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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↗

bioRxiv2017

PET studies of the glial cell marker TSPO in psychosis patients - a meta-analysis using individual participant data

BackgroundAccumulating evidence suggests that the immune system may be an important target for new treatment approaches in schizophrenia. Positron emission tomography (PET) and radioligands binding to the translocator protein (TSPO), which is expressed in glial cells in brain including immune cells, represents a potential method for patient stratification and treatment monitoring. This study examined if patients with first episode psychosis and schizophrenia had altered TSPO levels as compared to healthy control subjects.\n\nMethodsPubMed was searched for studies comparing patients with psychosis to healthy controls using second-generation TSPO radioligands. The outcome measure was distribution volume (VT), an index of TSPO levels, in frontal cortex (FC), temporal cortex (TC) and hippocampus (HIP). Bayes factors (BF) were applied to examine the relative support for higher, lower or no-change of TSPO levels in patients as compared to healthy controls.\n\nResultsFive studies, with 75 patients with first-episode psychosis or schizophrenia and 77 healthy control subjects were included. BF showed strong support for lower patient VT relative to no-change (all BF>32) or relative to an increase (all BF>422) in all brain regions. From the posterior distributions, mean patient-control differences in standardized VT values were -0.48 for FC (95% credible interval (CredInt)=-0.88 to -0.09), -0.47 for TC (CredInt=-0.87 to -0.07) and -0.63 for HIP (CredInt=-1.00 to -0.25).\n\nDiscussionThe observed reduction of TPSO in compared to control subjects in patients may correspond to altered function or lower density of brain immune cells. Future studies should focus on investigating the underlying biological mechanisms and their relevance for treatment.

immunology↗

bioRxiv2017

Evolutionary plasticity in the innate immune function of Akirin

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NF{kappa}B. Although nematodes lack NF{kappa}B, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways.

immunology↗

bioRxiv2017

Matrix-corrected mass spectrometry enables sensitive detection of food allergens and reveals widespread soy contamination of processed foods

The frequent use of precautionary food allergen labeling statements such as \"may contain\" poses challenges to allergic individuals who rely on such labeling to determine whether a food is safe to consume. We developed a multiplexed liquid chromatography-mass spectrometry assay targeting 14 common allergens in order to survey how frequently these precautionary statements indicate allergen contamination and to assess whether variations in precautionary phrasing affect the likelihood of allergen contamination. A survey of 84 foods revealed how scheduled multiple reaction monitoring (MRM) transition interference derived from complex and heterogeneous sample matrices hinders sensitive analyte detection. As a solution, we developed MAtrix-Dependent Interference Correction (MADIC), an approach to sensitively detect trace peptide quantities through interference identification and stringent peptide quality control criteria. Applying this method, we find frequent contamination of soy in breads and corn flour, and observe additional instances of food contamination with tree nuts, wheat, milk, and egg. In some of these cases, the food had no precautionary labeling for the offending allergen. We also find that only 10% of warning labels are indicative of contamination, and that products with \"same facility\" precautionary labeling are not necessarily less likely to contain trace amounts of allergens than products labeled \"may contain.\"

immunology↗

bioRxiv2017

Legionella protection and vaccination mediated by Mucosal Associated Invariant T (MAIT) cells

Mucosal associated invariant T (MAIT) cells recognize conserved microbial metabolites from riboflavin synthesis. Striking evolutionary conservation and pulmonary abundance implicate them in antibacterial host defense, yet their roles in protection against clinically significant pathogens are unknown. Murine Legionella infection induced MR1-dependent MAIT cell activation and rapid pulmonary accumulation of MAIT cells associated with immune protection detectable in fully immunocompetent host animals. MAIT cell protection was more evident in mice lacking CD4+ cells, whilst profoundly immunodeficient RAG2-/-{gamma}C-/- mice were substantially rescued from uniformly lethal Legionella infection by adoptively-transferred MAIT cells. This protection was dependent on MR1, IFN-{gamma} and GM-CSF, but not IL-17, TNF- or perforin. Protection was enhanced and observed earlier post-infection in mice that were Ag-primed to boost MAIT cells before infection. Our findings define a significant role for MAIT cells in protection against a major human pathogen and indicate a potential role for vaccination to enhance MAIT cell immunity.

immunology↗

bioRxiv2017

High-resolution repertoire analysis of Tfr and Tfh cells reveals unexpectedly high diversities indicating a bystander activation of follicular T cells

T follicular helper (Tfh) and regulatory (Tfr) cells regulate B cell activation and ultimately antibody production. While concordant results show that Tfh cells are specific for the immunizing antigens, limited and even controversial results have been reported regarding the specificity of Tfr cells. Here we used high-throughput T cell receptor (TCR) sequencing to address this issue. We observed that although the Tfh- and Tfr-cell repertoires are less diverse than those of effector (Teff) and regulatory T (Treg) cells, they still represent thousands of clonotypes after immunization with a single antigen. T-cell receptor beta variable (TRBV) gene usage distinguishes both follicular T cells (Tfol) from non-Tfol cells, as well as helper (Teff and Tfh) vs. regulatory (Treg and Tfr) cells. Analysis of the sharing of clonotypes between samples revealed that a specific response to the immunizing antigen can only be detected in Tfh cells immunized with a non-self-antigen and Tfr cells immunized with a self-antigen. Finally, the Tfr TCR repertoire is more similar to that of Tregs than to that of Tfh or Teff cells. Altogether, our results highlight a bystander Tfol-cell activation during antigenic response in the germinal centres and support the Treg cell origin of Tfr cells.\n\nSignificance StatementFollicular helper T (Tfh) cells promote high-affinity antibody production by B cells while follicular regulatory T (Tfr) cells represses it. The question of the specificity of follicular T (Tfol) cells is of utmost importance in the understanding of the antibody response specificity and our work is the first to analysed the global Tfol TCR repertoire in wild type mice. This allowed us not only to portray the overall global structure of these repertoires, but also to substantiate the fact that Tfr cells respond to self-antigen while Tfh cells respond to non self-antigen, a still controversial issue. Importantly, our work revealed an unexpected bystander activation of Tfol cells. We think and discuss that it has a general significance in immune responses and possibly immunopathologies.

immunology↗