ABSTRACT
Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll-like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist GS-9620 induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5- to 2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are under way to determine if GS-9620 can target HIV reservoirs.IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is persistently maintained in reservoirs of latently infected cells. Consequently, lifelong therapy is required to maintain viral suppression. Ultimately, new therapies that specifically target and eliminate the latent HIV reservoir are needed. Toll-like receptor agonists are potent enhancers of innate antiviral immunity that can also improve the adaptive immune response. Here, we show that a highly selective TLR7 agonist, GS-9620, activated HIV from peripheral blood mononuclear cells isolated from HIV-infected individuals with suppressed infection. GS-9620 also improved immune effector functions that specifically targeted HIV-infected cells. Previously published studies on the compound in other chronic viral infections show that it can effectively induce immune activation at safe and tolerable clinical doses. Together, the results of these studies suggest that GS-9620 may be useful for treating HIV-infected individuals on suppressive antiretroviral therapy.
Subject(s)
HIV Infections/virology , HIV/immunology , HIV/physiology , Immunologic Factors/pharmacology , Pteridines/pharmacology , Toll-Like Receptor 7/agonists , Virus Activation/drug effects , Anti-Retroviral Agents/therapeutic use , Antibody-Dependent Cell Cytotoxicity/drug effects , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/drug effects , Humans , Interferon Type I/metabolism , Lymphocyte Activation/drug effects , Phagocytes/drug effects , Phagocytes/physiology , RNA, Viral/blood , Viral LoadABSTRACT
Several prophylactic vaccines targeting herpes simplex virus 2 (HSV-2) have failed in the clinic to demonstrate sustained depression of viral shedding or protection from recurrences. Although these vaccines have generated high titers of neutralizing antibodies (NAbs), their induction of robust CD8 T cells has largely been unreported, even though evidence for the importance of HSV-2 antigen-specific CD8 T cells is mounting in animal models and in translational studies involving subjects with active HSV-2-specific immune responses. We developed a subunit vaccine composed of the NAb targets gD and gB and the novel T cell antigen and tegument protein UL40, and we compared this vaccine to a whole-inactivated-virus vaccine (formaldehyde-inactivated HSV-2 [FI-HSV-2]). We evaluated different formulations in combination with several Th1-inducing Toll-like receptor (TLR) agonists in vivo In mice, the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide formulated in a squalene-based oil-in-water emulsion promoted most robust, functional HSV-2 antigen-specific CD8 T cell responses and high titers of neutralizing antibodies, demonstrating its superiority to vaccines adjuvanted by monophosphoryl lipid A (MPL)-alum. We further established that FI-HSV-2 alone or in combination with adjuvants as well as adjuvanted subunit vaccines were successful in the induction of NAbs and T cell responses in guinea pigs. These immunological responses were coincident with a suppression of vaginal HSV-2 shedding, low lesion scores, and a reduction in latent HSV-2 DNA in dorsal root ganglia to undetectable levels. These data support the further preclinical and clinical development of prophylactic HSV-2 vaccines that contain appropriate antigen and adjuvant components responsible for programming elevated CD8 T cell responses.IMPORTANCE Millions of people worldwide are infected with herpes simplex virus 2 (HSV-2), and to date, an efficacious prophylactic vaccine has not met the rigors of clinical trials. Attempts to develop a vaccine have focused primarily on glycoproteins necessary for HSV-2 entry as target antigens and to which the dominant neutralizing antibody response is directed during natural infection. Individuals with asymptomatic infection have exhibited T cell responses against specific HSV-2 antigens not observed in symptomatic individuals. We describe for the first time the immunogenicity profile in animal models of UL40, a novel HSV-2 T cell antigen that has been correlated with asymptomatic HSV-2 disease. Additionally, vaccine candidates adjuvanted by a robust formulation of the CpG oligonucleotide delivered in emulsion were superior to unadjuvanted or MPL-alum-adjuvanted formulations at eliciting a robust cell-mediated immune response and blocking the establishment of a latent viral reservoir in the guinea pig challenge model of HSV-2 infection.
Subject(s)
Adjuvants, Immunologic/pharmacology , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , CD8-Positive T-Lymphocytes/immunology , Herpes Simplex Virus Vaccines/immunology , Herpes Simplex/prevention & control , Herpesvirus 2, Human/immunology , Virus Latency/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , Disease Models, Animal , Female , Glycoproteins/immunology , Guinea Pigs , Herpes Simplex/immunology , Herpes Simplex/virology , Herpesvirus 2, Human/physiology , Immunity, Cellular/immunology , Mice , Mice, Inbred C57BL , Oligodeoxyribonucleotides/immunology , Toll-Like Receptor 9/immunology , Vaccines, Subunit/immunology , Viral Envelope Proteins/immunologyABSTRACT
Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.
Subject(s)
Antiviral Agents/pharmacology , CD4-Positive T-Lymphocytes/virology , HIV Infections/immunology , Proteins/pharmacology , T-Lymphocytes, Cytotoxic/immunology , Virus Latency/drug effects , Enzyme-Linked Immunosorbent Assay , Enzyme-Linked Immunospot Assay , Flow Cytometry , Humans , Polymerase Chain Reaction , Recombinant Fusion Proteins , Virus Activation/drug effectsABSTRACT
HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs.
Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV Infections/immunology , HIV-1/drug effects , Leukocytes, Mononuclear/virology , T-Lymphocytes, Cytotoxic/virology , CD4 Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , HIV Antibodies/immunology , HIV Antibodies/therapeutic use , HIV Infections/therapy , HIV-1/immunology , Humans , Leukocytes, Mononuclear/immunology , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.
Subject(s)
Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Lentivirus/genetics , Sindbis Virus/genetics , Viral Envelope Proteins/genetics , Genetic Vectors/administration & dosage , HEK293 Cells , Humans , Immunity, Cellular/immunology , Tissue DistributionABSTRACT
BACKGROUND & AIMS: Silymarin, an extract from the seeds of the milk thistle plant Silybum marianum, has been used for centuries for the treatment of chronic liver diseases. Despite common use by patients with hepatitis C in the United States, its clinical efficacy remains uncertain. The goal of this study was to determine whether silymarin has in vitro effects on immune function that might have implications for its potential effect on hepatitis C virus (HCV)-induced liver disease. METHODS: Freshly isolated peripheral blood mononuclear cells (PBMC) and T cells from HCV-infected and uninfected subjects were tested in vitro for responses to nonspecific and antigenic stimulation in the presence and absence of a standardized preparation of silymarin (MK001). RESULTS: Minimal MK001 toxicity on PBMC was found at concentrations between 5 and 40 microg/mL. MK001 dose dependently inhibited the proliferation and secretion of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-2 by PBMC stimulated with anti-CD3. In addition, MK001 inhibited proliferation by CD4(+) T cells to HCV, Candida, and tetanus protein antigens and by HLA-A2/HCV 1406-1415-specific CD8(+) T cells to allogeneic stimulation. MK001 inhibited T-cell TNF-alpha and IFN-gamma cytokine secretion to tetanus and Candida protein antigens. Finally, MK001 inhibited nuclear factor-kappaB transcriptional activation after T-cell receptor-mediated stimulation of Jurkat T cells, consistent with its ability to inhibit Jurkat T-cell proliferation and secretion of IL-2. CONCLUSIONS: Silymarin's ability to inhibit the proliferation and proinflammatory cytokine secretion of T cells, combined with its previously described antiviral effect, suggests a possible mechanism of action that could lead to clinical benefit during HCV infection.
Subject(s)
Cell Proliferation/drug effects , Cytokines/metabolism , Hepatitis C/metabolism , Hepatitis C/pathology , Silymarin/pharmacology , T-Lymphocytes/drug effects , Antioxidants/pharmacology , Case-Control Studies , Cell Line , Cells, Cultured , Dose-Response Relationship, Drug , Hepacivirus , Humans , In Vitro Techniques , Interferon-gamma/metabolism , Interleukin-2/metabolism , Jurkat Cells , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The virological synapse (VS) is a specialized molecular structure that facilitates the transfer of certain lymphotropic viruses into uninfected T cells. However, the role of the VS in the transfer of nonlymphotropic viruses into T cells is unknown. Herpes simplex virus (HSV) has been shown in vitro to infect T cells and modulate T-cell receptor function, thereby suppressing T-cell antiviral function. However, whether such infection of T cells occurs in vivo is unknown. Here, we examined whether T-cell infection could be observed in human HSV disease and investigated the mechanism of HSV entry into T cells. We found that HSV-infected T cells were readily detectable during human disease, suggesting that infection and modulation of T-cell function plays a role in human immunopathology. HSV infection of both CD4(+) and CD8(+) T cells occurred much more efficiently via direct cell-to-cell spread from infected fibroblasts than by cell-free virus. Activation of T cells increased their permissivity to HSV infection. Cell-to-cell spread to T cells did not require HSV glycoproteins E and I (gE and gI), which are critical for cell-to-cell spread between epithelial cells. Transfer of HSV to T cells required gD, and the four known entry receptors appear to be contributing to viral entry, with a dominant role for the herpesvirus entry mediator and nectin-1. VS-like structures enriched in activated lymphocyte function-associated antigen 1 (LFA-1) were observed at the point of contact between HSV-infected fibroblasts and T cells. Consistent with spread occurring via the VS, transfer of HSV was increased by activation of LFA-1, and cell-to-cell spread could be inhibited by antibodies to LFA-1 or gD. Taken together, these results constitute the first demonstration of VS-dependent cell-to-cell spread for a predominantly nonlymphotropic virus. Furthermore, they support an important role for infection and immunomodulation of T cells in clinical human disease. Targeting of the VS might allow selective immunopotentiation during infections with HSV or other nonlymphotropic viruses.
Subject(s)
Herpes Simplex/virology , Herpesvirus 1, Human/physiology , T-Lymphocytes/virology , Virus Internalization , Animals , Cell Adhesion Molecules/metabolism , Chlorocebus aethiops , Fibroblasts/virology , Herpes Simplex/immunology , Herpesvirus 1, Human/immunology , Humans , Jurkat Cells , Lymphocyte Activation , Mice , Nectins , Receptors, Tumor Necrosis Factor, Member 14/metabolism , Vero Cells , Viral Envelope Proteins/metabolismABSTRACT
The Us5 gene of herpes simplex virus (HSV) encodes glycoprotein J (gJ). The only previously reported function of gJ was its ability to inhibit apoptosis. However, the mechanism by which gJ prevents apoptosis is not understood, and it is not known whether gJ mediates additional cellular effects. In this study, we evaluated the expression, localization, and cellular effects of Us5/gJ. Us5 was first expressed 4 h after infection. gJ was detectable at 6 h and was expressed in glycosylated and unglycosylated forms. Us5 was regulated as a late gene, with partial dependency on DNA replication for expression. Us5 expression was delayed in the absence of ICP22; furthermore, expression of Us5 in trans protected cells from apoptosis induced by an HSV mutant with deletion of ICP27, suggesting that the antiapoptotic effects of ICP22 and ICP27 are mediated in part through effects on gJ expression. Within HSV-infected or Us5-transfected cells, gJ was distributed widely, especially to the endoplasmic reticulum, trans-Golgi network, and early endosomes. gJ interacted with F(o)F(1) ATP synthase subunit 6 by a yeast two-hybrid screen and had strong antiapoptotic effects, which were mediated by protein rather than mRNA. Antiapoptotic activity required the extracellular and transmembrane domains of gJ, but not the intracellular domain. Consistent with inhibition of F(o)F(1) ATP synthase function, Us5 was required for HSV-induced reactive oxygen species (ROS) formation, and gJ was sufficient to induce ROS in Us5-transfected cells. Thus, HSV gJ is a multifunctional protein, modulating other cellular processes in addition to inhibition of apoptosis.
Subject(s)
Organelles/chemistry , Reactive Oxygen Species/metabolism , Simplexvirus/immunology , Viral Envelope Proteins/immunology , Viral Envelope Proteins/metabolism , Animals , Apoptosis , Cell Line , Chlorocebus aethiops , Gene Expression Regulation, Viral , Glycosylation , Humans , Protein Binding , Proton-Translocating ATPases/metabolism , Time Factors , Two-Hybrid System Techniques , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/geneticsABSTRACT
OBJECTIVE: To define the relationships between molecular measures of viral persistence in blood (i.e., plasma viremia, cellular HIV-1 DNA, and mRNA) and expressed or inducible virus from resting CD4 T cells of individuals on suppressive antiretroviral therapy. DESIGN: We compared molecular measurements of HIV-1 in plasma and in uncultured peripheral blood mononuclear cells (PBMCs) to the levels of virions produced by either unstimulated or phorbol myristate acetate and ionomycin (PMA/iono)-stimulated PBMC or resting CD4 T cells from 21 donors on suppressive antiretroviral therapy. RESULTS: We found that unstimulated virion release from cultured resting CD4 T cells was positively correlated with the levels of plasma viremia in vivo (Spearman rhoâ=â0.67, Pâ=â0.0017). We also found that levels of both cellular HIV-1 DNA and unspliced HIV-1 mRNA per million uncultured PBMC were positively correlated with the levels of inducible virion release from both PMA/iono-stimulated PBMC (total HIV-1 DNA: rhoâ=â0.64, Pâ=â0.0017; unspliced HIV-1 RNA: rhoâ=â0.77, Pâ<â0.001) and PMA/iono-stimulated resting CD4 T cells (total HIV-1 DNA: rhoâ=â0.75, Pâ<â0.001; unspliced HIV-1 RNA: rhoâ=â0.75, Pâ<â0.001). CONCLUSION: These results show for the first time that there are strong associations between in-vivo measures of HIV-1 persistence and ex-vivo measures of spontaneous and inducible virus production from cultured PBMC and resting CD4 T cells. Findings from this study provide insight into the biology of HIV-1 persistence and suggest methods to guide the evaluation of clinical strategies to reduce the size of the viral reservoir.
Subject(s)
Anti-Retroviral Agents/administration & dosage , Biomarkers/blood , DNA, Viral/blood , HIV Infections/drug therapy , HIV Infections/virology , RNA, Viral/blood , Sustained Virologic Response , Adult , Aged , Cells, Cultured , Cross-Sectional Studies , Female , Humans , Ionomycin/metabolism , Leukocytes, Mononuclear/virology , Male , Middle Aged , Tetradecanoylphorbol Acetate/metabolism , Virus Activation/drug effectsSubject(s)
Antibodies/immunology , Caspases/immunology , Caspases/metabolism , Cytotoxicity, Immunologic , T-Lymphocytes/immunology , Caspase 3 , Chromium Radioisotopes , Enzyme Activation , Flow Cytometry , Humans , Minor Histocompatibility Antigens/immunology , Minor Histocompatibility Antigens/metabolismABSTRACT
BACKGROUND: Non-invasive diagnostic assays to evaluate mitochondrial toxicity could have significant clinical utility for HIV-infected individuals on antiretroviral therapy (ART). METHODS: This study compared the ratio of mitochondrial to nuclear DNA determined by quantitative polymerase chain reaction (qPCR) to the ratio of mitochondrial to nuclear-encoded proteins by flow cytometry, in peripheral blood mononuclear cells from 73 HIV-infected individuals with and without risk factors for mitochondrial toxicity. RESULTS: PCR detected similar mitochondrial/nuclear DNA in HIV-infected individuals without a history of ART, and those receiving ART with lipodystrophy, lipoatrophy, or a history of suspected lactic acidosis. However, the ratio was significantly greater in ART-untreated compared to those receiving either stavudine or didanosine. In contrast, flow cytometry did not detect any differences in mitochondrial/nuclear protein (Lin et al., Cytometry B 2009;76B:181-190). There was no correlation between the assays (rho = -0.05, P = 0.65). CONCLUSIONS: Assessment of the mitochondrial/nuclear DNA ratio by qPCR performed better than the mitochondrial/nuclear-encoded protein ratio by flow cytometry to detect adverse effects of nucleoside analogs on mitochondria.
Subject(s)
DNA, Mitochondrial/analysis , HIV Infections/diagnosis , Mitochondria/pathology , Mitochondrial Proteins/analysis , Acidosis, Lactic , Adult , Anti-HIV Agents/therapeutic use , Antiretroviral Therapy, Highly Active , Didanosine/therapeutic use , Female , Flow Cytometry , HIV Infections/blood , Humans , Leukocytes, Mononuclear/cytology , Lipodystrophy , Male , Middle Aged , Real-Time Polymerase Chain Reaction , Stavudine/therapeutic useABSTRACT
BACKGROUND: Real-time PCR has quantified decreased mitochondrial DNA levels in association with nucleoside reverse transcriptase inhibitor (NRTI) therapy of HIV-infected populations. However, real-time PCR is best suited to distinguish log differences in an analyte. In an effort to monitor individuals in more detail, we developed a flow cytometric assay to gauge mitochondrial function. METHODS: Flow cytometric quantification of a mitochondrial DNA-encoded mitochondrial protein (cytochrome c oxidase subunit I (COX-I)) and a nuclear DNA-encoded mitochondrial protein [ATP synthase subunit D (Sub-D)] was optimized and validated. RESULTS: Intra-assay and interassay variability was low using peripheral blood mononuclear cells (PBMCs) (CV of 6.15% for COX-I and 7.11% Sub-D, and 9.38% and 9.83% for COX-I and Sub-D, respectively). Mitochondrial protein depletion was evident with in vitro treatment of cells with ethidium bromide (EtBr) and zalcitabine (ddC). Mitochondrial protein expression in 40 healthy adults clustered tightly. Depletion of mitochondrial protein, however, was neither detected in cryopreserved PBMC from NRTI-treated children (n = 9) nor in adults with a history of symptoms consistent with mitochondrial toxicity or ongoing treatment with didanosine (ddI) or stavudine (d4T) (n = 51). CONCLUSIONS: A validated flow cytometric assay allows simultaneous detection of mitochondrial DNA and nuclear DNA encoded proteins at the single cell level, offering a method to monitor for mitochondrial function. Prospective studies are required to evaluate whether mitochondrial protein loss is observed in at-risk patients prior to the onset of symptoms from mitochondrial dysfunction.
Subject(s)
Ethidium/toxicity , Leukocytes, Mononuclear/cytology , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Zalcitabine/toxicity , Adenosine Triphosphatases/metabolism , Adult , Child , Child, Preschool , DNA, Mitochondrial/genetics , Dose-Response Relationship, Drug , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Flow Cytometry , Humans , Infant , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/enzymology , Middle Aged , Mitochondria/enzymology , Mitochondrial Proteins/genetics , Reproducibility of Results , Sensitivity and Specificity , Young AdultABSTRACT
Herpes simplex virus (HSV)-specific T cells are essential for viral clearance. However, T cells do not prevent HSV latent infection or reactivation, suggesting that HSV has the potential to modulate T-cell function. T-cell receptor (TCR) stimulation is a potent and specific means of activating T cells. To investigate how HSV affects T-cell function, we have analyzed how HSV affects TCR-stimulated intracellular signaling and cytokine synthesis in mock-infected and HSV-infected T cells. Mock-infected T cells stimulated through the TCR synthesized a broad range of cytokines that included the proinflammatory cytokines tumor necrosis factor alpha, gamma interferon, and interleukin-2. In contrast, HSV-infected T cells stimulated through the TCR selectively synthesized interleukin-10, a cytokine that suppresses cellular immunity and favors viral replication. To achieve selective interleukin-10 synthesis, HSV differentially affected TCR signaling pathways. HSV inhibited TCR-stimulated formation of the linker for activation of the T-cell signaling complex, and HSV inhibited TCR-stimulated NF-kappaB activation. At the same time, HSV activated the p38 and JNK mitogen-activated protein kinases as well as the downstream transcription factors ATF-2 and c-Jun. HSV did not inhibit TCR-stimulated activation of STAT3, a transcription factor involved in interleukin-10 synthesis. The activation of p38 was required for interleukin-10 synthesis in HSV-infected T cells. The ability of HSV to differentially target intracellular signaling pathways and transform an activating stimulus into an immunosuppressive response represents a novel strategy for pathogen-mediated immune modulation. Selective, TCR-stimulated interleukin-10 synthesis may play an important role in HSV pathogenesis.
Subject(s)
Interleukin-10/biosynthesis , Receptors, Antigen, T-Cell/metabolism , Simplexvirus/immunology , T-Lymphocytes/immunology , T-Lymphocytes/virology , p38 Mitogen-Activated Protein Kinases/metabolism , Activating Transcription Factor 2/metabolism , Cell Line , Humans , MAP Kinase Kinase 4/antagonists & inhibitors , MAP Kinase Kinase 4/metabolism , MicroRNAs , Phosphorylation , Receptors, Antigen, T-Cell/agonists , STAT3 Transcription Factor/metabolism , Signal Transduction , Viral Proteins/antagonists & inhibitors , Viral Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
T cells are an essential component of the immune response against herpes simplex virus (HSV) infection. We previously reported that incubation of T cells with HSV-infected fibroblasts inhibits subsequent T cell antigen receptor signal transduction. In the current study, we found that incubation of T cells with HSV-infected fibroblasts also leads to apoptosis in exposed T cells. Apoptosis was observed in Jurkat cells, a T cell leukemia line, and also in CD4(+) cells isolated from human peripheral blood mononuclear cells. Direct infection of these cells with HSV also resulted in apoptosis. Clinical isolates of both HSV type 1 and 2 induced apoptosis in infected T cells at comparable levels to cells infected with laboratory strains of HSV, suggesting an immune evasion mechanism that may be clinically relevant. Further understanding of these viral immune evasion mechanisms could be exploited for better management of HSV infection.
Subject(s)
Apoptosis/physiology , B-Lymphocytes/immunology , Herpesvirus 1, Human/physiology , Herpesvirus 2, Human/physiology , Receptors, Antigen, B-Cell/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Animals , B-Lymphocytes/cytology , Chlorocebus aethiops , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/virology , Humans , Jurkat Cells , Phosphorylation , Signal Transduction , T-Lymphocytes/cytology , Vero CellsABSTRACT
T lymphocytes are an essential component of the immune response against HSV infection. We previously reported that T cells became functionally impaired or inactivated after contacting HSV-infected fibroblasts. In our current study, we investigate the mechanisms of inactivation. We report that HSV-infected fibroblasts or HSV alone can inactivate T cells by profoundly inhibiting TCR signal transduction. Inactivation requires HSV penetration into T cells but not de novo transcription or translation. In HSV-inactivated T cells stimulated through the TCR, phosphorylation of Zap70 occurs normally. However, TCR signaling is inhibited at linker for activation of T cells (LAT) and at steps distal to LAT in the TCR signal cascade including inhibition of calcium flux and inhibition of multiple MAPK. Inactivation of T cells by HSV leads to the reduced phosphorylation of LAT at tyrosine residues critical for TCR signal propagation. Treatment of T cells with tyrosine phosphatase inhibitors attenuates inactivation by HSV, and stimulus with a mitogen that bypasses LAT phosphorylation overcomes inactivation. Our findings elucidate a potentially novel method of viral immune evasion that could be exploited to better manage HSV infection, aid in vaccine design, or allow targeted manipulation of T cell function.
Subject(s)
Receptors, Antigen, T-Cell/antagonists & inhibitors , Signal Transduction/immunology , Simplexvirus/immunology , T-Lymphocytes/virology , Calcium Signaling/physiology , Cell Line, Transformed , Clone Cells , Cytokines/biosynthesis , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/physiology , Fibroblasts/virology , Glycoproteins/metabolism , Humans , Ionomycin/pharmacology , Jurkat Cells , Lymphocyte Activation/immunology , Phosphorylation , Receptors, Antigen, T-Cell/physiology , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor, Member 14 , Receptors, Virus/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Viral Envelope Proteins/metabolism , ZAP-70 Protein-Tyrosine Kinase/metabolismABSTRACT
To facilitate the study of the mechanisms of breast cancer metastasis we have previously characterized a pair of breast tumor cell lines that originate from the same breast tumor cell line MDA-MB-435, but which have diametrically opposite metastatic capabilities. These cell lines constitute a stable and accessible experimental system for the identification of metastasis-related genes and for the study of their role in the process of metastasis. In this study, we used a combination of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) fingerprinting and cDNA arrays (here termed "RAP-array") to identify genes differentially expressed with respect to metastatic phenotype. RAP-PCR was used to generate radioactive probes of reduced complexity for hybridization to nylon membranes containing 588 cDNAs of known identity. Single RAP-PCR fingerprint probes hybridized from 61 (10.4%) to 116 (19.7%) of the filter array targets, with a signal detection overlap of approximately 21%. A total of 344 (57%) of the 588 target genes were detected by five single RAP-PCR fingerprints. The advantage of using reduced complexity probes was highlighted by the fact that the combination of RAP probes before hybridization compromised the overall detection rate by up to 40%. Sequential application of RAP-PCR probes allowed the screening of a greater, and an alternative fraction of the transcript population than was achieved with a radiolabeled total cDNA probe. Verification by quantitative reverse transcriptase-PCR confirmed significantly increased expression of keratin 9 (>100-fold) in nonmetastatic breast tumor cells and of CD70 (fivefold) in metastatic cells. The differential expression of keratin 9 and CD70 was maintained between cells grown as primary xenografts in athymic mice. The RAP-array method enabled the detection of genes not revealed using other screening methods and that are candidates for further investigation in the context of metastatic phenotype.
Subject(s)
Antigens, CD , Breast Neoplasms/pathology , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Oligonucleotide Array Sequence Analysis/methods , Polymerase Chain Reaction/methods , Animals , CD27 Ligand , Cell Line, Tumor , DNA Fingerprinting , DNA Primers , Humans , Image Processing, Computer-Assisted , In Situ Hybridization , Keratins/biosynthesis , Membrane Proteins/biosynthesis , Mice , Models, Theoretical , Neoplasm Metastasis/pathology , Sensitivity and SpecificityABSTRACT
Numerous cell-to-cell signals tightly regulate CTL function. Human fibroblasts infected with HSV type 1 or 2 can generate such a signal and inactivate human CTL. Inactivated CTL lose their ability to release cytotoxic granules and synthesize cytokines when triggered through the TCR. Inactivation requires cell-to-cell contact between CTL and HSV-infected cells. However, inactivated CTL are not infected with HSV. The inactivation of CTL is sustainable, as CTL function remains impaired when the CTL are removed from the HSV-infected cells. IL-2 treatment does not alter inactivation, and the inactivated phenotype is not transferable between CTL, distinguishing this phenotype from traditional anergy and T regulatory cell models. CTL inactivated by HSV-infected cells are not apoptotic, and the inactivated state can be overcome by phorbol ester stimulation, suggesting that inactivated CTL are viable and that the signaling block is specific to the TCR. HSV-infected cells require the expression of U(S)3, a viral protein kinase, to transmit the inactivating signal. Elucidation of the molecular nature of this signaling pathway may allow targeted manipulation of CTL function.