Sharrón L Manuel

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.

Dendritic cells in autoimmune diseases and neuroinflammatory disorders.

Dendritic cells are the most potent antigen presenting cells and have long been recognized as key regulators of the immune system, linking both stimulatory and inhibitory components of normal immunity. While DCs are fully characterized with respect to primary and secondary immune responses, their unique role in coordinating central and peripheral tolerance is just emerging. It is increasingly evident that the failure of DCs ability to maintain tolerance can lead to autoimmune and/or inflammatory diseases. However, existing literature highlighting participation of DCs in several autoimmune phenomena is scattered and remains underappreciated. This review is a comprehensive account of current knowledge characterizing the role of DCs in various autoimmune diseases including psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Sjogrens syndrome, and diabetes. Additionally, it provides a rare description of DCs participation in various neuroinflammatory disorders including multiple sclerosis, HAM/TSP, Alzheimer disease and prion-associated diseases. Finally, a detailed description of the possible mechanisms of DC involvement in regulating immune response towards self versus non-self is discussed. Overall, the goal of this review is to establish DCs in the interface of tolerance and autoimmunity and generate a global interest in this field in order to exploit DC potential for the therapy of inflammatory diseases.

Presentation of human T cell leukemia virus type 1 (HTLV-1) Tax protein by dendritic cells: the underlying mechanism of HTLV-1-associated neuroinflammatory disease

HTLV‐1 is the etiologic agent of a debilitating neurologic disorder, HAM/TSP. This disease features a robust immune response including the oligoclonal expansion of CD8+ CTLs specific for the viral oncoprotein Tax. The key pathogenic process resulting in the proliferation of CTLs and the presentation of Tax peptide remains uncharacterized. We have investigated the role of APCs, particularly DCs, in priming of the anti‐Tax CTL response under in vitro and in vivo conditions. We investigated two routes (direct vs. indirect) of Tax presentation using live virus, infected primary CD4+/CD25+ T cells, and the CD4+ T cell line (C8166, a HTLV‐1‐mutated line that only expresses Tax). Our results indicated that DCs are capable of priming a pronounced Tax‐specific CTL response in cell cultures consisting of naïve PBLs as well as in HLA‐A*0201 transgenic mice (line HHD II). DCs were able to direct the presentation of Tax successfully through infected T cells, live virus, and cell‐free Tax. These observations were comparable with those made with a known stimulant of DC maturation, a combination of CD40L and IFN‐γ. Our studies clearly establish a role for this important immune cell component in HTLV‐1 immuno/neuropathogenesis and suggest that modulation of DC functions could be an important tool for therapeutic interventions.

Unique Cytokine/Chemokine Signatures for HIV-1 and HCV Mono-infection versus Co-infection as Determined by the Luminex® Analyses

Liver disease caused by HIV-1/HCV co-infection is characterized by the inflammation and cell-death. The co-existence of these two chronic viral infections also alters the cytokine production in vivo. The ability to visualize changes in cytokine networks with the onset and progression of disease or treatment is critical to advance our understanding of the immune response to pathogens. The recent Luminex® technology has revolutionized the simultaneous detection and quantitation of several cytokines and chemokines in clinical samples that are generally available in small quantities. We have applied this technology to analyze the plasma samples from patients who have either HIV-1 or HCV mono-infection or HIV-1/HCV co-infection and monitored the presence of 23 cytokines and chemokines. Of these, 8 (IFN-α2, IL-2, IL-3, IL-6, IL-8, IL-12p70, IL-15 and RANTES) cytokines were expressed at higher levels in the co-infected individuals. Interestingly, in case of HIV-1 mono-infected individuals, the levels of the proinflammatory cytokines IFN-γ and TNF-α were increased. Standard correlation clustering of the normalized data demonstrated unique plasma cytokine signatures for HIV-1/HCV co-infected individuals. These signatures were characterized not only by an up regulation of the aforementioned antiviral mediators but also by a marked down regulation in the chemokines Eotaxin and MIP-1α when compared to mono-infected individuals. Luminex®- based analyses have proven to be a powerful tool for therapeutic immunomonitoring, but may have an even greater impact in the discovery of the underlying immune response at all phases of infection. The study presented herein has potential to offer insight into the underlying mechanisms of immunopathogenesis of HIV-1/HCV co-infection.

Depletion of Dendritic Cells Enhances Susceptibility to Cell-Free Infection of Human T Cell Leukemia Virus Type 1 in CD11c-Diphtheria Toxin Receptor Transgenic Mice

Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1–associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. The genesis of these diseases is believed to be associated with the route (mucosa versus blood) and mode (cell-free versus cell-associated) of primary infection as well as the modulation of dendritic cell (DC) functions. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Moloney murine leukemia virus to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-diphtheria toxin receptor transgenic mouse model system that permits conditional transient depletion of CD11c+ DCs. We infected these transgenic mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free but not cell-associated HTLV-1 in both CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA in the non-CD4 fraction. Moreover, depletion of DCs significantly dampened the cellular immune response (IFN-γ+CD8+ T cells) against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of adult T cell leukemia and HTLV-1–associated myelopathy/tropical spastic paraparesis after the initial infection.

Lack of Recall Response to Tax in ATL and HAM/TSP Patients But Not in Asymptomatic Carriers of Human T-cell Leukemia Virus Type 1

Purpose & MethodsThe immunopathogenic mechanisms responsible for debilitating neurodegenerative and oncologic diseases associated with human T-cell leukemia virus type 1 (HTLV-1) are not fully understood. Quality of cytotoxic T lymphocytes (CTLs) is being increasingly associated with the outcome of persistent HTLV-1 infection. In this respect, a patient cohort (from HTLV-1 endemic region) consisting of seronegative controls (controls), asymptomatic carriers (ACs), and patients with adult T-cell leukemia (ATL) or HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) was analyzed for CD8+ T cells polyfunctionality in response to the viral antigen Tax.ResultsCompared to ACs, ATL and HAM/TSP patients had lower frequency and polyfunctionality of CTLs in response to Tax suggesting dysfunction of CD8+ T cells in these individuals. As an underlying mechanism, programmed death-1 (PD-1) receptor was found to be highly unregulated in Tax-responsive as well as total CD8+ T cells from ATL and HAM/TSP but not from ACs and directly correlated with the lack of polyfunctionality in these individuals. Further, PD-1 expression showed a direct whereas MIP-1α expression had an indirect correlation with the proviral load providing new insights about the immunopathogenesis of HTLV-associated diseases. Additionally, we identified key cytokine signatures defining the immune activation status of clinical samples by the luminex assay.ConclusionsCollectively, our findings suggest that reconstitution of fully functional CTLs, stimulation of MIP-1α expression, and/or blockade of the PD-1 pathway are potential approaches for immunotherapy / therapeutic vaccine against HTLV-mediated diseases.

An altered maturation and adhesion phenotype of dendritic cells in diseased individuals compared to asymptomatic carriers of human T-cell leukemia virus type 1

As critical effectors of antiviral immune response, dendritic cells (DCs) are implicated to play an important role in determining the outcome of HTLV-1 infection. However, a complete understanding of their role in any disease pathogenesis requires extensive assessment of the phenotypic and functional state of DCs. To enable this, we developed a polychromatic antibody cocktail comprising key phenotypic and functional markers of DCs and applied it in a patient cohort from the HTLV-1 endemic region, Jamaica, consisted of seronegative controls, asymptomatic carriers (ACs), ATL, and HAM/TSP patients. This ex vivo analyses included two major subsets of blood DCs, myeloid and plasmacytoid (mDCs and pDCs, respectively). The comparative analyses of results demonstrated a decreased pDC frequency in both ATL and HAM/TSP patients as compared to ACs and seronegative controls. Similarly, CD86 expression on both mDCs and pDCs was significantly higher in HAM/TSP (but not ATL) patients compared to ACs. Interestingly, HLA-DR expression was significantly lower on pDCs of patients as compared to carriers, whereas for mDCs, only the HAM/TSP group had significantly lower expression of HLA-DR. Unlike HAM/TSP individuals, ATL individuals had significantly higher HLA-ABC expression on mDCs compared to ACs. Finally, both mDCs and pDCs of HAM/TSP patients had significantly higher PD-L1 expression compared to ACs. Overall, this study suggests that DCs are differentially regulated between patients (ATL and HAM/TSP) and carriers of HTLV-1 and could provide an important tool to understand HTLV-1 immunopathogenesis during infection and disease.

Depletion of dendritic cells enhances susceptibility to cell-free infection of human T cell leukemia virus type 1 in CD11c-diphtheria toxin receptor transgenic mice

HTLV-1 is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. The genesis of these diseases is believed to be associated with the route (mucosa versus blood) and mode (cell-free versus cell-associated) of primary infection as well as the modulation of dendritic cell (DC) functions. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Moloney murine leukemia virus to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-diphtheria toxin receptor transgenic mouse model system that permits conditional transient depletion of CD11c(+) DCs. We infected these transgenic mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free but not cell-associated HTLV-1 in both CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA in the non-CD4 fraction. Moreover, depletion of DCs significantly dampened the cellular immune response against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of adult T cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis after the initial infection.

Dynamics of dendritic cells and T cells in HTLV-1-associated neuroinflammatory disease: implications in immunomodulatory therapies and diagnostic tools

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), is a debilitating neurodegenerative disease characterized by a robust immune response including the oligoclonal expansion of cytotoxic T lymphocytes (CTLs) specific for the viral oncoprotein Tax. However, the underlying mechanism resulting in the disease process is currently unknown. The CTL response is affected by many factors including the efficiency of epitope processing and presentation. In this respect, dendritic cells (DCs), the most potent antigen presenting cells, have long been recognized as key regulators of the immune system. We have previously demonstrated that DCs are capable of priming a pronounced Tax-specific CTL response in naive PBLs and in HLA-A2 transgenic mice. Since DCs are such crucial cells of the immune system, an extensive assessment of their function and interaction with T cells in HAM/TSP is critical. Therefore, utilizing a newly standardized DC and pre-standardized T cell polychromatic antibody cocktails, we have investigated the immune activation of these cells in HTLV-1 infected samples from the Jamaican region including the seronegative controls, asymptomatic carriers (ACs), and HAM/TSP patients. The extensive immune cell profiling was compared to the matched proviral loads and Tax mRNA levels leading to the identification of unique signatures distinguishing ACs from HAM/TSP patients. Collectively, these studies possess great potential to enable immune cell monitoring and development of diagnostic and therapeutic strategies for the HTLV-associated neuroinflammatory disease.

Lack of recall response to Tax in ATL and HAM/TSP patients but not in asymptomatic carriers of human T-cell leukemia virus type 1

Purpose & Methods The immunopathogenic mechanisms responsible for debilitating neurodegenerative and oncologic diseases associated with human T-cell leukemia virus type 1 (HTLV-1) are not fully understood. Quality of cytotoxic T lymphocytes (CTLs) is being increasingly associated with the outcome of persistent HTLV-1 infection. In this respect, a patient cohort (from HTLV-1 endemic region) consisting of seronegative controls (controls), asymptomatic carriers (ACs), and patients with adult T-cell leukemia (ATL) or HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) was analyzed for CD8+ T cells polyfunctionality in response to the viral antigen Tax.