Cells release a variety of membrane-enveloped vesicles, including microvesicles, exosomes and apoptotic bodies, the content of which differs according to the cell’s (patho)physiological state. These extracellular vesicles (EVs) are increasingly recognised to have important roles in intercellular communication, including immunomodulatory effects. EVs released from virus-infected cells can transfer viral as well as host cell proteins, mRNA/miRNAs, lipids and a variety of small molecular mediators between cells. They may enhance or restrict viral spread, acting either directly (e.g. EVs released from lentivirus-infected cells contain cGAMP, an innate messenger that activates induction of an antiviral state), or indirectly via effects on the host immune response. They can act as a source of viral proteins/peptides for antigen presentation, and modulate the functional capacity of immune cell subsets in multiple ways. Viruses including HIV enhance EV release from the cells they infect – and in macrophages/dendritic cells (DCs) HIV exploits the exosome production pathway for virion biogenesis. Large quantities of EVs are present in the circulation of HIV-infected individuals (even after commencement of anti-retroviral therapy), and are thought to contribute to viral pathogenesis – but their biological roles are poorly characterised. A better understanding of how EVs impact on HIV replication and pathogenesis could inform the development of novel strategies to control infection or prevent the development of chronic immune pathologies in infected individuals.
Aims of this project will be:
1. To define the subtypes and composition of EVs released from CD4 T cells, macrophages and DCs following in vitro exposure to HIV, and of EVs present in the plasma of HIV+ subjects at different stages of infection. Approaches employed: vesicle fractionation, high-sensitivity mass-spectrometry and RNASeq
2. To determine the role of EV subtypes in providing a source of viral proteins/peptides for DC-mediated cross-presentation, and dissect the importance of EVs in transfer of pre-formed peptide-MHC complexes to DCs/CD4 T cells. Approaches employed: immunopeptidomic analyses, super-resolution microscopy and in vitro presentation/priming assays
3. To address the impact of EV subtypes, particularly exosomes, on the activation state and functional profile of DCs and subsets of CD4 T cells including CD4 Treg cells, and define the mechanisms by which these effects are mediated. Approaches employed: Multiparameter flow cytometry based immunoassays, Luminex bead-based arrays, molecular immunological techniques.
The student will work within a small team of postdocs and other students addressing complementary aspects of HIV immunology/ immunobiology. They will receive training in all the techniques involved in the project, including high-sensitivity proteomics/immunoproteomic analysis methods, RNASeq, multiparameter flow cytometry and a breadth of immunological assays. In the co-supervisor’s laboratory, they will also employ cutting-edge high-resolution microscopy techniques to visualise EV-mediated transfer of peptide-MHC complexes and other molecules between cells. More generic research training, e.g. in experimental design, data interpretation, statistical analysis and presentation and writing skills will also be provided.
The student will take part in group meetings and journal clubs and will be encouraged to attend seminars given by internal and external speakers. They will also have the opportunity to attend and present their data and national and international meetings.
Project reference number: 963
|Professor Persephone Borrow||NDM Research Building||Oxford University, NDM Research Building||GBRfirstname.lastname@example.org|
|Dr Marco Fritzsche||Weatherall Institute of Molecular Medicine||Oxford University, Weatherall Institute of Molecular Medicine||GBR|
The release of membrane-bound vesicles from cells is being increasingly recognized as a mechanism of intercellular communication. Extracellular vesicles (EVs) or exosomes are produced by virus-infected cells and are thought to be involved in intercellular communication between infected and uninfected cells. Viruses, in particular oncogenic viruses and viruses that establish chronic infections, have been shown to modulate the production and content of EVs. Viral microRNAs, proteins and even entire virions can be incorporated into EVs, which can affect the immune recognition of viruses or modulate neighbouring cells. In this Review, we discuss the roles that EVs have during viral infection to either promote or restrict viral replication in target cells. We will also discuss our current understanding of the molecular mechanisms that underlie these roles, the potential consequences for the infected host and possible future diagnostic applications. Hide abstract
Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART. Surprisingly, these vesicles contained the viral accessory proteins Nef and Vpu, which are assumed to be not expressed under efficient ART, as well as pro-inflammatory effectors, including activated ADAM17. HIV pEV were characterized by the presence of activated αvβ3 and absence of CD81 and Tsg101. Correlating with immune activation, peripheral monocytes ingested large amounts of pEV, giving rise to an increased population of CD1c(+) CD14(+) cells that secreted inflammatory cytokines. Importantly, the pro-inflammatory content, particularly ADAM17 activity, correlated with low T cell counts. Preliminary evidence suggested that HIV pEV derived from peripheral mononuclear cells and from an unknown myeloid cell population. In summary we propose an important role of pro-inflammatory pEV in chronic HIV infection due to ongoing viral Nef activity. Hide abstract
Cyclic GMP-AMP synthase (cGAS) detects cytosolic DNA during virus infection and induces an antiviral state. cGAS signals by synthesis of a second messenger, cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING). We show that cGAMP is incorporated into viral particles, including lentivirus and herpesvirus virions, when these are produced in cGAS-expressing cells. Virions transferred cGAMP to newly infected cells and triggered a STING-dependent antiviral program. These effects were independent of exosomes and viral nucleic acids. Our results reveal a way by which a signal for innate immunity is transferred between cells, potentially accelerating and broadening antiviral responses. Moreover, infection of dendritic cells with cGAMP-loaded lentiviruses enhanced their activation. Loading viral vectors with cGAMP therefore holds promise for vaccine development. Hide abstract
Acute HIV-1 infection results in dysregulated immunity, which contributes to poor control of viral infection. DCs are key regulators of both adaptive and innate immune responses needed for controlling HIV-1, and we surmised that factors elicited during acute HIV-1 infection might impede DC function. We derived immature DCs from healthy donor peripheral blood monocytes and treated them with plasma from uninfected control donors and donors with acute HIV-1 infections. We found that the plasma from patients with HIV specifically inhibited DC function. This suppression was mediated by elevated apoptotic microparticles derived from dying cells during acute HIV-1 infection. Apoptotic microparticles bound to and inhibited DCs through the hyaluronate receptor CD44. These data suggest that targeting this CD44-mediated inhibition by apoptotic microparticles could be a novel strategy to potentiate DC activation of HIV-specific immunity. Hide abstract