytes from peripheral blood resulted in a reduction of these markers and reduced priming of NK cells during infection. This was complemented by the immunoproteomic changes observed. Mechanistically, ZIKV infection preferentially counterbalances monocyte and/or NK-cell activity, with implications for targeted cytokine immunotherapies.

immunology↗

bioRxiv2017

Deferiprone, an iron chelator, is preventive and therapeutic in experimental crescentic glomerulonephritis

Crescentic glomerulonephritis represents the most severe form of antibody-mediated glomerulonephritis. It is an important cause of renal dysfunction worldwide and there is a need for more effective treatment. Deferiprone, an orally active iron chelator, is widely used in patients with thalassemia. Here we present the preventive and therapeutic effects of deferiprone in experimental crescentic glomerulonephritis. Nephrotoxic nephritis was induced in Wistar Kyoto rats, and preventive treatment with deferiprone substantially lowered glomerular crescent formation by 84%, with 70% reduction in proteinuria. In established glomerulonephritis, deferiprone treatment effectively halted glomerular inflammation, reversed progression of proteinuria, and prevented deterioration of renal function. Deferiprone reduced glomerular inflammatory cell proliferation in vivo. It was internalised by monocyte/macrophages and inhibited their proliferation in vitro, without showing cellular toxicity. Interestingly, deferiprone showed a neutralizing effect on superoxide anions, and prevented the expression of monocyte chemoattractant protein-1 and matrix metalloproteinase 9, 12 and 14, by primary macrophages. These results suggest that deferiprone partly exerts its renal protective effect through inhibition of monocyte/macrophage proliferation and function by iron-chelating and anti-oxidant properties, respectively. We conclude that deferiprone is an effective treatment in a severe and reproducible model of antibody-mediated glomerular inflammation that resembles human crescentic glomerulonephritis, indicating its therapeutic potential.

immunology↗