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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Necrosis triggered by Mycobacterium tuberculosis alters macrophage triglyceride metabolism and inflammatory response in a DGAT1-dependent manner

Tuberculosis granulomas represent a site of controlling bacterial dissemination at the cost of host tissue damage. Within the heterogenous array of TB granulomas, some contain triglyceride (TG) rich foamy macrophages, the etiology and function of which remains largely unexplained. Here we show that necrosis of tuberculosis lesions and M. tuberculosis (Mtb) infected macrophages elicits a bystander response of triglyceride storage. We elucidate a role for the RD1 region of mycobacterial genome to be a key player in this phenomenon. TG storage in necrosis associated foamy macrophages promoted the pro-inflammatory state of macrophages while silencing expression of diacylglycerol O-acyltransferase (DGAT1) suppressed expression of pro-inflammatory genes. Surprisingly, acute infection with Mtb led to lipolysis of host TG, rather than synthesis, suggesting mobilization of triglyceride stored within macrophage lipid droplets to be involved in the inflammatory response to infection. Our data is likely to open new avenues for management of the inflammatory response during infection.

immunology↗

bioRxiv2017

Endoplasmic reticulum stress engenders immune-resistant, latent pancreatic cancer metastases

Patients who have had their primary pancreatic ductal adenocarcinoma (PDA) surgically resected often develop metastatic disease, exemplifying the problem of latent metastases. Livers from patients and mice with PDA contained single, disseminated cancer cells (DCCs) with an unusual phenotype of being cytokeratin-19 (CK19)- and MHC class I (MHCI)-. We created a mouse model to determine how DCCs develop, their relationship to metastatic latency, and the role of immunity. Intra-portal injection of immunogenic PDA cells into pre-immunized mice seeded livers only with single, non-replicating DCCs lacking MHCI and CK19; naive recipients had macro-metastases. Transcriptomic analysis of PDA cells with the DCC phenotype demonstrated an endoplasmic reticulum (ER) stress response. Relieving ER stress with a chemical chaperone, in combination with T cell-depletion, stimulated outgrowth of macro-metastatic lesions containing PDA cells expressing MHCI and CK19. The ER stress response is the cell-autonomous reaction that enables DCCs to escape immunity and establish latent metastases.\n\nOne sentence summaryLatent pancreatic cancer metastases are created when T cells select disseminated cancer cells in which immune resistance and quiescence have been imposed by endoplasmic stress.

immunology↗

bioRxiv2017

Toxoplasma gondii microneme proteins 1 and 4 bind to Toll-like receptors 2 and 4 N-glycans triggering innate immune response

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex, secreted on the parasite surface and function to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages to produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-{gamma} in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.\n\nAUTHOR SUMMARYToxoplasmosis is caused by the protozoan Toxoplasma gondii, belonging to the Apicomplexa phylum. This phylum comprises important parasites able to infect a broad diversity of animals, including humans. A particularity of T. gondii is its ability to invade virtually any nucleated cell of all warm-blooded animals through an active process, which depends on the secretion of adhesin proteins. These proteins are discharged by specialized organelles localized in the parasite apical region, and termed micronemes and rhoptries. We show in this study that two microneme proteins from T. gondii utilize their adhesion activity to stimulate innate immunity. These microneme proteins, denoted MIC1 and MIC4, recognize specific sugars on receptors expressed on the surface of mammalian immune cells. This binding activates these innate immune cells to secrete cytokines, which promotes efficient host defense mechanisms against the parasite and regulate their pathogenesis. This activity promotes a chronic infection by controlling parasite replication during acute infection.

immunology↗

bioRxiv2017

Progressive change in killer-like receptor and GPR56 expression defines cytokine production of human CD4+ T memory cells

Memory T cells mount an accelerated response upon re-challenge but are heterogeneous in phenotype and function. Traditionally memory T cells were classified into central memory, effector memory and terminally differentiated effector memory (TEMRA) cells based on expression of CCR7 and CD45RA. Functional heterogeneity even within these subsets demonstrated the need for more suitable markers. We applied bulk and single gene expression profiling of human CD4+ memory T cells and identified surface markers, KLRB1, KLRG1, GPR56 and KLRF1, allowing classification into \"low\", \"high\" or \"exhausted\" cytokine producers. In contrast to common understanding KLRG1 expression was not associated with exhaustion and highest production of multiple cytokines was observed in KLRB1+KLRG1+GPR56+ T cells. Only additional KLRF1 expression was associated with a decline in cytokine production. The superiority of KLRF1 to define exhausted cytokine producers compared to classical TEMRA identification was best exemplified for intrahepatic T cells in patients with inflammatory liver diseases.

immunology↗

bioRxiv2017

A novel combination of cyclophosphamide and anti-ICOS mAb prevents tumor growth by affecting regulatory T cells in mice with a human immune system

Mice reconstituted with a human immune system and bearing human tumors represent a promising model for developing novel cancer immunotherapies. Here, we used mass cytometry and multi-parametric flow cytometry to characterize human leukocytes infiltrating a human breast cancer tumor model in immunocompromised NOD.SCID.{gamma}c-null mice reconstituted with a human immune system and compared it to samples of breast cancer patients. We observed highly activated human CD4+ and CD8+ T cells in the tumor, as well as minor subsets of innate immune cells in both settings. We also report that ICOS+ CD4+ regulatory T cells (Treg) were enriched in the tumor relative to the periphery in humanized mice and patients, providing a target to affect Treg and tumor growth. Indeed, administration of a neutralizing mAb to human ICOS reduced Treg proportions and numbers and improved CD4+ T cell proliferation in humanized mice. Moreover, a combination of the anti-ICOS mAb with cyclophosphamide reduced tumor growth, and that was associated with an improved CD8 to Treg ratio. However, depletion of human CD8+ T cells only marginally affected tumor control whereas depletion of murine myeloid cells abrogated the effect of the combination therapy. Altogether, our results indicate that a combination of anti-ICOS mAb and chemotherapy controls tumor growth in humanized mice and highlight the crucial implication of innate immunity in treatment efficacy, opening new perspectives for the treatment of breast cancer.\n\nOne sentence summaryICOS expressed on Tregs is a promising target to improve tumor immunity in humans\n\nAbbreviations

immunology↗

bioRxiv2017

Evaluating the Role of Cell-free Fetal DNA in Inflammation and Spontaneous Preterm Birth.

Preterm birth is the leading cause of neonatal mortality. While spontaneous preterm birth (sPTB) is the cause of over 70% of PTB, the pathogenesis behind sPTB remains unclear. Cell-free fetal DNA (cff-DNA) originates from the placenta and is increased in women who develop PTB. It has been demonstrated that fetal DNA is hypomethylated and is pro-inflammatory. The pro-inflammatory properties of placental-derived DNA, the effects of placental inflammation on the production of cff-DNA, and its significance in the pathogenesis of PTB are unknown.\n\nUsing a human placental explant model, we analysed the effect of lipopolysaccharide (LPS) stimulation on cff-DNA production, and used the cff-DNA generated by these explants to examine the methylation profile and in-vitro pro-inflammatory properties of cff-DNA. LPS caused significant production of TNF- from placental explants, but did not significantly increase the cff-DNA production. Placental-derived cff-DNA, was found to have a small proportion of unmethylated CpG motifs, but was more similar to adult DNA than to more highly unmethylated E-coli DNA. However, cff-DNA did not elicit production of inflammatory cytokines (IL-6, IL-8, TNF- and CXCL10) by peripheral blood mononuclear cells from pregnant women. Furthermore, in contrast to LPS, intra-uterine injections of mouse placental DNA did not decrease time to delivery in an in-vivo mouse PTB model compared to control animals.\n\nThis study demonstrates that placental inflammation does not increase the production of cff-DNA in placental explants, and cff-DNA alone is not sufficient to elicit an inflammatory response in human PBMC cultures ex-vivo. It also shows that mouse placental DNA does not cause PTB in-vivo. This suggests that cff-DNA might be predominantly an effect of parturition and not a principal causative agent.

immunology↗

bioRxiv2017

A novel strategy to express different antigens from one modified vaccinia Ankara vaccine vector

To enhance global control of encephalitis and hepatitis caused by rabies-(RABV), Japanese encephalitis-(JEV), hepatitis B-virus (HBV), and enterovirus 71 (EV71) novel immunisation strategies are needed. Therefore, a multipathogen modified Vaccinia Ankara vector, expressing antigens from the above pathogens, was constructed. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one the RABV-HBV pathogen sequence were generated. To ensure similar expression of the antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA. Protein expression using this co-infection model of expression was demonstrated in vitro. To investigate the co-infection model of antigen delivery in vivo, a murine immunogenicity study was performed using the MVA-RABV-HBV recombinant. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected.

immunology↗

bioRxiv2017

Aging Of Antiviral CD8+ Memory T Cells Fosters Increased Survival, Metabolic Adaptations And Lymphoid Tissue Homing

Aging of established antiviral T cell memory fosters a series of progressive adaptations that paradoxically improve rather than compromise protective CD8+T cell immunity. We now provide evidence that this gradual evolution, the pace of which is contingent on the precise context of the primary response, also impinges on the molecular mechanisms that regulate CD8+ memory T cell (CD8+TM) homeostasis. Over time, CD8+TM become more resistant to apoptosis and acquire enhanced cytokine responsiveness without adjusting their homeostatic proliferation rates; concurrent metabolic adaptations promote increased CD8+TM quiescence and fitness but also impart the re-acquisition of a partial effector-like metabolic profile; and a gradual redistribution of aging CD8+TM from blood and nonlymphoid tissues to lymphatic organs results in CD8+TM accumulations in bone marrow, splenic white pulp and particularly lymph nodes. Altogether, these data demonstrate how temporal alterations of fundamental homeostatic determinants converge to render aged CD8+TM poised for greater recall responses.\n\nABBREVIATIONST cell subsets

immunology↗

bioRxiv2017

Stage-specific ISG expression reveals functional convergence of type I and II IFNs in SIV infection

AbstractInterferons play a major role in controlling viral infections including HIV/SIV infections. Persistent up-regulation of interferon-stimulated-genes (ISGs) is associated with chronic immune activation and progression in SIV/HIV infections, but the respective contribution of different IFNs is unclear. We analyzed the expression of annotated IFN-induced genes in SIV-infected macaques to decrypt the respective roles of type-I (,{beta}) and type-II ({gamma}) IFNs. Both IFN types were induced in lymph nodes during early stage of primary infection. Induction of type-II IFN persisted during the chronic phase, in contrast to undetectable induction of type-I IFN. Interferome-based analysis of ISGs revealed that at both acute and chronic infection phases most differentially expressed ISGs were inducible by both type-I and type-II IFNs and displayed the highest increases, indicating strong convergence and synergy between type-I and type-II IFNs. The analysis of functional signatures of ISG expression revealed temporal changes in IFN expression patterns identifying phase-specific ISGs. These results suggest that IFN-{gamma} strongly contribute to shape ISG upregulation in addition to type-I IFN and may contribute to progression.

immunology↗

bioRxiv2017

Systematic identification of cancer-specific MHC-binding peptides with RAVEN

Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Recurrent somatic mutations in the exome can create highly specific neo-antigens. However, especially pediatric cancers are oligo-mutated and hardly exhibit recurrent neo-antigens. Yet, immunogenic peptides encoded by cancer-specific genes (CSGs), which are virtually not expressed in normal tissues, may enable a targeted immunotherapy of such cancers. Here, we describe an algorithm and provide a user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues), which automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from publicly available gene expression data. We applied RAVEN to a dataset assembled from more than 2,700 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on sarcomas and pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs. As a proof-of-concept we identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated by qRT-PCR in cancer cell lines and by immunohistochemistry in a comprehensive tissue-microarray comprising 412 samples. Subsequently, RAVEN identified likely immunogenic peptides encoded by these CSGs by predicting the affinity to MHCs. Putative highly affine peptides were automatically crosschecked with the UniProt protein-database to exclude sequence identity with abundantly expressed proteins. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed similar kinetics to a very immunogenic influenza control peptide.\n\nCollectively, we provide a comprehensive, exquisitely curated and validated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer entities. In addition, we developed an intuitive and freely available software to easily apply our algorithm to any gene expression dataset (https://github.com/JSGerke/RAVENsoftware). We anticipate that our peptide libraries and software constitute a rich resource to accelerate the development of novel immunotherapies.

immunology↗

bioRxiv2017

Antibodies set boundaries limiting microbial metabolite penetration and the resultant mammalian host response

Although the mammalian microbiota is well-contained within the intestine and on other body surfaces, it profoundly shapes development and metabolism of almost every host organ, presumably through pervasive microbial metabolite penetration. The challenge is that most metabolites can be of both host and microbial origin. We developed a model to distinguish between microbial and host metabolites by stable isotope tracing using fully 13C-labelled live non-replicating Escherichia coli, differentiating 12C and 13C isotopes with high-resolution mass spectrometry. Hundreds of microbial compounds penetrated across 23 host tissues and fluids after intestinal exposure: subsequent 12C host metabolome signatures included lipidemia, reduced glycolysis and inflammation. Mucosal barrier maturation with transient microbial exposure increased early clearance of penetrant bacterial metabolites from the small intestine into the urine, independently of antibody induction. Induced antibodies curtailed microbial metabolite exposure at the intestinal surface, by accelerating intestinal bacterial transit into the colon where metabolite transport mechanisms are limiting.

immunology↗

bioRxiv2017

Sepsis: Partial least squares structural equation modelling (PLS-SEM) suggests a critical role for anti-inflammatory responses in clinical severity

BackgorundDespite major advances in medicine, Sepsis remains one of the major killers in critical care wards around the world. For several years it was widely believed that an early pro-inflammatory host response is followed by an overwhelming anti-inflammatory phase. The hypo-inflammatory status, termed as Compensatory anti-inflammatory response syndrome (CARS), was proposed to be the primary cause of sepsis-associated mortality. However, this paradiam changed in recent years since there was little evidence to support the linear model of host response and pathogenesis in sepsis. Currently held view is that both inflammatory and anti-inflammatory host responses are stimulated in an overlapping manner. In this study a robust statistical model to study the complex interplay of host cytokines in human sepsis has been developed to evaluate host responses in sepsis that contribute significantly to clinical pathology.\n\nMethodsTwentyseven cytokines/ chemokines were quantified in 139 sepsis patients and multivariate analysis of variance (MANOVA) was performed to assess differences in host responses in different categories of clinical severity. Partial least squares regression based structural equation modelling (PLS-SEM) was used to assess interactions between different groups of cytokines and their contribution to clinical pathology. An array of 23 cytokines was analysed in a mouse model of endotoxemia and a similar mathematical model was constructed.\n\nResultsThe results of MANOVA demonstrated the ability of combined cytokine response to discriminate sepsis patients according to clinical severity or outcome. Structural equation modelling revealed strong positive association between inflammatory and anti-inflammatory cytokines. In human sepsis, anti-inflammatory cytokines emerged as a significant entity associated with clinical severity as assessed by APACHE II scores.\n\nConclusionPLS-SEM modeling of cytokine responses and APACHE II score in human sepsis revealed that anti-inflammatory molecules contribute significantly towards clinical severity. More critically, the model offers emperical evidence for failures of clinical trials conducted during the last two decades in which antagonists of inflammatory host responses for human Sepsis were used for sepsis. The model also provides credence to the notion that inflammatory and anti-inflammatory host responses occur concurrently in both experimental endotoxemia and in human sepsis.

immunology↗

bioRxiv2017

Kinetics of HIV-Specific CTL Responses Plays a Minimal Role in Determining HIV Escape Dynamics

Cytotoxic T lymphocytes (CTLs) have been suggested to play an important role in controlling human immunodeficiency virus (HIV-1 or simply HIV) infection. HIV, due to its high mutation rate, can evade recognition of T cell responses variants that can not be recognized by HIV-specific CTLs. Although HIV escape from CTL responses has been well documented, factors contributing to the timing and the rate of viral escape from T cells have not been fully elucidated. Fitness costs associated with escape and magnitude of the epitope-specific T cell response are generally considered to be the key in determining timing of HIV escape. Several previous analyses generally ignored the kinetics of T cell responses in predicting viral escape by either considering constant or maximal T cell response; several studies also considered escape from different T cell responses to be independent. Here we focus our analysis on data from two patients from a recent study with relatively frequent measurements of both virus sequences and HIV-specific T cell response to determine impact of CTL kinetics on viral escape. In contrast with our expectation we found that including temporal dynamics of epitope-specific T cell response did not improve the quality of fit of different models to escape data. We also found that for well sampled escape data the estimates of the model parameters including T cell killing efficacy did not strongly depend on the underlying model for escapes: models assuming independent, sequential, or concurrent escapes from multiple CTL responses gave similar estimates for CTL killing efficacy. Interestingly, the model assuming sequential escapes (i.e., escapes occurring along a defined pathway) was unable to accurately describe data on escapes occurring rapidly within a short-time window, suggesting that some of model assumptions must be violated for such escapes. Our results thus suggest that the current sparse measurements of temporal CTL dynamics in blood bear little quantitative information to improve predictions of HIV escape kinetics. More frequent measurements using more sensitive techniques and sampling in secondary lymphoid tissues may allow to better understand whether and how CTL kinetics impacts viral escape.

immunology↗

bioRxiv2017

Improved prediction of Bovine Leucocyte Antigens (BoLA) presented ligands by use of MS eluted ligands and in-vitro binding data; impact for the identification T cell epitopes

Peptide binding to MHC class I molecules is the single most selective step in antigen presentation and the strongest single correlate to peptide cellular immunogenicity. The cost of experimentally characterizing the rules of peptide presentation for a given MHC-I molecule is extensive, and predictors of peptide-MHC interactions constitute an attractive alternative.\n\nRecently, an increasing amount of MHC presented peptides identified by mass spectrometry (MS ligands) has been published. Handling and interpretation of MS ligand data is in general challenging due to the poly-specificity nature of the data. We here outline a general pipeline for dealing with this challenge, and accurately annotate ligands to the relevant MHC-I molecule they were eluted from by use of GibbsClustering and binding motif information inferred from in-silico models. We illustrate the approach here in the context of MHCI molecules (BoLA) of cattle. Next, we demonstrate how such annotated BoLA MS ligand data can readily be integrated with in-vitro binding affinity data in a prediction model with very high and unprecedented performance for identification of BoLA-I restricted T cell epitopes.\n\nThe approach has here been applied to the BoLA-I system, but the pipeline is readily applicable to MHC systems in other species.

immunology↗

bioRxiv2017

Method for identification of condition-associated public antigen receptor sequences

Diverse repertoires of hypervariable immunoglobulin receptors (TCR and BCR) recognize antigens in the adaptive immune system. The development of immunoglobulin receptor repertoire sequencing methods makes it possible to perform repertoire-wide disease association studies of antigen receptor sequences. We developed a statistical framework for associating receptors to disease from only a small cohort of patients, with no need for a control cohort. Our method successfully identifies previously validated Cytomegalovirus and type 1 diabetes responsive receptors.

immunology↗

bioRxiv2017

Development of a high-throughput bead based assay system to measure HIV-1 specific immune signatures in clinical samples

The monitoring and assessment of a broadly neutralizing antibody (bnAb) based HIV-1 vaccine require detailed measurements of HIV-1 binding antibody responses to support the detection of correlates of protection. Here we describe the development of a flexible, high-throughput microsphere based multiplex assay system that allows monitoring complex binding antibody signatures. Studying a panel of 13 HIV-1 antigens in a parallel assessment of different IgG subclasses (IgG1, IgG2 and IgG3) we demonstrate the potential of our strategy. The technical advances we describe include means to improve antigen reactivity using directed neutravidin-biotin immobilization of antigens and biotin saturation to reduce background. A particular emphasis of our study was to provide tools for the assessment of reproducibility and stability of the assay system and strategies to control for variations allowing the application in high-throughput assays, where reliability of single measurements needs to be guaranteed.

immunology↗

bioRxiv2017

An unbiased reconstruction of the T helper cell type 2 differentiation network

T helper type 2 (Th2) cells are important regulators of our adaptive immune response, particularly the response against parasites, and have relevance for auto-immunity as well as tumour progression. This classic T helper type has been studied intensively, but not systematically. Using newly developed, genome-wide retroviral CRISPR knock-out (KO) technology, combined with RNA-seq, ATAC-seq and ChIP-seq, we have dissected the regulatory circuitry governing differentiation in these cells. During Th2 activation/differentiation approximately 4000 genes are perturbed, with at least 200 genes specifically associated with the Th2 program in mouse and human. We confirm previously known Th2 driver genes and have discovered several novel genes, including transcription factors, metabolic genes and potential receptors/cytokines, critical for Th2 function. Our study provides an atlas for, but not limited to, the Th2 regulatory network, pinpointing the key players of Th2 differentiation.

immunology↗

bioRxiv2017

IRF4 haploinsufficiency in a family with Whipples disease

The pathogenesis of Whipples disease (WD) remains largely unknown, as WD strikes only a very small minority of the individuals infected with Tropheryma whipplei (Tw). Asymptomatic carriage of Tw is less rare. We studied a large multiplex French kindred, containing four otherwise healthy WD patients (mean age: 76.7 years) and five healthy carriers of Tw (mean age: 55 years). We used a strategy combining genome-wide linkage analysis and whole-exome sequencing to test the hypothesis that WD is inherited in an autosomal dominant (AD) manner, with age-dependent incomplete penetrance. WD was linked to 12 genomic regions covering 27 megabases in the four patients. These regions contained only one very rare non-synonymous variation: the R98W variant of IRF4. The five Tw carriers were heterozygous for R98W. Interferon regulatory factor 4 (IRF4) is a transcription factor with pleiotropic roles in immunity. We showed that R98W was a loss-of-function allele, like only five other exceedingly rare IRF4 alleles of a total of 39 rare and common non-synonymous alleles tested. Furthermore, heterozygosity for R98W led to a distinctive pattern of transcription in leukocytes following stimulation with BCG or Tw. Finally, we found that IRF4 had evolved under purifying selection and that R98W was not dominant-negative, suggesting that the IRF4 deficiency in this kindred was due to haploinsufficiency. Overall, haploinsufficiency at the IRF4 locus selectively underlies WD in this multiplex kindred. This deficiency displays AD inheritance with incomplete penetrance, and chronic carriage probably precedes WD by several decades in Tw-infected heterozygotes.

immunology↗