The Evolution of Dendritic Cell Immunotherapy against HIV-1 Infection: Improvements and Outlook.

Dendritic cells (DC) are key phagocytic cells that play crucial roles in both the innate and adaptive immune responses against the human immunodeficiency virus type 1 (HIV-1). By processing and presenting pathogen-derived antigens, dendritic cells initiate a directed response against infected cells. They activate the adaptive immune system upon recognition of pathogen-associated molecular patterns (PAMPs) on infected cells. During the course of HIV-1 infection, a successful adaptive (cytotoxic CD8+ T-cell) response is necessary for preventing the progression and spread of infection in a variety of cells. Dendritic cells have thus been recognized as a valuable tool in the development of immunotherapeutic approaches and vaccines effective against HIV-1. The advancements in dendritic cell vaccines in cancers have paved the way for applications of this form of immunotherapy to HIV-1 infection. Clinical trials with patients infected with HIV-1 who are well-suppressed by antiretroviral therapy (ART) were recently performed to assess the efficacy of DC vaccines, with the goal of mounting an HIV-1 antigen-specific T-cell response, ideally to clear infection and eliminate the need for long-term ART. This review summarizes and compares methods and efficacies of a number of DC vaccine trials utilizing autologous dendritic cells loaded with HIV-1 antigens. The potential for advancement and novel strategies of improving efficacy of this type of immunotherapy is also discussed.

Modeling the pathology, immune responses, and kinetics of HSV-1 replication in the lip scarification model.

The lip scarification model of herpes simplex virus type 1 (HSV-1) infection can be used to study acute infection in the orofacial tissue and the establishment of viral latency. In this study, mice were inoculated with HSV-1 and tissue harvested during the acute phase of infection. Clinical presentation of classical open sores on the lip of infected mice was observed. We defined the histopathology, disease scores, and immune infiltration of the lower lip during the formation and resolution of the clinical lesions. Finally, the kinetics of virus replication and transport of viral genomes to the trigeminal ganglia were established. With the virological and pathologic events of acute infection defined, the HSV-1 lip scarification model can now be used to study primary HSV-1 infection, invasion of the trigeminal ganglia, and establishment of latency.

Animal models of herpes simplex virus immunity and pathogenesis.

Herpes simplex viruses are ubiquitous human pathogens represented by two distinct serotypes: herpes simplex virus (HSV) type 1 (HSV-1); and HSV type 2 (HSV-2). In the general population, adult seropositivity rates approach 90% for HSV-1 and 20-25% for HSV-2. These viruses cause significant morbidity, primarily as mucosal membrane lesions in the form of facial cold sores and genital ulcers, with much less common but more severe manifestations causing death from encephalitis. HSV infections in humans are difficult to study in many cases because many primary infections are asymptomatic. Moreover, the neurotropic properties of HSV make it much more difficult to study the immune mechanisms controlling reactivation of latent infection within the corresponding sensory ganglia and crossover into the central nervous system of infected humans. This is because samples from the nervous system can only be routinely obtained at the time of autopsy. Thus, animal models have been developed whose use has led to a better understanding of multiple aspects of HSV biology, molecular biology, pathogenesis, disease, and immunity. The course of HSV infection in a spectrum of animal models depends on important experimental parameters including animal species, age, and genotype; route of infection; and viral serotype, strain, and dose. This review summarizes the animal models most commonly used to study HSV pathogenesis and its establishment, maintenance, and reactivation from latency. It focuses particularly on the immune response to HSV during acute primary infection and the initial invasion of the ganglion with comparisons to the events governing maintenance of viral latency.

Immunological control of herpes simplex virus infections.

Herpes simplex virus type 1 (HSV-1) is capable of causing a latent infection in sensory neurons that lasts for the lifetime of the host. The primary infection is resolved following the induction of the innate immune response that controls replication of the virus until the adaptive immune response can clear the active infection. HSV-1-specific CD8(+) T cells survey the ganglionic regions containing latently infected neurons and participate in preventing reactivation of HSV from latency. The long-term residence and migration dynamics of the T cells in the trigeminal ganglia appear to distinguish them from the traditional memory T cell subsets. Recently described tissue resident memory (TRM) T cells establish residence and survive for long periods in peripheral tissue compartments following antigen exposure. This review focuses on the immune system response to HSV-1 infection. Particular emphasis is placed on the evidence pointing to the HSV-1-specific CD8(+) T cells in the trigeminal belonging to the TRM class of memory T cells and the role of TRM cells in virus infection, pathogenesis, latency, and disease.

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.

Cytomegalovirus infection leads to microvascular dysfunction and exacerbates hypercholesterolemia-induced responses.

Cytomegalovirus (CMV) persistently infects more than 60% of the worldwide population. In immunocompetent hosts, it has been implicated in several diseases, including cardiovascular disease, possibly through the induction of inflammatory pathways. Cardiovascular risk factors promote an inflammatory phenotype in the microvasculature long before clinical disease is evident. This study determined whether CMV also impairs microvascular homeostasis and synergizes with hypercholesterolemia to exaggerate these responses. Intravital microscopy was used to assess endothelium-dependent and -independent arteriolar vasodilation and venular leukocyte and platelet adhesion in mice after injection with either mock inoculum or murine CMV (mCMV). Mice were fed a normal (ND) or high-cholesterol (HC) diet beginning at 5 weeks postinfection (p.i.), or a HC diet for the final 4 weeks of infection. mCMV-ND mice exhibited impaired endothelium-dependent vasodilation versus mock-ND at 9 and 12 weeks and endothelium-independent arteriolar dysfunction by 24 weeks. Transient mild leukocyte adhesion occurred in mCMV-ND venules at 7 and 21 weeks p.i. HC alone caused temporary arteriolar dysfunction and venular leukocyte and platelet recruitment, which were exaggerated and prolonged by mCMV infection. The time of introduction of HC after mCMV infection determined whether mCMV+HC led to worse venular inflammation than either factor alone. These findings reveal a proinflammatory influence of persistent mCMV on the microvasculature, and suggest that mCMV infection enhances microvasculature susceptibility to both inflammatory and thrombogenic responses caused by hypercholesterolemia.

CD4+ T cells are required for the priming of CD8+ T cells following infection with herpes simplex virus type 1.

The role of CD4(+) helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4(+) T cells are needed for the generation of the protective HSV-1-specific CD8(+)-T-cell response. This study examined the contribution of CD4(+) T cells in the generation of the primary CD8(+)-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8(+)-T-cell response generated in the draining lymph nodes of CD4(+)-T-cell-depleted C57BL/6 mice and B6-MHC-II(-/-) mice is quantitatively and qualitatively distinct from the CD8(+) T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8(+) T cells express comparable levels of the activation marker CD44 in mice lacking CD4(+) T cells and normal mice. In contrast, CD8(+) T cells generated in the absence of CD4(+) T cells express the interleukin 2 receptor alpha-chain (CD25) at lower levels. Importantly, the CD8(+) T cells in the CD4(+)-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8(+) T cells is diminished in the absence of CD4(+)-T-cell help. These results suggest that CD4(+)-T-cell help is essential for the generation of fully functional CD8(+) T cells during the primary response to HSV-1 infection.

Dendritic cells are required for optimal activation of natural killer functions following primary infection with herpes simplex virus type 1.

Natural killer (NK) cells play an important role in the optimal clearance of herpes simplex virus type 1 (HSV-1) infection in mice. Activated NK cells function via cytokine secretion or direct cytolysis of target cells; dendritic cells (DCs) are thought to make critical contributions in the activation of both of these functions. Yet, the magnitude and physiological relevance of DC-mediated NK cell activation in vivo is not completely understood. To examine the contribution of DC help in regulating NK cell functions after infection with HSV-1, we utilized a transgenic mouse model that allows the transient ablation of DCs. Using this approach, it was found that the gamma interferon (IFN-gamma) expression potential of NK cells is quantitatively and qualitatively impaired in the absence of DCs. With regard to priming of NK cytolytic functions, the ablation of DCs did not significantly affect cytotoxic protein expression by NK cells. An in vivo cytolytic assay did, however, reveal impairments in the magnitude of NK cell cytotoxicity. Overall, this study provides direct evidence that functional DCs are required for optimal IFN-gamma expression and cytolytic function by NK cells following infection with HSV-1.

Characterization of age-related changes in natural killer cells during primary influenza infection in mice.

The current investigation examined the importance of natural killer (NK) cells during the innate immune response to primary influenza infection in young and aged mice. Young (6-8 weeks) and aged (22 months) C57BL/6 mice were infected intranasally with influenza A virus, and NK cell-mediated cytotoxicity was determined in lung and spleen during the first 4 days of infection. Aged mice demonstrated both a decrease in influenza-inducible NK activity and a reduction in the percentage and number of NK1.1+ cells in response to primary influenza infection, relative to young mice. In order to further establish a role for NK cells in controlling influenza infection, young mice were depleted of NK cells in vivo by injecting rabbit anit-NK1.1 antibody 2 days and 1 day prior to influenza infection. Young mice depleted of NK cells exhibited increased weight loss and lung virus titers during the course of infection, compared to young mice infected with influenza virus. These data indicate that NK cell function is impaired in response to primary influenza infection in aged mice. More importantly, these results underscore the essential role of NK cells in controlling virus titers in lung during the early course of influenza infection, regardless of age.

Memory CD8+ T cells require CD28 costimulation.

CD8(+) T cells are a critical component of the adaptive immune response against infections and tumors. A current paradigm in immunology is that naive CD8(+) T cells require CD28 costimulation, whereas memory CD8(+) T cells do not. We show here, however, that during viral infections of mice, costimulation is required in vivo for the reactivation of memory CD8(+) T cells. In the absence of CD28 costimulation, secondary CD8(+) T cell responses are greatly reduced and this impairs viral clearance. The failure of CD8(+) T cells to expand in the absence of CD28 costimulation is CD4(+) T cell help independent and is accompanied by a failure to down-regulate Bcl-2 and by cell cycle arrest. This requirement for CD28 costimulation was shown in both influenza A and HSV infections. Thus, contrary to current dogma, memory CD8(+) T cells require CD28 costimulation to generate maximal secondary responses against pathogens. Importantly, this CD28 requirement was shown in the context of real infections were multiple other cytokines and costimulators may be up-regulated. Our findings have important implications for pathogens, such as HIV and measles virus, and tumors that evade the immune response by failing to provide CD28 costimulation. These findings also raise questions about the efficacy of CD8(+) T cell-based vaccines against such pathogens and tumors.

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 ablation of CD11c-positive dendritic cells increases susceptibility to herpes simplex virus type 1 infection and diminishes NK and T-cell responses.

The precise role of each of the seven individual CD11c+ dendritic cell subsets (DCs) identified to date in the response to viral infections is not known. DCs serve as critical links between the innate and adaptive immune responses against many pathogens, including herpes simplex virus type 1 (HSV-1). The role of DCs as mediators of resistance to HSV-1 infection was investigated using CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice, in which DCs can be transiently depleted in vivo by treatment with low doses of DT. We show that ablation of DCs led to enhanced susceptibility to HSV-1 infection in the highly resistant C57BL/6 mouse strain. Specifically, we showed that the depletion of DCs led to increased viral spread into the nervous system, resulting in an increased rate of morbidity and mortality. Furthermore, we showed that ablation of DCs impaired the optimal activation of NK cells and CD4+ and CD8+ T cells in response to HSV-1. These data demonstrated that DCs were essential not only in the optimal activation of the acquired T-cell response to HSV-1 but also that DCs were crucial for innate resistance to HSV-1 infection.

Cytokine profiles and long-term virus-specific antibodies following immunization of CBA mice with equine herpesvirus 1 and viral glycoprotein D.

Equine herpesvirus 1 (EHV-1)-specific antibody-secreting cells (ASC) isolated from the lung and spleen of mice at 12 months after immunization with attenuated EHV-1 KyA, heat-killed KyA, or recombinant viral glycoprotein D (rgD) assessed by ELISPOT showed a three- to fivefold increase in three immunoglobulin isotypes at 3 days post-challenge with pathogenic EHV-1 RacL11 as compared to control mice. ELISPOT assays demonstrated a high frequency of cells secreting proinflammatory tumor necrosis factor-alpha (TNF-alpha), interferon gamma (IFN-gamma), and interleukin 4 (IL-4) in the lungs in response to infection with KyA or RacL11 or immunization with rgD. Cytokine production elicited by EHV-1 KyA or RacL11 infection revealed similar frequencies of EHV-1-specific IFN-gamma and IL-4 spot forming cells in the mediastinal lymph nodes and spleen. However, KyA induced significantly greater amounts of IFN-gamma producing cells in the lungs than did RacL11. Intranasal immunization with KyA or rgD induced long-term immunity that provided protection against pathogenic EHV-1 challenge infection at 12 months post-immunization. Overall, the data indicate that immunization with infectious KyA or rgD induces significant levels of cytokines, virus-specific ASC in the lungs and spleen, and long-term virus specific B-cell responses.

The role of T-lymphocytes in hypercholesterolemia-induced leukocyte-endothelial interactions.

OBJECTIVE: Hypercholesterolemia promotes the adhesion of leukocytes to vascular endothelium in large and microscopic blood vessels. Lymphocytes that can modulate endothelial cell adhesion molecule expression have been implicated in the altered structure and function of large arterial vessels associated with chronic hypercholesterolemia. This study assesses the contribution of CD4(+) and CD8(+) T-cells to acute inflammatory responses observed in the microcirculation of hypercholesterolemic mice. METHODS: Intravital microscopy was used to quantify baseline leukocyte-endothelial cell interactions in cremasteric postcapillary venules of wild-type (WT) and severe combined immunodeficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. A group of SCID-HC mice received splenocytes from WT-HC mice (WT-->SCID). Separate WT-HC groups were depleted of neutrophils or CD4(+) and/or CD8(+) T-cells. RESULTS: WT-HC mice, compared with WT-ND, exhibited exaggerated leukocyte adherence and emigration. These leukocytes were predominantly granulocytes. These responses were absent in neutropenic WT-HC mice. SCID-HC mice also showed significantly less leukocyte adherence and emigration than WT-HC mice. Elevated leukocyte adherence and emigration were restored in WT-->SCID mice, despite a continued absence of circulating blood lymphocytes. Selective depletion of either CD4(+) or CD8(+) cell populations attenuated HC-induced leukocyte adhesion but not emigration. However, simultaneous depletion of both CD4(+) and CD8(+) cells attenuated both leukocyte adhesion and emigration to ND levels. DISCUSSION: These findings indicate that both CD4(+) and CD8(+) T-cells contribute to granulocyte adhesion and emigration elicited in postcapillary venules by hypercholesterolemia.