Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis.

In colorectal cancer patients, a high density of cytotoxic CD8+ T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/ß-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8+ T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8+ T cells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.

Wilms' tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP.

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKÆ´ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.

Author Correction: Targeting cardiac fibrosis with engineered T cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting cardiac fibrosis with engineered T cells.

Fibrosis is observed in nearly every form of myocardial disease1. Upon injury, cardiac fibroblasts in the heart begin to remodel the myocardium by depositing excess extracellular matrix, resulting in increased stiffness and reduced compliance of the tissue. Excessive cardiac fibrosis is an important factor in the progression of various forms of cardiac disease and heart failure2. However, clinical interventions and therapies that target fibrosis remain limited3. Here we demonstrate the efficacy of redirected T cell immunotherapy to specifically target pathological cardiac fibrosis in mice. We find that cardiac fibroblasts that express a xenogeneic antigen can be effectively targeted and ablated by adoptive transfer of antigen-specific CD8+ T cells. Through expression analysis of the gene signatures of cardiac fibroblasts obtained from healthy and diseased human hearts, we identify an endogenous target of cardiac fibroblasts-fibroblast activation protein. Adoptive transfer of T cells that express a chimeric antigen receptor against fibroblast activation protein results in a significant reduction in cardiac fibrosis and restoration of function after injury in mice. These results provide proof-of-principle for the development of immunotherapeutic drugs for the treatment of cardiac disease.

CXCL1 confers a survival advantage in Kaposi's sarcoma-associated herpesvirus-infected human endothelial cells through STAT3 phosphorylation.

Many cytokines produced by Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells have been shown to participate in the pathogenesis of KSHV. Determination of the exact role of cytokines in Kaposi's sarcoma (KS) pathogenesis is limited, however, by the difficulty to manipulate the target genes in human endothelial cells. In this study, we sought to elucidate the role of cytokines in KSHV-infected human immortalized endothelial cell line (HuARLT cells) by knockout (KO) of the corresponding target genes using the CRISPR/Cas9 system. The cytokine production profile of KSHV-infected HuARLT cells was analyzed using a protein array, and several cytokines were found to be highly upregulated following KSHV infection. This study focused on CXCL1, which was investigated by knocked out in HuARLT cells. KSHV-infected CXCL1 KO cells underwent increased cell death compared to KSHV-infected wild-type (WT) cells and mock-infected CXCL1 KO cells. Lytic replication was not observed in KSHV-infected WT nor CXCL1 KO cells. Phosphorylation of STAT3 was significantly suppressed in KSHV-infected CXCL1 KO cells. Additionally, inhibitors of STAT3 and CXCL1 induced cell death in KSHV-infected endothelial cells. Our results show that CXCL1 production is required for the survival of KSHV-infected endothelial cells, and the CXCL1 to STAT3 phosphorylation signaling pathway may be a therapeutic target for KS.

Synthetic rewiring and boosting type I interferon responses for visualization and counteracting viral infections.

Mammalian first line of defense against viruses is accomplished by the interferon (IFN) system. Viruses have evolved numerous mechanisms to reduce the IFN action allowing them to invade the host and/or to establish latency. We generated an IFN responsive intracellular hub by integrating the synthetic transactivator tTA into the chromosomal Mx2 locus for IFN-based activation of tTA dependent expression modules. The additional implementation of a synthetic amplifier module with positive feedback even allowed for monitoring and reacting to infections of viruses that can antagonize the IFN system. Low and transient IFN amounts are sufficient to trigger these amplifier cells. This gives rise to higher and sustained-but optionally de-activatable-expression even when the initial stimulus has faded out. Amplification of the IFN response induced by IFN suppressing viruses is sufficient to protect cells from infection. Together, this interfaced sensor/actuator system provides a toolbox for robust sensing and counteracting viral infections.

Targeting Kaposi's Sarcoma-Associated Herpesvirus ORF21 Tyrosine Kinase and Viral Lytic Reactivation by Tyrosine Kinase Inhibitors Approved for Clinical Use.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of three human malignancies: Kaposi's sarcoma, primary effusion lymphoma, and the plasma cell variant of multicentric Castleman disease. Previous research has shown that several cellular tyrosine kinases play crucial roles during several steps in the virus replication cycle. Two KSHV proteins also have protein kinase function: open reading frame (ORF) 36 encodes a serine-threonine kinase, while ORF21 encodes a thymidine kinase (TK), which has recently been found to be an efficient tyrosine kinase. In this study, we explore the role of the ORF21 tyrosine kinase function in KSHV lytic replication. By generating a recombinant KSHV mutant with an enzymatically inactive ORF21 protein, we show that the tyrosine kinase function of ORF21/TK is not required for the progression of the lytic replication in tissue culture but that it is essential for the phosphorylation and activation to toxic moieties of the antiviral drugs zidovudine and brivudine. In addition, we identify several tyrosine kinase inhibitors, already in clinical use against human malignancies, which potently inhibit not only ORF21 TK kinase function but also viral lytic reactivation and the development of KSHV-infected endothelial tumors in mice. Since they target both cellular tyrosine kinases and a viral kinase, some of these compounds might find a use in the treatment of KSHV-associated malignancies.IMPORTANCE Our findings address the role of KSHV ORF21 as a tyrosine kinase during lytic replication and the activation of prodrugs in KSHV-infected cells. We also show the potential of selected clinically approved tyrosine kinase inhibitors to inhibit KSHV TK, KSHV lytic replication, infectious virion release, and the development of an endothelial tumor. Since they target both cellular tyrosine kinases supporting productive viral replication and a viral kinase, these drugs, which are already approved for clinical use, may be suitable for repurposing for the treatment of KSHV-related tumors in AIDS patients or transplant recipients.

Model-based analysis of influenza A virus replication in genetically engineered cell lines elucidates the impact of host cell factors on key kinetic parameters of virus growth.

The best measure to limit spread of contagious diseases caused by influenza A viruses (IAVs) is annual vaccination. The growing global demand for low-cost vaccines requires the establishment of high-yield production processes. One possible option to address this challenge is the engineering of novel vaccine producer cell lines by manipulating gene expression of host cell factors relevant for virus replication. To support detailed characterization of engineered cell lines, we fitted an ordinary differential equation (ODE)-based model of intracellular IAV replication previously established by our group to experimental data obtained from infection studies in human A549 cells. Model predictions indicate that steps of viral RNA synthesis, their regulation and particle assembly and virus budding are promising targets for cell line engineering. The importance of these steps was confirmed in four of five single gene overexpression cell lines (SGOs) that showed small, but reproducible changes in early dynamics of RNA synthesis and virus release. Model-based analysis suggests, however, that overexpression of the selected host cell factors negatively influences specific RNA synthesis rates. Still, virus yield was rescued by an increase in the virus release rate. Based on parameter estimations obtained for SGOs, we predicted that there is a potential benefit associated with overexpressing multiple host cell genes in one cell line, which was validated experimentally. Overall, this model-based study on IAV replication in engineered cell lines provides a step forward in the dynamic and quantitative characterization of IAV-host cell interactions. Furthermore, it suggests targets for gene editing and indicates that overexpression of multiple host cell factors may be beneficial for the design of novel producer cell lines.

Neoantigen Expression in Steady-State Langerhans Cells Induces CTL Tolerance.

The skin hosts a variety of dendritic cells (DCs), which act as professional APC to control cutaneous immunity. Langerhans cells (LCs) are the only DC subset in the healthy epidermis. However, due to the complexity of the skin DC network, their relative contribution to either immune activation or immune tolerance is still not entirely understood. To specifically study the function of LCs in vivo, without altering the DC subset composition in the skin, we have generated transgenic mouse models for tamoxifen-inducible de novo expression of Ags in LCs but no other langerin+ DCs. Therefore, this system allows for LC-restricted Ag presentation to T cells. Presentation of nonsecreted OVA (GFPOVA) by steady-state LCs resulted in transient activation of endogenous CTL in transgenic mice. However, when these mice were challenged with OVA by gene gun immunization in the contraction phase of the primary CTL response they did not respond with a recall of CTL memory but, instead, with robust Ag-specific CTL tolerance. We found regulatory T cells (Tregs) enriched in the skin of tolerized mice, and depletion of Tregs or adoptive experiments revealed that Tregs were critically involved in CTL tolerance. By contrast, when OVA was presented by activated LCs, a recallable CTL memory response developed in transgenic mice. Thus, neoantigen presentation by epidermal LCs results in either robust CTL tolerance or CTL memory, and this decision-making depends on the activation state of the presenting LCs.

Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.

p120 Catenin-Mediated Stabilization of E-Cadherin Is Essential for Primitive Endoderm Specification.

E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.

Mesenteric lymph node stromal cell-derived extracellular vesicles contribute to peripheral de novo induction of Foxp3+ regulatory T cells.

Intestinal regulatory T cells (Tregs) are fundamental in peripheral tolerance toward commensals and food-borne antigens. Accordingly, gut-draining mesenteric lymph nodes (mLNs) represent a site of efficient peripheral de novo Treg induction when compared to skin-draining peripheral LNs (pLNs), and we had recently shown that LN stromal cells substantially contribute to this process. Here, we aimed to unravel the underlying molecular mechanisms and generated immortalized fibroblastic reticular cell lines (iFRCs) from mLNs and pLNs, allowing unlimited investigation of this rare stromal cell subset. In line with our previous findings, mLN-iFRCs showed a higher Treg-inducing capacity when compared to pLN-iFRCs. RNA-seq analysis focusing on secreted molecules revealed a more tolerogenic phenotype of mLN- as compared to pLN-iFRCs. Remarkably, mLN-iFRCs produced substantial numbers of microvesicles (MVs) that carried elevated levels of TGF-ß when compared to pLN-iFRC-derived MVs, and these novel players of intercellular communication were shown to be responsible for the tolerogenic properties of mLN-iFRCs. Thus, stromal cells originating from mLNs contribute to peripheral tolerance by fostering de novo Treg induction using TGF-ß-carrying MVs. This finding provides novel insights into the subcellular/molecular mechanisms of de novo Treg induction and might serve as promising tool for future therapeutic applications to treat inflammatory disorders.

TLR9-Mediated Conditioning of Liver Environment Is Essential for Successful Intrahepatic Immunotherapy and Effective Memory Recall.

Immune defense against hepatotropic viruses such as hepatitis B (HBV) and hepatitis C (HCV) poses a major challenge for therapeutic approaches. Intrahepatic cytotoxic CD8 T cells that are crucial for an immune response against these viruses often become exhausted resulting in chronic infection. We elucidated the T cell response upon therapeutic vaccination in inducible transgenic mouse models in which variable percentages of antigen-expressing hepatocytes can be adjusted, providing mosaic antigen distribution and reflecting the varying viral antigen loads observed in patients. Vaccination-induced endogenous CD8 T cells could eliminate low antigen loads in liver but were functionally impaired if confronted with elevated antigen loads. Strikingly, only by conditioning the liver environment with TLR9 ligand prior and early after peripheral vaccination, successful immunization against high intrahepatic antigen density with its elimination was achieved. Moreover, TLR9 immunomodulation was also indispensable for functional memory recall after high frequency antigen challenge. Together, the results indicate that TLR9-mediated conditioning of liver environment during therapeutic vaccination or antigen reoccurrence is crucial for an efficacious intrahepatic T cell response.

Proliferation status defines functional properties of endothelial cells.

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi's sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.

Murine alveolar epithelial cells and their lentivirus-mediated immortalisation.

In this study, we describe the isolation and immortalisation of primary murine alveolar epithelial cells (mAEpC), as well as their epithelial differentiation and barrier properties when grown on Transwell® inserts. Like human alveolar epithelial cells (hAEpC), mAEpC transdifferentiate in vitro from an alveolar type II (ATII) phenotype to an ATI-like phenotype and exhibit features of the air-blood barrier, such as the establishment of a thin monolayer with functional tight junctions (TJs). This is demonstrated by the expression of TJ proteins (ZO-1 and occludin) and the development of high transepithelial electrical resistance (TEER), peaking at 1800Ω ·cm². Transport across the air-blood barrier, for general toxicity assessments or preclinical drug development, is typically studied in mice. The aim of this work was the generation of novel immortalised murine lung cell lines, to help meet Three Rs requirements in experimental testing and research. To achieve this goal, we lentivirally transduced mAEpC of two different mouse strains with a library of 33 proliferation-promoting genes. With this immortalisation approach, we obtained two murine alveolar epithelial lentivirus-immortalised (mAELVi) cell lines. Both showed similar TJ protein localisation, but exhibited less prominent barrier properties (TEERmax ~250Ω·cm²) when compared to their primary counterparts. While mAEpC demonstrated their suitability for use in the assessment of paracellular transport rates, mAELVi cells could potentially replace mice for the prediction of acute inhalation toxicity during early ADMET studies.

Evidence that hepatitis B virus replication in mouse cells is limited by the lack of a host cell dependency factor.

BACKGROUND & AIMS: Hepatitis B virus (HBV) is a major human pathogen restricted to hepatocytes. Expression of the specific receptor human sodium taurocholate cotransporting polypeptide (hNTCP) in mouse hepatocytes renders them susceptible to hepatitis delta virus (HDV), a satellite of HBV; however, HBV remains restricted at an early stage of replication. This study aims at clarifying whether this restriction is caused by the lack of a dependency factor or the activity of a restriction factor. METHODS: Six hNTCP-expressing mouse and human cell lines were generated and functionally characterized. By fusion with replication-supporting but non-infectable HepG2 cells, we analysed the ability of these heterokaryonic cells to fully support HBV replication by HBcAg expression and HBsAg/HBeAg secretion. RESULTS: While hNTCP expression in three mouse cell lines and the non-hepatic human HeLa cells conferred susceptibility to HDV, HBV replication was still restricted. Upon fusion of refractive cells to HepG2 cells, all heterokaryonic cells supported receptor-mediated infection with HBV. hNTCP was provided by the mouse cells and replication competence came from the HepG2 cell line. Transfection of a covalently closed circular DNA (cccDNA)-like molecule into non-susceptible cells promoted gene expression, indicating that the limiting step is upstream of cccDNA formation. CONCLUSIONS: In addition to the expression of hNTCP, establishment of HBV infection in mouse and non-hepatocytic human cell lines requires supplementation with a dependency factor and is not limited by a restriction factor. This result opens new avenues for the development of a fully permissive immunocompetent HBV mouse model.

Impaired antiviral response of adenovirus-transformed cell lines supports virus replication.

Activation of the innate immune response represents one of the most important cellular mechanisms to limit virus replication and spread in cell culture. Here, we examined the effect of adenoviral gene expression on the antiviral response in adenovirus-transformed cell lines; HEK293, HEK293SF and AGE1.HN. We demonstrate that the expression of the early region protein 1A in these cell lines impairs their ability to activate antiviral genes by the IFN pathway. This property may help in the isolation of newly emerging viruses and the propagation of interferon-sensitive virus strains.

Reversible silencing of cytomegalovirus genomes by type I interferon governs virus latency.

Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNß blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNß is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNß. Finally, IFNß prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNß is consistent with the establishment of CMV latency.

Uncoupling of the dynamics of host-pathogen interaction uncovers new mechanisms of viral interferon antagonism at the single-cell level.

Antiviral defence in mammals is mediated through type-I interferons (IFNs). Viruses antagonise this process through expression of IFN antagonist proteins (IAPs). Understanding and modelling of viral escape mechanisms and the dynamics of IAP action has the potential to facilitate the development of specific and safe drugs. Here, we describe the dynamics of interference by selected viral IAPs, NS1 from Influenza A virus and NS3/4A from Hepatitis C virus. We used Tet-inducible IAP gene expression to uncouple this process from virus-driven dynamics. Stochastic activation of the IFN-ß gene required the use of single-cell live imaging to define the efficacy of the inhibitors during the virus-induced signalling processes. We found significant correlation between the onset of IAP expression and halted IFN-ß expression in cells where IFN-ß induction had already occurred. These data indicate that IAPs not only prevent antiviral signalling prior to IFN-ß induction, but can also stop the antiviral response even after it has been activated. We found reduced NF-κB activation to be the underlying mechanism by which activated IFN expression can be blocked. This work demonstrates a new mechanism by which viruses can antagonise the IFN response.

Stable Expression by Lentiviral Transduction of Cells.

Lentiviral gene transfer represents a versatile and powerful method for genetic transduction of many cell lines and primary cells including "hard-to-transfect" cells. As a consequence of the integration of the recombinant lentiviral vector into the cellular genome, the transgene is stably maintained, and long-term producing cells are established. Here, we describe the current state of the art and give details for lab-scale production of lentiviral vectors as well as for infection and titration of the viral vectors.