scholarly journals Maraviroc as a potential HIV-1 latency-reversing agent in cell line models and ex vivo CD4 T cells

2020 ◽  
Author(s):  
Ilaria Vicenti ◽  
Filippo Dragoni ◽  
Martina Monti ◽  
Claudia Maria Trombetta ◽  
Alessia Giannini ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Reversal Potential ◽  
Patient Specific ◽  
Luciferase Expression ◽  
Target Cells ◽  
Reversal Effect ◽  
Hiv 1

Recent studies have suggested that the CCR5 antagonist maraviroc (MVC) may exert an HIV-1 latency reversal effect. This study aimed at defining MVC-mediated induction of HIV-1 in three cell line latency models and in ex vivo CD4 T cells from six patients with suppressed viraemia. HIV-1 induction was evaluated in TZM-bl cells by measuring HIV-1 LTR-driven luciferase expression, and in ACH-2 and U1 latently infected cell lines by measuring cell-free (CFR) and cell-associated (CAR) HIV-1 RNA by qPCR. NF-κB p65 was quantified in nuclear extracts by immunodetection. In ex vivo CD4 T cells, CAR, CFR and cell-associated DNA (CAD) were quantified at baseline and 1–7–14 days post-induction (T1, T7, T14). At T7 and T14, the infectivity of the CD4 T cells co-cultured with MOLT-4/CCR5 target cells was evaluated in the TZM-bl assay (TZA). Results were expressed as fold activation (FA) with respect to untreated cells. No LTR activation was observed in TZM-bl cells at any MVC concentration. NF-κB activation was only modestly upregulated (1.6±0.4) in TZM-bl cells with 5 µM MVC. Significant FA of HIV-1 expression was only detected at 80 µM MVC, namely on HIV-1 CFR in U1 (3.1±0.9; P=0.034) and ACH-2 cells (3.9±1.4; P=0.037). CFR was only weakly stimulated at 20 µM in ACH-2 (1.7±1.0 FA) cells and at 5 µM in U1 cells (1.9±0.5 FA). Although no consistent pattern of MVC-mediated activation was observed in ex vivo experiments, substantial FA values were detected sparsely on individual samples with different parameters. Notably, in one sample, MVC stimulated all parameters at T7 (2.3±0.2 CAD, 6.8±3.7 CAR, 18.7±16.7 CFR, 7.3±0.2 TZA). In conclusion, MVC variably induces HIV-1 production in some cell line models not previously used to test its latency reversal potential. In ex vivo CD4 T cells, MVC may exert patient-specific HIV-1 induction; however, clinically relevant patterns, if any, remain to be defined.

scholarly journals Th1/17 Polarization of CD4 T Cells Supports HIV-1 Persistence during Antiretroviral Therapy

2015 ◽  
Vol 89 (22) ◽  
pp. 11284-11293 ◽  
Author(s):  
Hong Sun ◽  
Dhohyung Kim ◽  
Xiaodong Li ◽  
Maja Kiselinova ◽  
Zhengyu Ouyang ◽  
...  
Keyword(s):  
T Cells ◽  
Antiretroviral Therapy ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Memory Cell ◽  
Viral Reservoir ◽  
Target Cells ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTThe ability to persist long term in latently infected CD4 T cells represents a characteristic feature of HIV-1 infection and the predominant barrier to efforts aiming at viral eradication and cure. Yet, increasing evidence suggests that only small subsets of CD4 T cells with specific developmental and maturational profiles are able to effectively support HIV-1 long-term persistence. Here, we analyzed how the functional polarization of CD4 T cells shapes and structures the reservoirs of HIV-1-infected cells. We found that CD4 T cells enriched for a Th1/17 polarization had elevated susceptibilities to HIV-1 infection inex vivoassays, harbored high levels of HIV-1 DNA in persons treated with antiretroviral therapy, and made a disproportionately increased contribution to the viral reservoir relative to their contribution to the CD4 T memory cell pool. Moreover, HIV-1 DNA levels in Th1/17 cells remained stable over many years of antiretroviral therapy, resulting in a progressively increasing contribution of these cells to the viral reservoir, and phylogenetic studies suggested preferential long-term persistence of identical viral sequences during prolonged antiretroviral treatment in this cell compartment. Together, these data suggest that Th1/17 CD4 T cells represent a preferred site for HIV-1 DNA long-term persistence in patients receiving antiretroviral therapy.IMPORTANCECurrent antiretroviral therapy is very effective in suppressing active HIV-1 replication but does not fully eliminate virally infected cells. The ability of HIV-1 to persist long term despite suppressive antiretroviral combination therapy represents a perplexing aspect of HIV-1 disease pathogenesis, since most HIV-1 target cells are activated, short-lived CD4 T cells. This study suggests that CD4 T helper cells with Th1/17 polarization have a preferential role as a long-term reservoir for HIV-1 infection during antiretroviral therapy, possibly because these cells may imitate some of the functional properties traditionally attributed to stem cells, such as the ability to persist for extremely long periods of time and to repopulate their own pool size through homeostatic self-renewal. These observations support the hypothesis that HIV-1 persistence is driven by small subsets of long-lasting stem cell-like CD4 T cells that may represent particularly promising targets for clinical strategies aiming at HIV-1 eradication and cure.

scholarly journals Optimizing CD4+ T-Cells Transduction Protocol for Gene Therapy of HIV-1 Infected Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4429-4429
Author(s):  
Amani Ouedrani ◽  
Lounes Djerroudi ◽  
Isabelle Hmitou ◽  
Marina Cavazzana ◽  
Fabien Touzot
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Cd4 T Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Effector Memory ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Hiv 1

Abstract Gene therapy represents an alternative and promising strategy that could provide a path to a curative therapy for HIV-1 infection. One approach involves the introduction of protective gene into a cell, thereby conferring protection against HIV. We plan to conduct an open label phase I/II gene therapy trial for HIV-1 infected patients presenting with lymphoma. The patients will received autologous hematopoietic stem cells transplantation with gene modified CD34+ cells and CD4+ T-cells. CD34+ and CD4+ will be ex vivo transduced by the LVsh5/C46 lentiviral vector (Cal-1, Calimmune, Inc. Tucson, USA). LVsh5/C46 is a SIN lentiviral vector that inhibits two crucial steps of CD4+ T cell infection by the HIV virus: (i) attachment of the virus to its target by downregulation of CCR5 via a short hairpin RNA, (ii) fusion of the virus to the target cell through expression of the C46 inhibitor. We developed a transduction process for CD4+ T-cells using the TransAct™ reagent (Miltenyi Biotec, Bergisch Gladbach , Germany) for CD4+ T-cells activation. Compared to previously published T-cells transduction protocols, the use of Miltenyi TransAct™ permits an equivalent efficacy of transduction - evaluated by measurement of vector copy number through quantitative PCR - without major phenotypic modification. Indeed, CD4+ T-cells ex vivo transduced after activation with the TransAct™ reagent display very few changes in their surface marker with conservation of naive (CCR7+CD62L+CD45RA+), central memory (CCR7+CD62L+CD45RA-) and effector memory (CCR7-CD62L-CD45RA-) subsets in superimposable proportions as initially. Moreover, expression of CD25 remains below 15-25% of cells suggesting a more "gentle " activation of the transduced CD4+ T-cells. Our transduction process had no significant impact in TCRβ repertoire diversity as evaluated by high-throughput sequencing and analyzis of diversity through the Gini-Simpson index or the Shannon index. Finally, transduced CD4 + T-cells retained the ability to to be primed towards the TH1, TH2 and TH17 pathways suggesting that the transduction protocol used did not alter the functional properties of the target cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inconsistent Reversal of HIV-1 Latency Ex Vivo by Antigens of HIV-1, CMV, and Other Infectious Agents

2020 ◽  
Author(s):  
Thomas Vollbrecht ◽  
Aaron O. Angerstein ◽  
Bryson Menke ◽  
Nikesh M. Kumar ◽  
Michelli Faria Oliveira ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Ex Vivo ◽  
Latent Reservoir ◽  
Antigen Specificity ◽  
Pcr Assay ◽  
Hiv 1

Abstract BackgroundA reservoir of replication-competent but latent virus is the main obstacle to a cure for HIV-infection. Much of this reservoir resides in memory CD4 T cells. We hypothesized that these cells can be reactivated with antigens from HIV and other common pathogens to reverse latency. ResultsWe obtained mononuclear cells from the peripheral blood of antiretroviral-treated patients with suppressed viremia. We tested pools of peptides and proteins derived from HIV and from other pathogens including CMV for their ability to reverse latency ex vivo by activation of memory responses. We assessed activation of the CD4 T cells by measuring the up-regulation of cell-surface CD69. We assessed HIV-expression using two assays: a real-time PCR assay for virion-associated viral RNA and a droplet digital PCR assay for cell-associated, multiply spliced viral mRNA. Reversal of latency occurred in a minority of cells from some participants, but no single antigen induced HIV-expression ex vivo consistently. When reversal of latency was induced by a specific peptide pool or protein, the extent was proportionally greater than that of T cell activation. ConclusionsIn this group of patients in whom antiretroviral therapy was started during chronic infection, the latent reservoir does not appear to consistently reside in CD4 T cells of a predominant antigen-specificity. Peptide-antigens reversed HIV-latency ex vivo with modest and variable activity. When latency was reversed by specific peptides or proteins, it was proportionally greater than the extent of T cell activation, suggesting partial enrichment of the latent reservoir in cells of specific antigen-reactivity.

scholarly journals HIV-1 Infection Ex Vivo Accelerates Measles Virus Infection by Upregulating Signaling Lymphocytic Activation Molecule (SLAM) in CD4+ T Cells

2012 ◽  
Vol 86 (13) ◽  
pp. 7227-7234
Author(s):  
Y.-y. Mitsuki ◽  
K. Terahara ◽  
K. Shibusawa ◽  
T. Yamamoto ◽  
T. Tsuchiya ◽  
...  
Keyword(s):  
T Cells ◽  
Virus Infection ◽  
Cd4 T Cells ◽  
Measles Virus ◽  
Ex Vivo ◽  
Signaling Lymphocytic Activation Molecule ◽  
Hiv 1

WT1-Specific T Cells Exhibiting a Non-Exhausted, Functional Phenotype Can Be Generated From The Natural Repertoire Of Healthy Donors For Clinical Use

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4504-4504 ◽  
Author(s):  
Sabine Schmied ◽  
Anne Richter ◽  
Mario Assenmacher ◽  
Juergen Schmitz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Target Cells ◽  
Clinical Use ◽  
Healthy Donors ◽  
Effector Cytokines

Background The Wilms tumor antigen 1 (WT1) is a self-antigen expressed at high levels in leukemic cells, but not in healthy tissue. As WT1 expression in leukemic cells drives leukemogenesis, it is a favorable target antigen for immunotherapy, e.g. adoptive transfer of allogeneic T cells, to prevent or treat leukemic relapse after stem cell transplantation (Cheever et al., Clin Cancer Res 2009;15(17)). WT1-specific CD8+ T cells have been detected in healthy individuals at low frequencies (Rezvani et al., Blood 2003;102). However, a comprehensive characterization of CD4+ and CD8+WT1-specific T cells is missing and the efficient expansion of a polyclonal WT1-reactive T cell population for clinical use has remained a major challenge. In this study we aim to directly ex vivo characterize WT1-specific T cells present in the blood of healthy donors at high-resolution and to develop a rapid method for the generation of functionally potent, polyclonal CD4+ and CD8+WT1-specific T cells for clinical use. Methods For direct ex vivo analysis of CD4+ WT1-specific T cells peripheral blood mononuclear cells (PBMC) of healthy blood donors were in vitro stimulated with a pool of overlapping peptides spanning the WT1 protein for 7 hours. Subsequently CD154 (CD40L)-expressing cells were magnetically enriched and flow cytometrically examined for expression of effector cytokines and their differentiation status. Presence and phenotype of CD8+ WT1-specific T cells have been studied after stimulation of presorted naïve and memory T cell populations with WT-1 peptide pool for 30 hours, magnetic enrichment of CD137+ (4-1BB) cells and subsequent staining using pMHCI-Tetramers. For the generation of polyclonal WT1-specific CD4+ and CD8+ T cells PBMC were in vitro activated with WT-1 peptide pool for 30 hours. CD137+cells were magnetically selected and expanded for 9 days in the presence of the cytokines IL-7, IL-15 and IL-21 at low doses. Expanded T cells were analyzed for their phenotype, the expression of co-stimulatory and exhaustion markers and were tested for their functionality and cytotoxicity by restimulation experiments with antigen-loaded target cells. Results Ex vivo frequencies of WT1-specific T cells are low, 1 to 10 WT1-specific CD154+ CD4+ T cells can be detected within 1x106 CD4+ T cells. In about 80% of healthy donors (n=15) a CD4+ memory response, accompanied by production of effector cytokines like IFNγ, TNFα and IL-2, against WT1 peptides is present. Additionally, in all donors naïve WT1-specific CD4+ T cells can be detected. In contrast, detected CD137+CD8+ WT1-reactive T cells exhibit a naïve phenotype (CD45RA+CCR7+) in all donors (n=5), no WT1-reactive CD8+T cells could be enriched from presorted memory T cells. To evaluate the usefulness of our improved short-term expansion protocol to generate potent WT1-specific T cell cultures for clinical use, we characterized CD137 enriched and expanded T cells. Notably, a high frequency of CD4+ and CD8+ T cells show specific reactivity against WT1-presenting autologous cells as detected by production of effector cytokines like IFNγ, TNFα and IL-2 after antigen-specific restimulation. Cytotoxic activity against antigen-loaded target cells could be shown by direct flow-cytometry-based cytotoxicity assays and antigen-specific upregulation of the degranulation marker CD107a. Stainings using multiple WT1-MHCI-tetramers furthermore confirmed antigen-specificity and suggested polyclonality within the CD8+T cell population. In contrast to previous expansion protocols our polyclonally expanded T cells exhibit a favourable, unexhausted memory phenotype, express co-stimulatory markers CD27 and CD28 and the IL7R-a chain (CD127) which has been shown to mark cells with stem T cell like properties. Furthermore exhaustion markers like CD279 (PD-1), CD178 (FasL) and CD57 are scarcely expressed. Conclusions Functional, polyclonal, CD4+ and CD8+ WT1-specific, reactive T cells can be efficiently enriched directly ex vivo from the natural repertoire by magnetic separation of T cells after antigen-specific stimulation. Phenotypic and functional characterization revealed a non-exhausted phenotype of expanded WT1-specific T cells, thereby suggesting good persistence and functionality of the obtained T cell product in vivo. Thus, our approach holds great potential for the GMP-compliant generation of WT1-specific T cells for future clinical use. Disclosures: Schmied: Miltenyi Biotec GmbH: Employment. Richter:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec: Employment.

scholarly journals Inhibition of HIV-1 Integration in Ex Vivo-Infected CD4 T Cells from Elite Controllers

2011 ◽  
Vol 85 (18) ◽  
pp. 9646-9650 ◽  
Author(s):  
M. J. Buzon ◽  
K. Seiss ◽  
R. Weiss ◽  
A. L. Brass ◽  
E. S. Rosenberg ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Elite Controllers ◽  
Hiv 1

scholarly journals HIV-1–induced activation of CD4+ T cells creates new targets for HIV-1 infection in human lymphoid tissue ex vivo

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 699-704 ◽  
Author(s):  
Angélique Biancotto ◽  
Sarah J. Iglehart ◽  
Christophe Vanpouille ◽  
Cristian E. Condack ◽  
Andrea Lisco ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Lymphoid Tissue ◽  
Ex Vivo ◽  
Lymphoid Tissues ◽  
Strong Positive Correlation ◽  
New Targets ◽  
Hla Dr ◽  
Human Lymphoid Tissue ◽  
Hiv 1

We demonstrate mechanisms by which HIV-1 appears to facilitate its own infection in ex vivo–infected human lymphoid tissue. In this system, HIV-1 readily infects various CD4+ T cells, but productive viral infection was supported predominantly by activated T cells expressing either CD25 or HLA-DR or both (CD25/HLA-DR) but not other activation markers: There was a strong positive correlation (r = 0.64, P = .001) between virus production and the number of CD25+/HLA-DR+ T cells. HIV-1 infection of lymphoid tissue was associated with activation of both HIV-1–infected and uninfected (bystanders) T cells. In these tissues, apoptosis was selectively increased in T cells expressing CD25/HLA-DR and p24gag but not in cells expressing either of these markers alone. In the course of HIV-1 infection, there was a significant increase in the number of activated (CD25+/HLA-DR+) T cells both infected and uninfected (bystander). By inducing T cells to express particular markers of activation that create new targets for infection, HIV-1 generates in ex vivo lymphoid tissues a vicious destructive circle of activation and infection. In vivo, such self-perpetuating cycle could contribute to HIV-1 disease.

scholarly journals Nef enhances HIV-1 replication and infectivity independently of SERINC3 and SERINC5 in CEM T cells

2020 ◽  
Author(s):  
Peter W. Ramirez ◽  
Aaron A. Angerstein ◽  
Marissa Suarez ◽  
Thomas Vollbrecht ◽  
Jared Wallace ◽  
...  
Keyword(s):  
Growth Rate ◽  
T Cells ◽  
T Cell ◽  
Viral Replication ◽  
Cell Line ◽  
Host Protein ◽  
Target Cells ◽  
Viral Fusion ◽  
Viral Growth ◽  
Hiv 1

AbstractThe lentiviral nef gene encodes several discrete activities aimed at co-opting or antagonizing cellular proteins and pathways to defeat host defenses and maintain persistent infection. Primary functions of Nef include downregulation of CD4 and MHC class-I from the cell surface, disruption or mimicry of T-cell receptor signaling, and enhancement of viral infectivity by counteraction of the host antiretroviral proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 incorporate into virions and inhibit viral fusion with target cells, decreasing infectivity. However, whether Nef’s counteraction of SERINC3 and SERINC5 is the cause of its positive influence on viral growth-rate in CD4-positive T cells is unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays robust nef-related infectivity and growth-rate phenotypes. As previously reported, viral replication was severely attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1ΔNef). This attenuated growth-rate phenotype was observed regardless of whether or not the coding regions of the serinc3 and serinc5 genes were intact. Moreover, knockout of serinc3 and serinc5 failed to restore the infectivity of HIV-1ΔNef virions produced from infected CEM cells in single-cycle replication experiments using CD4-positive HeLa cells as targets. Taken together, our results corroborate a recent study using another T-lymphoid cell line (MOLT-3) and suggest that Nef modulates a still unidentified host protein(s) to enhance viral growth rate and infectivity in CD4-positive T cells.ImportanceHIV-1 Nef is a major pathogenicity factor in vivo. A well-described activity of Nef is the enhancement of virion-infectivity and viral propagation in vitro. The infectivity-effect has been attributed to Nef’s ability to prevent the cellular, antiretroviral proteins SERINC3 and SERINC5 from incorporating into viral particles. While the activity of the SERINCs as inhibitors of retroviral infectivity has been well-documented, the role these proteins play in controlling HIV-1 replication is less clear. We report here that genetic disruption of SERINC3 and SERINC5 rescues neither viral replication-rate nor the infectivity of cell-free virions produced from CD4-positive T cells of the CEM lymphoblastoid line infected with viruses lacking Nef. This indicates that failure to modulate SERINC3 and SERINC5 is not the cause of the virologic attenuation of nef-negative HIV-1 observed using this system.

scholarly journals Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells

2019 ◽  
Author(s):  
Birgitta Lindqvist ◽  
Sara Svensson Akusjarvi ◽  
Anders Sonnerborg ◽  
Marios Dimitriou ◽  
J. Peter Svensson
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Ex Vivo ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Active Transcription ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir. The reservoir that reinstates an active replication comprises only cells with intact provirus that can be reactivated. We confirmed that latently infected cells from patients exhibited active transcription throughout the provirus. To find transcriptional determinants, we characterized the establishment and maintenance of viral latency during proviral chromatin maturation in cultures of primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (i.e., H3K27ac) remained detectable, even after prolonged proviral silencing. After T-cell activation, the proviral activation occurred uniquely in cells with H3K27ac-marked proviruses. Our observations suggested that, after transient proviral activation, cells were actively returned to latency.

scholarly journals Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb and reverses HIV-1 latency

2019 ◽  
Author(s):  
Mateusz Stoszko ◽  
Abdullah M.S. Al-Hatmi ◽  
Anton Skriba ◽  
Michael Roling ◽  
Enrico Ne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Reversal Agents ◽  
Latently Infected ◽  
Infected Cells ◽  
Therapeutic Compounds ◽  
Hiv 1

AbstractA leading pharmacological strategy towards HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with Latency Reversal Agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs we used fungal secondary metabolites (extrolites) as a source of bio-active molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the P-TEFb inhibitory 7SK snRNP complex to be significantly reduced upon GTX treatment of independent donor CD4+T cells. GTX disrupted 7SK snRNP, releasing active P-TEFb, which then phosphorylated RNA Pol II CTD, inducing HIV transcription. Our data highlight the power of combining a medium throughput bioassay, mycology and orthogonal mass spectrometry to identify novel potentially therapeutic compounds.

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