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1.  HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection 
EBioMedicine  2016;6:103-113.
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.
Highlights
•Viremic and non-viremic HIV patients harbor high levels of plasma extracellular vesicles.•Besides inflammatory factors they contain Nef and Vpu, hinting at ongoing viral activity despite efficient ART.•The level of Nef vesicles correlates with immunactivation and low CD4 levels in chronic HIV infection.
Lee et al. found high levels of extracellular vesicles in plasma (pEV) in HIV infection that did not decline under treatment and analyzed a possible correlation with HIV pathogenesis. The pEV contained inflammatory factors and HIV proteins. This was unexpected as viral replication is efficiently suppressed by treatment. The pEV content and viral proteins correlated stringently with symptoms of chronic HIV disease, including low T cell count and increased inflammation. Analyzing the cytokine pattern of HIV pEV it seemed that they were secreted by a so far unknown cell compartment. Suppressing pEV secretion may greatly improve the treatment of chronic HIV infection.
doi:10.1016/j.ebiom.2016.03.004
PMCID: PMC4856776  PMID: 27211553
Extracellular vesicles; HIV; Nef; ADAM17; Vpu; HIV reservoir; Chronic HIV infection
2.  Exosomes from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Cells License Quiescent CD4+ T Lymphocytes To Replicate HIV-1 through a Nef- and ADAM17-Dependent Mechanism 
Journal of Virology  2014;88(19):11529-11539.
ABSTRACT
Resting CD4+ T lymphocytes resist human immunodeficiency virus (HIV) infection. Here, we provide evidence that exosomes from HIV-1-infected cells render resting human primary CD4+ T lymphocytes permissive to HIV-1 replication. These results were obtained with transwell cocultures of HIV-1-infected cells with quiescent CD4+ T lymphocytes in the presence of inhibitors of exosome release and were confirmed using exosomes purified from supernatants of HIV-1-infected primary CD4+ T lymphocytes. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4+ T lymphocytes. We also identified a Nef domain important for the effects we observed, i.e., the 62EEEE65 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-tumor necrosis factor alpha (TNF-α) in its mature form, associates with exosomes from HIV-1-infected cells, and plays a key role in the HIV-1 replication in quiescent CD4+ T lymphocytes. Treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in resting CD4+ T lymphocytes. TNF-α is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNF-α antibodies blocked the HIV-1 replication. The data presented here are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4+ T lymphocytes, thus stimulating viral spread.
IMPORTANCE Overall, our findings support the idea that HIV evolved to usurp the exosome-based intercellular communication network to favor its spread in infected hosts.
doi:10.1128/JVI.01712-14
PMCID: PMC4178784  PMID: 25056899
3.  Immunogenic and tolerogenic effects of the chimeric IL-2-diphtheria toxin cytocidal agent Ontak® on CD25+ cells 
Oncoimmunology  2014;3:e28223.
Ontak®, a conjugate between IL-2 and a diphtheria toxin fragment, was recently investigated in cancer clinical trials aiming to kill CD25+ regulatory T cells (Tregs). We found that the activity of Ontak® was more complex on Tregs and conventional T cells (Tconvs) than anticipated, including a novel strong influence on dendritic cells (DCs).
doi:10.4161/onci.28223
PMCID: PMC4091105  PMID: 25050193
Ontak; dendritic cells; T cells; regulatory T cells; interleukin-2 receptor
5.  DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells 
Immunome Research  2010;6:10.
Background
The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs).
Results
Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules.
Conclusions
The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.
doi:10.1186/1745-7580-6-10
PMCID: PMC3000836  PMID: 21092113
6.  Nef Alleles from Human Immunodeficiency Virus Type 1-Infected Long-Term-Nonprogressor Hemophiliacs with or without Late Disease Progression Are Defective in Enhancing Virus Replication and CD4 Down-Regulation▿  
Journal of Virology  2006;80(21):10663-10674.
Infection with human immunodeficiency virus (HIV)-encoding defective nef variants may contribute to a relatively benign course of disease in a minority of long-term nonprogressors (LTNP). We have examined the functions of nef alleles from six individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Three out of six individuals have progressed to HIV disease (late progressors [LP]), whereas the three remainders have maintained their LTNP status at least up to 2003. The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells of all six individuals in 1995 and 1998. The proportion of sequences containing mutations not yielding Nef expression significantly diminished in 1998 versus that in 1995. Several previously defined functional regions of intact nef alleles were highly conserved. However, the major variant obtained in 1998 from plasma RNA of five out of six individuals significantly reduced HIV infectivity/replication and impaired Nef-mediated CD4 but not major histocompatibility complex class I antigen down-modulation from the cell surface. Thus, functional alterations of the nef gene are present in both LP and LTNP, suggesting that Nef defectiveness in vitro is not necessarily associated with the long-term maintenance of LTNP status. Of interest is the fact that isolates from three out of three LP showed a dual CCR5/CXCR4 coreceptor use (R5X4), in contrast to those from LTNP, which were exclusively R5. Thus, in vivo evolution of gp120 Env to CXCR4 use appears to be associated with HIV disease progression in individuals infected with nef-defective viruses.
doi:10.1128/JVI.02621-05
PMCID: PMC1641799  PMID: 16943296
7.  Nef from Human Immunodeficiency Virus Type 1F12 Inhibits Viral Production and Infectivity 
Journal of Virology  2001;75(14):6601-6608.
Human immunodeficiency virus type 1F12 (HIV-1F12) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55Gag precursor in the presence of Nef from HIV-1F12. Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1NL4-3 also created a dominant-negative Nef protein. Effects of Nef from HIV-1F12 on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.
doi:10.1128/JVI.75.14.6601-6608.2001
PMCID: PMC114383  PMID: 11413327
8.  Direct In Vitro Binding of Full-Length Human Immunodeficiency Virus Type 1 Nef Protein to CD4 Cytoplasmic Domain 
Journal of Virology  2001;75(8):3960-3964.
The Nef protein of the simian and human immunodeficiency viruses is known to directly bind and downregulate the CD4 receptor. Although the molecular mechanism is well understood, direct binding of Nef and CD4 is difficult to demonstrate and is believed to be of low affinity. Applying nuclear magnetic resonance and fluorescence spectroscopy, we biophysically reevaluated the CD4-Nef complex and found the dissociation constant to be in the submicromolar range. We conclude that additional, so far disregarded residues in the N terminus of Nef are important for interaction with CD4.
doi:10.1128/JVI.75.8.3960-3964.2001
PMCID: PMC114886  PMID: 11264384
9.  Induction of Fas Ligand Expression by HIV Involves the Interaction of Nef with the T Cell Receptor ζ Chain  
The Journal of Experimental Medicine  1999;189(9):1489-1496.
During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas–Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4+ and CD8+ T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the ζ chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR ζ chain. Conformation for the importance of ζ for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of ζ but not CD3ε was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.
PMCID: PMC2193060  PMID: 10224289
Jurkat; immunoreceptor tyrosine-based activation motif; Nef-associated kinase; activation-induced cell death; apoptosis

Results 1-9 (9)