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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 757 records · Page 42Linked to original sources

bioRxiv2018

Polyfunctional tumor-reactive T cells are effectively expanded from non-small cell lung cancers, and correlate with an immune-engaged T cell profile in situ

Non-small cell lung cancer (NSCLC) is the second most prevalent type of cancer. With the current treatment regimens, the mortality rate remains high. Therefore, better therapeutic approaches are necessary. NSCLCs generally possess many genetic mutations and are well infiltrated by T cells (TIL), making TIL therapy an attractive option. Here we show that T cells from treatment naive, stage I-IVa NSCLC tumors can effectively be isolated and expanded, with similar efficiency as from normal lung tissue. Importantly, 76% (13/17) of tested TIL products isolates from NSCLC lesions exhibited clear reactivity against primary tumor digests, with 0.5%-30% of T cells producing the inflammatory cytokine Interferon (IFN)-{gamma}. Both CD4+ and CD8+ T cells displayed tumor reactivity. The cytokine production correlated well with CD137 and CD40L expression. Furthermore, almost half (7/17) of the TIL products contained polyfunctional T cells that produced Tumor Necrosis Factor (TNF)- and/or IL-2 in addition to IFN-{gamma}, a hallmark of effective immune responses. Tumor-reactivity in the TIL products correlated with high percentages of CD103+CD69+CD8+ T cell infiltrates in the tumor lesions, with PD-1hiCD4+ T cells, and with FoxP3+CD25+CD4+ regulatory T cell infiltrates, suggesting that the composition of T cell infiltrates may predict the level of tumor reactivity. In conclusion, the effective generation of tumor-reactive and polyfunctional TIL products implies that TIL therapy will be a successful treatment regimen for NSCLC patients.

immunology↗

bioRxiv2018

High diversity, turnover, and structural constraints characterize TCR α and β repertoire selection

Recognition modes of individual T-cell receptors (TCR) are well studied, but how TCR repertoires are selected during acute through persistent human virus infections is less clear. Here, we show that persistent EBV-specific clonotypes account for only 9% of unique clonotypes but are highly expanded in acute infectious mononucleosis, and have distinct antigen-specific public features that drive selection into convalescence. The other 91% of highly diverse unique clonotypes disappear and are replaced in convalescence by equally diverse \"de-novo\" clonotypes. These broad fluctuating repertoires lend plasticity to antigen recognition and potentially protect against T-cell clonal loss and viral escape.

immunology↗

bioRxiv2018

The N-end rule E3 ligase UBR2 activates Nlrp1b inflammasomes

Innate immunity relies on the formation of different inflammasomes to initiate immune responses. The recognition of diverse infection and other danger signals by innate immune receptors trigger caspase-1 activation that induces pyroptosis. Anthrax lethal factor (LF) is a secreted bacterial protease that known to potently activate Nlrp1b inflammasomes in mouse macrophages, but the molecular mechanism underlying LF-induced Nlrp1b activation remains unknown. We here carried out both a mouse genome-wide siRNA screen and a CRISPR/Cas9 knockout screen seeking to identify genes that participate in Nlrp1b activation triggered by LF treatment. We found that the N-end rule pathway E3 ligase UBR2 is required for Nlrp1b activation and a ubiquitin conjugating E2 enzyme E2O is also involved in this process via its physically interaction with UBR2. We show that LF triggers activation of Nlrp1b by initiating the degradation of the N-terminal fragment of Nlrp1b itself that produced via an auto-cleavage process. This study deepens our understanding of innate immunity defense against bacterial infection by elucidating the functional role of UBR2-mediated N-end rule pathway in LF-induced Nlrp1b activation.

immunology↗

bioRxiv2018

Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen

Throughout the last decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g. respiratory syncytial virus (RSV), influenza, dengue and others) have resisted vaccine development efforts, largely due to the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B-cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the Palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies.

immunology↗

bioRxiv2018

A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy

Tumors can evade natural killer (NK) cells by activating inhibitory pathways. We therefore have developed a highly efficient CRISPR/Cas9-based method for editing the genome of peripheral blood human NK cells (PB-NKs) to knock out ADAM17 and PD1 or knock-in genes using recombinant AAV6. Our method allows editing of PB-NKs at efficiencies reaching 90%, equivalent to methods reported for primary human T cells. Moreover, we demonstrate that ADAM17 and PD1 KO PB-NKs have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Our platform represents a feasible method for generating engineered NK cells as a universal therapeutic for cancer immunotherapy.

immunology↗

bioRxiv2018

Immune regeneration in irradiated mice is not impaired by the absence of DPP9 enzymatic activity

The ubiquitous intracellular protease dipeptidyl peptidase 9 (DPP9) has roles in antigen presentation and B cell signaling. To investigate the importance of DPP9 in immune regeneration, primary and secondary chimeric mice were created in irradiated recipients using fetal liver cells and adult bone marrow cells, respectively, using wild-type (WT) and DPP9 gene-knockin (DPP9S729A) enzyme-inactive mice. Immune cell reconstitution was assessed at 6 and 16 weeks post-transplant. Primary chimeric mice successfully regenerated neutrophils, natural killer, T and B cells, irrespective of donor cell genotype. There were no significant differences in total myeloid cell or neutrophil numbers between DPP9-WT and DPP9S729A-reconstituted mice. There were fewer B cells in the DPP9S729A-origin compared to DPP9 WT-origin mice in primary chimeric mice. In secondary chimeric mice, cells of DPP9S729A-origin cells displayed enhanced engraftment compared to WT. However, we observed no differences in myeloid or lymphoid lineage reconstitution between WT and DPP9S729A donors, indicating that hematopoietic stem cell (HSC) engraftment and self-renewal is not diminished by the absence of DPP9 enzymatic activity. This is the first report on transplantation of bone marrow cells that lack DPP9 enzymatic activity.

immunology↗

bioRxiv2018

Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor

The pivotal task of the immune system is to distinguish between self and foreign antigens. The kinetic proofreading model (KPR) proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the ligand-TCR interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B to selectively control the dynamics of ligand binding to the TCR by light. Combining experiments with mathematical modeling we find that the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating the KPR hypothesis.\n\nOne Sentence SummaryThe half-life of the ligand-T cell receptor complex determines T cell activation.

immunology↗

bioRxiv2018

Type I Interferon regulates IgE-mediated enhancement of rhinovirus-induced Th2 priming by primary human monocytes

Rhinovirus infections are linked to the development and exacerbation of allergic diseases including allergic asthma. IgE, another contributor to atopic disease pathogenesis, has been shown to regulate dendritic cell antiviral functions and influence T cell priming by monocytes. We previously demonstrated that IgE-mediated stimulation of monocytes alters multiple cellular functions including cytokine secretion, phagocytosis, and influenza-induced Th1 priming. In this study, we investigate the effects of IgE-mediated allergic stimulation on monocyte-driven, RV-induced T cell priming utilizing primary human monocyte-T cell co-cultures. We demonstrate that IgE crosslinking of RV-exposed monocytes enhances monocyte-driven Th2 priming. This increase in RV-induced Th2 differentiation was regulated by IgE-mediated inhibition of type I interferon and induction of IL-10. These findings suggest an additional mechanism by which two clinically significant risk factors for allergic disease exacerbations - IgE-mediated stimulation and rhinovirus infection, may synergistically promote Th2 differentiation and allergic inflammation.

immunology↗

bioRxiv2018

Expression of a recombinant high affinity IgG Fc receptor by engineered NK cells as a docking platform for therapeutic mAbs to target cancer cells

Anti-tumor mAbs are the most widely used and characterized cancer immunotherapy agents. Despite having a significant impact on some malignancies, most cancer patients respond poorly or develop resistance to this therapy. A known mechanism of action of these therapeutic mAbs is antibody-dependent cell-mediated cytotoxicity (ADCC), which is a primarily effector function of NK cells. CD16A on human NK cells has an exclusive role in binding to tumor-bound IgG antibodies. Though CD16A is a potent activating receptor, it is a low affinity Fc{gamma}R and its cell surface levels can be rapidly downregulated by a proteolytic process involving ADAM17 upon NK cell activation, which are likely to limit the efficacy of tumor-targeting therapeutic mAbs in the tumor environment. We sought to enhance NK cell binding to anti-tumor mAbs by engineering these cells with a recombinant Fc{gamma}R consisting of the extracellular region of CD64, the highest affinity IgG Fc receptor expressed by leukocytes, and the transmembrane and cytoplasmic regions of CD16A. This novel recombinant Fc{gamma}R (CD64/16A) was expressed in the human NK cell line NK92 and in induced pluripotent stem cells from which primary NK cells were derived. CD64/16A also lacked the ADAM17 cleavage region in CD16A and it was not rapidly downregulated in expression following NK cell activation during ADCC. CD64/16A on NK cells facilitated conjugation to antibody-treated tumor cells, ADCC, and cytokine production, demonstrating functional activity by its two components. Unlike NK cells expressing CD16A, CD64/16A captured soluble therapeutic mAbs and the modified NK cells mediated tumor cell killing. Hence, CD64/16A could potentially be used as a docking platform on engineered NK cells for therapeutic mAbs and IgG Fc chimeric proteins, allowing for switchable targeting elements, and a novel cancer cellular therapy.

immunology↗

bioRxiv2018

Light-based tuning of ligand half-life supports kinetic proofreading model of T cell activation

T cells are thought to discriminate stimulatory versus non-stimulatory ligands by converting small changes in ligand binding half-life to large changes in cell activation. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, most notably the stability of the receptor-ligand interaction under load. Here we develop an optogenetic approach to specifically tune the binding half-life of a light-responsive ligand to a chimeric antigen receptor without changing other binding parameters. By simultaneously manipulating binding half-life while controlling for receptor occupancy, we find that signaling is strongly gated by ligand binding half-life. Our results provide direct evidence of kinetic proofreading in ligand discrimination by T cells.\n\nOne Sentence SummaryDirect control of ligand binding half-life with light shows that lifetime, not occupancy, dominates T cell activation.

immunology↗

bioRxiv2018

Endogenous retroviruses transcriptional modulation after severe infection, trauma and burn.

Although human endogenous retroviruses (HERVs) expression is a growing subject of interest, no study focused before on specific endogenous retroviruses loci activation in severely injured patients. Yet, HERV reactivation is observed in immunity compromised settings like some cancers and auto-immune diseases. Our objective was to assess the transcriptional modulation of HERVs in burn, trauma and septic shock patients. We analyzed HERV transcriptome with microarray data from whole blood samples of a burn cohort (n=30), a trauma cohort (n=105) and 2 septic shock cohorts (n=28, n=51), and healthy volunteers (HV, n=60). We described expression of the 337 probesets targeting HERV from U133 plus 2.0 microarray in each dataset and then we compared HERVs transcriptional modulation of patients compared to healthy volunteers. Although all 4 cohorts contained very severe patients, the majority of the 337 HERVs was not expressed (around 74% in mean). Each cohort had differentially expressed probesets in patients compared to HV (from 19 to 46). Strikingly, 5 HERVs were in common in all types of severely injured patients, with 4 being up-modulated in patients. We highlighted co-expressed profiles between HERV and nearby gene as well as autonomous HERV expression. We suggest an inflammatory-specific HERV transcriptional response, and importantly, we introduce that the HERVs close to immunity-related genes might have a role on its expression.

immunology↗

bioRxiv2018

A novel approach reveals that HLA class 1 single antigen bead-signatures provide a means of high-accuracy pre-transplant risk assessment of acute cellular rejection

Acute cellular rejection (ACR) is associated with complications after kidney transplantation, such as graft dysfunction and graft loss. Early risk assessment is therefore critical for the improvement of transplantation outcomes. In this work, we retrospectively analyzed a pre-transplant HLA antigen bead assay data set that was acquired by the e:KID consortium as part of a systems medicine approach. The data set included single antigen bead (SAB) reactivity profiles of 52 low-risk graft recipients (negative complement dependent cytotoxicity crossmatch, PRA<30%) who showed detectable pre-transplant anti-HLA 1 antibodies. To assess whether the reactivity profiles provide a means for ACR risk assessment, we established a novel approach which differs from standard approaches in two aspects: the use of quantitative continuous data and the use of a multiparameter classification method. Remarkably, it achieved significant prediction of the 38 graft recipients who experienced ACR with a balanced accuracy of 82.7% (sensitivity=76.5%, specificity= 88.9%). The resultant classifier achieved one of the highest prediction accuracies in the literature for pre-transplant risk assessment of ACR. Importantly, it can facilitate risk assessment in non-sensitized patients who lack donor-specific antibodies. As the classifier is based on continuous data and includes weak signals, our results emphasize that not only strong but also weak binding interactions of antibodies and HLA 1 antigens contain predictive information.

immunology↗

bioRxiv2018

Innate Immune Priming by cGAS as a Preparatory Countermeasure Against RNA Virus Infection

The detection of nucleic acids by pattern recognition receptors is an ancient and conserved component of the innate immune system. Notably, RNA virus genomes are sensed by mammalian cytosolic RIG-I-like receptors, thereby activating interferon-stimulated gene (ISG) expression to restrict viral replication. However, recent evidence indicates that the cGAS-STING DNA sensing pathway also protects against RNA viruses. So far, the mechanisms responsible for DNA sensing of RNA viruses, which replicate without known DNA intermediates, remain unclear. By using cGAS gene knockout and reconstitution in human and mouse cell cultures, we discovered that DNA sensing and cGAMP synthase activities are required for cGAS-mediated restriction of vesicular stomatitis virus and Sindbis virus. The level of cGAMP produced in response to RNA virus infection was below the threshold of detection, suggesting that only transient and/or low levels of cGAMP are produced during RNA virus infections. To clarify the DNA ligands that activate cGAS activity, we confirmed that cGAS binds mitochondrial DNA in the cytosol of both uninfected and infected cells; however, the amount of cGAS-associated mitochondrial DNA did not change in response to virus infection. Rather, a variety of pre-existing cytosolic DNAs, including mitochondrial DNA and endogenous cDNAs, may serve as stimuli for basal cGAS activation. Importantly, cGAS knockout and reconstitution experiments demonstrated that cGAS drives low-level ISG expression at steady state. We propose that cGAS-STING restricts RNA viruses by promoting a preparatory immune activation state within cells, likely primed by endogenous cellular DNA ligands.\n\nImportanceMany medically important RNA viruses are restricted by the cGAS-STING DNA-sensing pathway of innate immune activation. Since these viruses replicate without DNA intermediates, it is unclear what DNA ligand(s) are responsible for triggering this pathway. We show here that cGASs DNA binding and signaling activities are required for RNA virus restriction, similar to the mechanisms by which it restricts DNA viruses. Furthermore, we confirmed that cGAS continuously binds host DNA, which was unaffected by RNA virus infection. Finally, cGAS expression correlated with the low-level expression of interferon-stimulated genes in uninfected cells, both in vitro and in vivo. We propose that cGAS-mediated sensing of endogenous DNA ligands contributes to RNA virus restriction by establishing a baseline of innate immune activation.

immunology↗

bioRxiv2018

Intestinal Serum Amyloid A suppresses systemic neutrophil activation and bactericidal activity in response to microbiota colonization

The intestinal microbiota influence diverse aspects of host physiology, including the development and function of myeloid lineages. Numerous host and microbial factors are known to poise neutrophils and other granulocytes for response to pathogens and danger signals, yet the mechanisms by which the intestinal microbiota regulate this process are largely unknown. Using gnotobiotic zebrafish, we identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization. Saa is a conserved secreted protein produced in the intestine and liver with described effects on neutrophils in vitro, however its in vivo functions are poorly defined. We engineered saa mutant zebrafish to test requirements for Saa on innate immunity in vivo. Zebrafish mutant for saa displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. At baseline, saa mutants exhibited moderate neutrophilia and altered neutrophil tissue distribution. Molecular and functional analyses of isolated neutrophils revealed that Saa suppresses expression of pro-inflammatory mRNAs and bactericidal activity. Saas effects on neutrophils depends on microbiota colonization, suggesting this protein mediates the microbiotas influence on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we generated transgenic zebrafish over-expressing saa in the intestine. Transgenic intestinal saa expression was sufficient to partially complement the neutrophil phenotypes in saa mutants. These results indicate Saa produced by the intestine in response to microbiota serves as a systemic signal to neutrophils to restrict aberrant activation, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to wounds.

immunology↗

bioRxiv2018

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor binding head domains

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy to decipher the mechanism of action of a potent HA head-directed monoclonal antibody bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, pre-fusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the pre-fusion conformation. The \"breathing\" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.\n\nAuthor SummaryA transiently exposed epitope on influenza H7 hemagglutinin represents a new target for neutralizing antibodies.

immunology↗

bioRxiv2018

IFN-γ selectively suppresses a subset ofTLR4-activated genes and enhancers to potentiate M1-like macrophage polarization

Complete polarization of macrophages towards an M1-like proinflammatory and antimicrobial state requires combined action of IFN-{gamma} and LPS. Synergistic activation of canonical inflammatory NF-{kappa}B target genes by IFN-{gamma} and LPS is well appreciated, but less is known about whether IFN-{gamma} negatively regulates components of the LPS response, and how this affects polarization. A combined transcriptomic and epigenomic approach revealed that IFN-{gamma} selectively abrogates LPS-induced feedback and select metabolic pathways by suppressing TLR4-mediated activation of gene enhancers. In contrast to superinduction of inflammatory genes via enhancers that harbor IRF sequences and bind STAT1, IFN-{gamma}-mediated repression targeted enhancers with STAT sequences that bound STAT3. TLR4-activated IFN-{gamma}-suppressed enhancers comprised two subsets distinguished by differential regulation of histone acetylation and recruitment of STAT3, CDK8 and cohesin, and were functionally inactivated by IFN-{gamma}. These findings reveal that IFN-{gamma} suppresses feedback inhibitory and metabolic components of the TLR response to achieve full M1 polarization, and provide insights into mechanisms by which IFN-{gamma} selectively inhibits TLR4-induced transcription.

immunology↗

bioRxiv2018

Highly mutated memory cells drive an inefficient secondary antibody response to a variant protein

A powerful vaccine against mutable viruses might induce memory antibodies that either strongly bound antigenic variants or that could rapidly undergo secondary affinity maturation to achieve this. We have recently shown after secondary immunization of mice with a widely variant protein (Burton et al. 2018) that IgM+ memory B-cells with few mutations supported an efficient secondary germinal centre (GC) and serum response, superior to a primary response to the same protein. Here, boosting with more closely related proteins produced a GC response dominated by highly mutated B-cells that failed, not efficiently improving serum avidity even in the presence of extra adjuvant, and that was worse than a primary response. This supports a hypothesis that over certain antigenic differences, a cross reactive, mutated, memory B-cell compartment can be an impediment to affinity maturation.

immunology↗

bioRxiv2018

TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer

TET enzymes are dioxygenases that promote DNA demethylation by oxidizing the methyl group of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here we report a close correspondence between 5hmC-marked regions, chromatin accessibility and enhancer activity in B cells, and a strong enrichment for consensus binding motifs for basic region-leucine zipper (bZIP) transcription factors at TET-responsive genomic regions. Functionally, Tet2 and Tet3 regulate class switch recombination (CSR) in murine B cells by enhancing expression of Aicda, encoding the cytidine deaminase AID essential for CSR. TET enzymes deposit 5hmC, demethylate and maintain chromatin accessibility at two TET-responsive elements, TetE1 and TetE2, located within a superenhancer in the Aicda locus. Transcriptional profiling identified BATF as the bZIP transcription factor involved in TET-dependent Aicda expression. 5hmC is not deposited at TetE1 in activated Batf-deficient B cells, indicating that BATF recruits TET proteins to the Aicda enhancer. Our data emphasize the importance of TET enzymes for bolstering AID expression, and highlight 5hmC as an epigenetic mark that captures enhancer dynamics during cell activation.

immunology↗