Regulation of human T-cell leukemia virus type 1 antisense promoter by myocyte enhancer factor-2C in the context of adult T-cell leukemia and lymphoma.

Adult T-cell leukemia and lymphoma (ATLL) is an intractable T-cell neoplasia caused by a retrovirus, namely human T-cell leukemia virus type 1 (HTLV-1). Patients suffering from ATLL present a poor prognosis and have a dearth of treatment options. In contrast to the sporadic expression of viral transactivator protein Tax present at the 5' promoter region long terminal repeats (LTR), HTLV-1 bZIP gene (HBZ) is encoded by 3'LTR (the antisense promoter) and maintains its constant expression in ATLL cells and patients. The antisense promoter is associated with selective retroviral gene expression and has been an understudied phenomenon. Herein, we delineate the activity of transcription factor MEF (myocyte enhancer factor)-2 family members, which were found to be enriched at the 3'LTR and play an important role in the pathogenesis of ATLL. Of the four MEF isoforms (A to D), MEF-2A and 2C were highly overexpressed in a wide array of ATLL cell lines and in acute ATLL patients. The activity of MEF-2 isoforms were determined by knockdown experiments that led to decreased cell proliferation and regulated cell cycle progression. High enrichment of MEF-2C was observed at the 3'LTR along with cofactors Menin and JunD resulting in binding of MEF-2C to HBZ at this region. Chemical inhibition of MEF-2 proteins resulted in the cytotoxicity of ATLL cells in vitro and reduction of proviral load in a humanized mouse model. Taken together, this study provides a novel mechanism of 3'LTR regulation and establishes MEF-2 signaling a potential target for therapeutic intervention for ATLL.

Human T cell leukemia virus type 1 and Zika virus: tale of two reemerging viruses with neuropathological sequelae of public health concern.

Human T cell leukemia virus type 1 (HTLV-1) and Zika virus (ZIKV) have been considered neglected viruses of low public health concern until recently when incidences of HTLV-1 and ZIKV were observed to be linked to serious immune-related disease and neurological complications. This review will discuss the epidemiology, genomic evolution, virus-host interactions, virulence factors, neuropathological sequelae, and current perspectives of these reemerging viruses. There are no FDA-approved therapeutics or vaccines against these viruses, and as such, it is important for clinical trials to focus on developing vaccines that can induce cell-mediated immune response to confer long-term protective immunity. Furthermore, attention should be paid to reducing the transmission of these viruses through unprotected sex, infected blood during sharing of contaminated needles, donated blood and organs, and vertical transmission from mother to baby via breastfeeding. There is an urgent need to re-evaluate repurposing current antiviral therapies as well as developing novel antiviral agents with enhanced efficacy due to the high morbidity rate associated with these two reemerging chronic viral diseases.

Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

BACKGROUND: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome. RESULTS: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-ß) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity. CONCLUSIONS: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

Effect of morphine and SIV on dendritic cell trafficking into the central nervous system of rhesus macaques.

Recruitment of immune cells such as monocytes/macrophages and dendritic cells (DCs) across the blood-brain barrier (BBB) has been documented in diseases involving neuroinflammation. Neuroinvasion by HIV leads to neurocognitive diseases and alters the permeability of the BBB. Likewise, many HIV patients use drugs of abuse such as morphine, which can further compromise the BBB. While the role of monocytes and macrophages in neuroAIDS is well established, research demonstrating the presence and role of DCs in the CNS during HIV infection has not been developed yet. In this respect, this study explored the presence of DCs in the brain parenchyma of rhesus macaques infected with a neurovirulent form of SIV (SIV mac239 R71/17E) and administered with morphine. Cells positive for DC markers including CD11c (integrin), macDC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin), CD83 (a maturation factor), and HLA-DR (MHC class II) were consistently found in the brain parenchyma of SIV-infected macaques as well as infected macaques on morphine. Control animals did not exhibit any DC presence in their brains. These results provide first evidence of DCs' relevance in NeuroAIDS vis-à-vis drugs of abuse and open new avenues of understanding and investigative HIV-CNS inflictions.

Targeting the C-type lectins-mediated host-pathogen interactions with dextran.

Dextran, the α-1,6-linked glucose polymer widely used in biology and medicine, promises new applications. Linear dextran applied as a blood plasma substitute demonstrates a high rate of biocompatibility. Dextran is present in foods, drugs, and vaccines and in most cases is applied as a biologically inert substance. In this review we analyze dextran's cellular uptake principles, receptor specificity and, therefore, its ability to interfere with pathogen-lectin interactions: a promising basis for new antimicrobial strategies. Dextran-binding receptors in humans include the DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) family receptors: DC-SIGN (CD209) and L-SIGN (the liver and lymphatic endothelium homologue of DC-SIGN), the mannose receptor (CD206), and langerin. These receptors take part in the uptake of pathogens by dendritic cells and macrophages and may also participate in the modulation of immune responses, mostly shown to be beneficial for pathogens per se rather than host(s). It is logical to predict that owing to receptor-specific interactions, dextran or its derivatives can interfere with these immune responses and improve infection outcome. Recent data support this hypothesis. We consider dextran a promising molecule for the development of lectin-glycan interaction-blocking molecules (such as DC-SIGN inhibitors) that could be applied in the treatment of diseases including tuberculosis, influenza, hepatitis B and C, human immunodeficiency virus infection and AIDS, etc. Dextran derivatives indeed change the pathology of infections dependent on DC-SIGN and mannose receptors. Complete knowledge of specific dextran-lectin interactions may also be important for development of future dextran applications in biological research and medicine.

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. RESULTS: Compared 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. CONCLUSIONS: Collectively, 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.

Murine FLT3 ligand-derived dendritic cell-mediated early immune responses are critical to controlling cell-free human T cell leukemia virus type 1 infection.

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. We observed previously that depletion of dendritic cells (DCs) in CD11c-diphtheria toxin receptor transgenic mice followed by infection with cell-free virus led to greater proviral and Tax mRNA loads and diminished cellular immune response compared with mice infected with cell-associated virus. To understand the significance of these in vivo results and explore the host-pathogen interaction between DCs and cell-free HTLV-1, we used FLT3 ligand-cultured mouse bone marrow-derived DCs (FL-DCs) and chimeric HTLV-1. Phenotypically, the FL-DCs upregulated expression of surface markers (CD80, CD86, and MHC class II) on infection; however, the level of MHC class I remained unchanged. We performed kinetic studies to understand viral entry, proviral integration, and expression of the viral protein Tax. Multiplex cytokine profiling revealed production of an array of proinflammatory cytokines and type 1 IFN (IFN-α) by FL-DCs treated with virus. Virus-matured FL-DCs stimulated proliferation of autologous CD3(+) T cells as shown by intracellular nuclear Ki67 staining and produced IFN-γ when cultured with infected FL-DCs. Gene expression studies using type 1 IFN-specific and DC-specific arrays revealed upregulation of IFN-stimulated genes, most cytokines, and transcription factors, but a distinct downregulation of many chemokines. Overall, these results highlight the critical early responses generated by FL-DCs on challenge with cell-free chimeric HTLV-1.

Apigenin improves cytotoxicity of antiretroviral drugs against HTLV-1 infected cells through the modulation of AhR signaling.

Objectives: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory autoimmune disease characterized by high levels of infected immortalized T cells in circulation, which makes it difficult for antiretroviral (ART) drugs to work effectively. In previous studies, we established that Apigenin, a flavonoid, can exert immunomodulatory effects to reduce neuroinflammation. Flavonoids are natural ligands for the aryl hydrocarbon receptor (AhR), which is a ligand activated endogenous receptor involved in the xenobiotic response. Consequently, we tested Apigenin's synergy in combination with ART against the survival of HTLV-1-infected cells. Methods: First, we established a direct protein-protein interaction between Apigenin and AhR. We then demonstrated that Apigenin and its derivative VY-3-68 enter activated T cells, drive nuclear shuttling of AhR, and modulate its signaling both at RNA and protein level. Results: In HTLV-1 producing cells with high AhR expression, Apigenin cooperates with ARTs such as Lopinavir (LPN) and Zidovudine (AZT), to impart cytotoxicity by exhibiting a major shift in IC50 that was reversed upon AhR knockdown. Mechanistically, Apigenin treatment led to an overall downregulation of NF-κB and several other pro-cancer genes involved in survival. Conclusions: This study suggest the potential combinatorial use of Apigenin with current first-line antiretrovirals for the benefit of patients affected by HTLV-1 associated pathologies.

Dendritic cell CNS recruitment correlates with disease severity in EAE via CCL2 chemotaxis at the blood-brain barrier through paracellular transmigration and ERK activation.

BACKGROUND: Transmigration of circulating dendritic cells (DCs) into the central nervous system (CNS) across the blood-brain barrier (BBB) has not thus far been investigated. An increase in immune cell infiltration across the BBB, uncontrolled activation and antigen presentation are influenced by chemokines. Chemokine ligand 2 (CCL2) is a potent chemoattractant known to be secreted by the BBB but has not been implicated in the recruitment of DCs specifically at the BBB. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by injection of MOG35-55 peptide and pertussis toxin intraperitoneally. Animals with increasing degree of EAE score were sacrificed and subjected to near-infrared and fluorescence imaging analysis to detect and localize the accumulation of CD11c+-labeled DCs with respect to CCL2 expression. To further characterize the direct effect of CCL2 in DC trafficking at the BBB, we utilized an in vitro BBB model consisting of human brain microvascular endothelial cells to compare migratory patterns of monocyte-derived dendritic cells, CD4+ and CD8+ T cells. Further, this model was used to image transmigration using fluorescence microcopy and to assess specific molecular signaling pathways involved in transmigration. RESULTS: Near-infrared imaging of DC transmigration correlated with the severity of inflammation during EAE. Ex vivo histology confirmed the presence of CCL2 in EAE lesions, with DCs emerging from perivascular spaces. DCs exhibited more efficient transmigration than T cells in BBB model studies. These observations correlated with transwell imaging, which indicated a paracellular versus transcellular pattern of migration by DCs and T cells. Moreover, at the molecular level, CCL2 seems to facilitate DC transmigration in an ERK1/2-dependent manner. CONCLUSION: CNS recruitment of DCs correlates with disease severity in EAE via CCL2 chemotaxis and paracellular transmigration across the BBB, which is facilitated by ERK activation. Overall, these comprehensive studies provide a state-of-the-art view of DCs within the CNS, elucidate their path across the BBB, and highlight potential mechanisms involved in CCL2-mediated DC trafficking.

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-gamma(+)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.

Can Soluble Immune Checkpoint Molecules on Exosomes Mediate Inflammation?

Immune checkpoints (ICPs) are major co-signaling pathways that trigger effector functions in immune cells, with isoforms that are either membrane bound, engaging in direct cell to cell activation locally, or soluble, acting at distant sites by circulating freely or potentially via extracellular vesicles (EVs). Exosomes are small EVs secreted by a variety of cells carrying various proteins and nucleic acids. They are distributed extensively through biological fluids and have major impacts on infectious diseases, cancer, and neuroinflammation. Similarly, ICPs play key roles in a variety of disease conditions and have been extensively utilized as a prognostic tool for various cancers. Herein, we explored if the association between exosomes and ICPs could be a significant contributor of inflammation, particularly in the setting of cancer, neuroinflammation and viral infections, wherein the up regulation in both exosomal proteins and ICPs correlate with immunosuppressive effects. The detailed literature review of existing data highlights the significance and complexity of these two important pathways in mediating cancer and potentiating neuroinflammation via modulating overall immune response. Cells increasingly secret exosomes in response to intracellular signals from invading pathogens or cancerous transformations. These exosomes can carry a variety of cargo including proteins, nucleic acids, cytokines, and receptors/ligands that have functional consequences on recipient cells. Illustration generated using BioRender software.

Apigenin Modulates Dendritic Cell Activities and Curbs Inflammation Via RelB Inhibition in the Context of Neuroinflammatory Diseases.

Neuroinflammation leads to tissue injury causing many of the clinical symptoms of Multiple Sclerosis, an autoimmune disorder of the central nervous system (CNS). While T cells, specifically Th1 and Th17 cells, are the ultimate effectors of this disease, dendritic cells (DCs) mediate T cell polarization, activation, etc. In our previous study, Apigenin, a natural flavonoid, has been shown to reduce EAE disease severity through amelioration of demyelination in the CNS as well as the sequestering of DCs and other myeloid cells in the periphery. Here, we show that Apigenin exerts its effects possibly through shifting DC modulated T cell responses from Th1 and Th17 type towards Treg directed responses evident through the decrease in T-bet, IFN-γ (Th1), IL-17 (Th17) and increase in IL-10, TGF-ß and FoxP3 (Treg) expression in cells from both normal human donors and EAE mice. RelB, an NF-κß pathway protein is central to DC maturation, its antigen presentation capabilities and DC-mediated T cell activation. Apigenin reduced mRNA and protein levels of RelB and also reduced its nuclear translocation. Additionally, siRNA-mediated silencing of RelB further potentiated the RelB-mediated effects of Apigenin thus confirming its role in Apigenin directed regulation of DC biology. These results provide key information about the molecular events controlled by Apigenin in its regulation of DC activity marking its potential as a therapy for neuroinflammatory disease. Graphical Abstract.

DC-SIGN mediates cell-free infection and transmission of human T-cell lymphotropic virus type 1 by dendritic cells.

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.

Potential Role of Flavonoids in Treating Chronic Inflammatory Diseases with a Special Focus on the Anti-Inflammatory Activity of Apigenin.

Inflammation has been reported to be intimately linked to the development or worsening of several non-infectious diseases. A number of chronic conditions such as cancer, diabetes, cardiovascular disorders, autoimmune diseases, and neurodegenerative disorders emerge as a result of tissue injury and genomic changes induced by constant low-grade inflammation in and around the affected tissue or organ. The existing therapies for most of these chronic conditions sometimes leave more debilitating effects than the disease itself, warranting the advent of safer, less toxic, and more cost-effective therapeutic alternatives for the patients. For centuries, flavonoids and their preparations have been used to treat various human illnesses, and their continual use has persevered throughout the ages. This review focuses on the anti-inflammatory actions of flavonoids against chronic illnesses such as cancer, diabetes, cardiovascular diseases, and neuroinflammation with a special focus on apigenin, a relatively less toxic and non-mutagenic flavonoid with remarkable pharmacodynamics. Additionally, inflammation in the central nervous system (CNS) due to diseases such as multiple sclerosis (MS) gives ready access to circulating lymphocytes, monocytes/macrophages, and dendritic cells (DCs), causing edema, further inflammation, and demyelination. As the dearth of safe anti-inflammatory therapies is dire in the case of CNS-related disorders, we reviewed the neuroprotective actions of apigenin and other flavonoids. Existing epidemiological and pre-clinical studies present considerable evidence in favor of developing apigenin as a natural alternative therapy against chronic inflammatory conditions.

MEF-2 isoforms' (A-D) roles in development and tumorigenesis.

Myocyte enhancer factor (MEF)-2 plays a critical role in proliferation, differentiation, and development of various cell types in a tissue specific manner. Four isoforms of MEF-2 (A-D) differentially participate in controlling the cell fate during the developmental phases of cardiac, muscle, vascular, immune and skeletal systems. Through their associations with various cellular factors MEF-2 isoforms can trigger alterations in complex protein networks and modulate various stages of cellular differentiation, proliferation, survival and apoptosis. The role of the MEF-2 family of transcription factors in the development has been investigated in various cell types, and the evolving alterations in this family of transcription factors have resulted in a diverse and wide spectrum of disease phenotypes, ranging from cancer to infection. This review provides a comprehensive account on MEF-2 isoforms (A-D) from their respective localization, signaling, role in development and tumorigenesis as well as their association with histone deacetylases (HDACs), which can be exploited for therapeutic intervention.

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-kappaB, 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.

In vivo and in vitro immunogenicity of novel MHC class I presented epitopes to confer protective immunity against chronic HTLV-1 infection.

Human T-cell leukemia virus type 1 (HTLV-1) has infected as many as 10 million people worldwide. While 90% are asymptomatic, 5% develop severe diseases including adult T-cell leukemia/lymphoka (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). No vaccine against HTLV-1 exists, and screening programs are not universal. However, patients with chronic HTLV-1 infection have high frequencies of HTLV-1-activated CD8+ T cells, and the two main HLA alleles (A2, A24) are present in 88% of infected individuals. We thus utilized an immunoproteomics approach to characterize MHC-I restricted epitopes presented by HLA-A2+, A24+ MT-2 and SLB-1 cell lines. Unlike traditional motif prediction algorithms, this approach identifies epitopes associated with cytotoxic T-cell responses in their naturally processed forms, minimizing differences in antigen processing and protein expression levels. Out of nine identified peptides, we confirmed six novel MHC-I restricted epitopes that were capable of binding HLA-A2 and HLA-A24 alleles and used in vitro and in vivo methods to generate CD8+ T cells specific for each of these peptides. MagPix MILLIPLEX data showed that in vitro generated epitope-specific CD8+ T cells secreted IFN-É£, granzyme B, MIP-1α, TNF-α, perforin and IL-10 when cultured in the presence of MT-2 cell line. Degranulation assay confirmed cytotoxic response through surface expression of CD107 on CD8+ T cells when cultured with MT-2 cells. A CD8+ T-cell killing assay indicated significant antiviral activity of CD8+ T cells specific against all identified peptides. In vivo generated CD8+ T cells similarly demonstrated immunogenicity on ELISpot, CD107 degranulation assay, and MagPix MILLIPLEX analysis. These epitopes are thus candidates for a therapeutic peptide-based vaccine against HTLV-1, and our results provide preclinical data for the advancement of such a vaccine.

Diminished TLR2-TLR9 mediated CD4+ T cell responses are associated with increased inflammation in intraocular tuberculosis.

Intraocular tuberculosis (IOTB) is amongst the leading causes of uveitis in tropical countries. Despite reports on involvement of proinflammatory cytokines, studies on innate immune responses in disease pathogenesis are lacking. Reports from animal models and patients with pulmonary tuberculosis indicate that defects in toll like receptor (TLR)2 and TLR9 signalling predispose them to tuberculosis. In this context, we investigated the role of TLR2, TLR4 and TLR9 in generation of CD4+ T effector (Teff) cell responses during IOTB. Firstly, the cells in vitreous fluids showed lower expression of TLR2 and TLR9 in IOTB as compared to non-uveitis and non-TB uveitis groups. Next, peripheral CD4+ Teff cells of subjects with IOTB showed decreased proliferative responses and lower induction of Tregs following TLR2 and TLR9 stimulation. Further, TLR9 ligation resulted in increased IFN-γ and IL-17a but decreased expression of IL-10 and TGF-ß. Lastly, lower expression of genes involved in TLR9 signalling after direct TLR9 ligation was observed in IOTB. Collectively, our results show that a subdued response to direct TLR2 and TLR9 stimulation in CD4+ T cells is associated with increased proinflammatory responses in IOTB. These findings reveal an important link between innate immune signalling and ensuing adaptive immune responses in IOTB with implications in other forms of extrapulmonary tuberculosis.

FDC:TFH Interactions within Cervical Lymph Nodes of SIV-Infected Rhesus Macaques.

Cerebrospinal fluid (CSF) drains via the lymphatic drainage pathway. This lymphatic pathway connects the central nervous system (CNS) to the cervical lymph node (CLN). As the CSF drains to CLN via the dural and nasal lymphatics, T cells and antigen presenting cells pass along the channels from the subarachnoid space through the cribriform plate. Human immunodeficiency virus (HIV) may also egress from the CNS along this pathway. As a result, HIV egressing from the CNS may accumulate within the CLN. Towards this objective, we analyzed CLNs isolated from rhesus macaques that were chronically-infected with simian immunodeficiency virus (SIV). We detected significant accumulation of SIV within the CLNs. SIV virion trapping was observed on follicular dendritic cells (FDCs) localized within the follicular regions of CLNs. In addition, SIV antigens formed immune complexes when FDCs interacted with B cells within the germinal centers. Subsequent interaction of these B cells with CD4+ T follicular helper cells (TFHs) resulted in infection of the latter. Of note, 73% to 90% of the TFHs cells within CLNs were positive for SIV p27 antigen. As such, it appears that not only do the FDCs retain SIV they also transmit them (via B cells) to TFHs within these CLNs. This interaction results in infection of TFHs in the CLNs. Based on these observations, we infer that FDCs within the CLNs have a novel role in SIV entrapment with implications for viral trafficking.

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, Sjogren's 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.