Jaya Ahuja

DC-SIGN Mediates Cell-Free Infection and Transmission of Human T-Cell Lymphotropic Virus Type 1 by Dendritic Cells

ABSTRACT Despite the susceptibility of dendritic cells (DCs) to human T-cell lymphotropic virus type 1 (HTLV-1) infection and the defined role of these cells in disease pathogenesis, the mechanisms of viral binding to DCs have not been fully delineated. Recently, a glucose transporter, GLUT-1, heparan sulfate proteoglycans (HSPGs), and neuropilin-1 (NRP-1) were demonstrated to facilitate HTLV-1 entry into T cells. DCs express their own array of antigen receptors, the most important being the DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) with respect to retrovirus binding. Consequently, the role of DC-SIGN and other HTLV-1 attachment factors was analyzed in viral binding, transmission, and productive infection using monocyte-derived DCs (MDDCs), blood myeloid DCs, and B-cell lines expressing DC-SIGN. The relative expression of DC-SIGN, GLUT-1, HSPGs, and NRP-1 first was examined on both DCs and B-cell lines. Although the inhibition of these molecules reduced viral binding, HTLV-1 transmission from DCs to T cells was mediated primarily by DC-SIGN. DC-SIGN also was shown to play a role in the infection of MDDCs as well as model B-cell lines. The HTLV-1 infection of MDDCs also was achieved in blood myeloid DCs following the enhancement of virus-induced interleukin-4 production and subsequent DC-SIGN expression in this cell population. This study represents the first comprehensive analysis of potential HTLV-1 receptors on DCs and strongly suggests that DC-SIGN plays a critical role in HTLV-1 binding, transmission, and infection, thereby providing an attractive target for the development of antiretroviral therapeutics and microbicides.

Modulation of dendritic cell maturation and function by the Tax protein of human T cell leukemia virus type 1

Human T cell leukemia virus type 1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by the generation of an intense CTL cell response directed against the viral transactivator protein Tax. In addition, patients diagnosed with HAM/TSP exhibit rapid activation and maturation of dendritic cells (DC), likely contributing to the robust, Tax‐specific CTL response. In this study, extracellular Tax has been shown to induce maturation and functional alterations in human monocyte‐derived DC, critical observations being confirmed in freshly isolated myeloid DC. Tax was shown to promote the production of proinflammatory cytokines and chemokines involved in the DC activation process in a dose‐ and time‐dependent manner. Furthermore, Tax induced the expression of DC activation (CD40, CD80, and CD86) and maturation (CD83) markers and enhanced the T cell proliferation capability of DC. Heat inactivation of Tax resulted in abrogation of these effects, indicating a requirement for the native structure of Tax, which was found to bind efficiently to the DC membrane and was internalized within a few hours, suggesting that extracellular Tax may possess an intracellular mechanism of action subsequent to entry. Finally, inhibitors of cellular signaling pathways, NF‐κB, protein kinase, tyrosine kinase, and phospholipase C, were shown to inhibit Tax‐mediated DC activation. This is the first study reporting the immunomodulatory effects of extracellular Tax in the DC compartment. These results suggest that DC, once exposed to Tax by uptake from the extracellular environment, can undergo activation, providing constant antigen presentation and costimulation to T cells, leading to the intense T cell proliferation and inflammatory responses underlying HAM/TSP.

Identification of Human T Cell Leukemia Virus Type 1 Tax Amino Acid Signals and Cellular Factors Involved in Secretion of the Viral Oncoprotein

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of a number of pathologic abnormalities, including adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The viral oncoprotein Tax has been implicated in the pathogenesis of these diseases. Recently, cell-free Tax was detected in the cerebrospinal fluid of HAM/TSP patients, implying that extracellular Tax may be relevant to neurologic disease. Additionally, the presence of a nuclear export signal within Tax and its active secretion has been demonstrated in vitro. However, the mechanism of Tax secretion remains to be established. Studies reported herein elucidate the process of Tax secretion and identify domains of Tax critical to its subcellular localization and secretion. Tax was shown to interact with a number of cellular secretory pathway proteins in both the model cell line BHK (baby hamster kidney)-21 and an HTLV-1-infected T cell line, C8166, physiologically relevant to HTLV-1-induced disease. Silencing of selected components of the secretory pathway affected Tax secretion, further confirming regulated secretion of Tax. Additionally, mutations in two putative secretory signals within Tax DHE and YTNI resulted in aberrant subcellular localization of Tax and significantly altered protein secretion. Together, these studies demonstrate that Tax secretion is a regulated event facilitated by its interactions with proteins of the cellular secretory pathway and the presence of secretory signals within the carboxyl-terminal domain of the protein.

Use of human antigen presenting cell gene array profiling to examine the effect of human T-cell leukemia virus type 1 Tax on primary human dendritic cells

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8+ cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11–19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naïve CD8+ T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease.

AP-1-directed human T cell leukemia virus type 1 viral gene expression during monocytic differentiation

Human T cell leukemia virus type 1 (HTLV‐1) has previously been shown to infect antigen‐presenting cells and their precursors in vivo. However, the role these important cell populations play in the pathogenesis of HTLV‐1‐associated myelopathy/tropical spastic paraparesis or adult T cell leukemia remains unresolved. To better understand how HTLV‐1 infection of these important cell populations may potentially impact disease progression, the regulation of HTLV‐1 viral gene expression in established monocytic cell lines was examined. U‐937 promonocytic cells transiently transfected with a HTLV‐1 long‐terminal repeat (LTR) luciferase construct were treated with phorbol 12‐myristate 13‐acetate (PMA) to induce cellular differentiation. PMA‐induced cellular differentiation resulted in activation of basal and Tax‐mediated transactivation of the HTLV‐1 LTR. In addition, electrophoretic mobility shift analyses demonstrated that PMA‐induced cellular differentiation induced DNA‐binding activity of cellular transcription factors to Tax‐responsive element 1 (TRE‐1) repeat II. Supershift analyses revealed that factors belonging to the activator protein 1 (AP‐1) family of basic region/leucine zipper proteins (Fra‐1, Fra‐2, JunB, and JunD) were induced to bind to TRE‐1 repeat II during cellular differentiation. Inhibition of AP‐1 DNA‐binding activity by overexpression of a dominant‐negative c‐Fos mutant (A‐Fos) in transient expression analyses resulted in severely decreased levels of HTLV‐1 LTR activation in PMA‐induced U‐937 cells. These results have suggested that following infection of peripheral blood monocytes, HTLV‐1 viral gene expression may become up‐regulated by AP‐1 during differentiation into macrophages or dendritic cells.

DC-SIGN as a Receptor for HTLV-1 Binding, Entry and Infection

Human T cell leukemia virus type 1 (HTLV-1) has been identified as the etiologic agent of adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Numerous studies have demonstrated that patients diagnosed with HAM/TSP exhibit rapid activation and maturation of dendritic cells (DCs) while ATL is associated with their maturation defect. In addition to T cells, HTLV-1 is known to infect DCs. HTLV1 infection of DCs could alter general DC function or the specific processing and/or presentation of HTLV-1-specific peptides, potentially playing a major role in the course of HTLV-1-associated disease. In this regard, we have demonstrated that an important antigen receptor on DCs, DC-SIGN serves as a receptor for HTLV-1 binding using a quantum dot-based fluorescent binding assay. We have also demonstrated that gene silencing of DC-SIGN inhibits the infection of DC in a DC/T cell co-infection system. Furthermore, expression of DC-SIGN in B cells enhances viral binding, integration, and infection. These investigations, which consider the involvement of DC surface molecules in HTLV-1 pathogenesis, are the first explorations of the intricate mechanisms that underlie the interactions between DCs and HTLV-1. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

HTLV-1 Tax Protein Induces the Secretion of Th1 Cytokines and β-Chemokines from Dendritic Cells

HTLV-1-associated myelopathy/tropical spastic paraparesis is characterized by highly stimulated immune response that includes elevated levels of inflammatory cytokines/ chemokines, and oligoclonal expansion of Tax-specific CD8+ cytotoxic T lymphocytes in the cerebrospinal fluid. Studies have shown that the HTLV-1 transactivator protein Tax is available for immune recognition by antigen presenting cells such as dendritic cells. In this study, we have shown that the treatment of monocyte-derived dendritic cells (MDDCs) with extracellular Tax induces the secretion of Th1 cytokines (IL-12, and TNF-α) and βchemokines (MIP-Iα, MIP-Iβ, and RANTES). A significant dose-dependent increase was observed with IL-12 (5-, 7-, and 24-fold) and TNF-α (5-, 6-, and 9.6-fold) with Tax treatment for 24 hr at concentrations of 0.1, 1, and 10 mg/ ml, respectively. All three chemokines exhibited both doseand time-dependent increase in the presence of Tax. More specifically, after 24 hr treatment with Tax (0.1, 1 and 10 μg/ml), MIP-1a was induced by 3.7-, 5.3-, and 6fold, MIP-1b by 2-, 3.7-, and 3.7-fold, and RANTES by 7-, 8-, and 20-fold, respectively. The mRNA expression of these cytokines/chemokines was confirmed by real time PCR and was in direct correlation with observations regarding their protein expression. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Activation and Maturation of Human Dendritic Cells by Extracellular Tax Protein of HTLV-1

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by the generation of an intense cytotoxic T cell (CTL) response directed against oncoprotein Tax. Previous studies have suggested that Tax may be available for immune recognition by dendritic cells (DCs). In this study, we have shown that purified Tax protein efficiently bound and localized to the cell membrane of monocyte-derived dendritic cells (MDDCs) and was internalized within a few hours. After uptake, Taxinduced expression of DC activation markers MHC class I and II, and costimulatory molecules as well as the DC maturation marker, CD83. Tax has also promoted the production of major immune-directing cytokines IL-12, TNF-α, and proinflammatory chemokines MIP-1α, MIP1β, and RANTES. The inhibitors of NF-κB have abrogated Tax-induced secretion of cytokines/chemokines indicating a role for NF-κB signaling in Tax-mediated immune response. Finally, Tax enhanced the allogenic and antigenspecific T cell proliferation capability of MDDCs. These results have indicated that extracellular Tax may selectively target MDDCs, be taken up by these cells and promote their maturation and antigen-presenting functions, driving a Th1-type immune response. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Human Antigen Presenting Cell Gene Array Profiling of the Effect of Human T Cell Leukemia Virus Type 1 Tax Protein on Dendritic Cells

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by a highly stimulated immune response including the oligoclonal expansion of CD8+ cytotoxic T lymphocytes specific for viral oncoprotein Tax. Studies have demonstrated that Tax may be available for immune recognition by dendritic cells (DCs). In this study, a pathway-specific human dendritic and antigen presenting cell gene array has been used to study the global transcriptional changes mediated by extracellular Tax on monocyte-derived dendritic cells (MDDCs). Of the 192 genes examined, approximately 100 genes were differentially expressed after extracellular exposure to Tax. These genes were functionally categorized as the genes involved in antigen uptake and presentation (MHC class II molecule: HLA-DMB, DC-SIGN), intracellular adhesion molecules (ICAM-1 and -2), T cell costimulatory molecule (ICOS), cell surface receptor (CD47, toll-like receptors: TLR-1, -3, -6, -9), and several cytokines, chemokines; and their receptors (IFN-g, IL-6, IL-12, TNF-α, IL-17, CCL5, CCL20, TNFSF4). Additionally, the expression of cellular signal transduction genes: ATF4, NFKB1, RELA, RELB (members of the NFkB family), G1P2 and G1P3 (interferon inducible) was also significantly altered. The expression of DC activation markers was confirmed by real-time PCR and was in direct correlation with the microarray observations. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005