Cholesterol Metabolism in Antigen-Presenting Cells and HIV-1 Trans-Infection of CD4+ T Cells.

Antiretroviral therapy (ART) provides an effective method for managing HIV-1 infection and preventing the onset of AIDS; however, it is ineffective against the reservoir of latent HIV-1 that persists predominantly in resting CD4+ T cells. Understanding the mechanisms that facilitate the persistence of the latent reservoir is key to developing an effective cure for HIV-1. Of particular importance in the establishment and maintenance of the latent viral reservoir is the intercellular transfer of HIV-1 from professional antigen-presenting cells (APCs-monocytes/macrophages, myeloid dendritic cells, and B lymphocytes) to CD4+ T cells, termed trans-infection. Whereas virus-to-cell HIV-1 cis infection is sensitive to ART, trans-infection is impervious to antiviral therapy. APCs from HIV-1-positive non-progressors (NPs) who control their HIV-1 infection in the absence of ART do not trans-infect CD4+ T cells. In this review, we focus on this unique property of NPs that we propose is driven by a genetically inherited, altered cholesterol metabolism in their APCs. We focus on cellular cholesterol homeostasis and the role of cholesterol metabolism in HIV-1 trans-infection, and notably, the link between cholesterol efflux and HIV-1 trans-infection in NPs.

B Lymphocytes, but Not Dendritic Cells, Efficiently HIV-1 Trans Infect Naive CD4+ T Cells: Implications for the Viral Reservoir.

Insight into the establishment and maintenance of HIV-1 infection in resting CD4+ T cell subsets is critical for the development of therapeutics targeting the HIV-1 reservoir. Although the frequency of HIV-1 infection, as quantified by the frequency of HIV-1 DNA, is lower in CD4+ naive T cells (TN) than in the memory T cell subsets, recent studies have shown that TN harbor a large pool of replication-competent virus. Interestingly, however, TN are highly resistant to direct (cis) HIV-1 infection in vitro, in particular to R5-tropic HIV-1, as TN do not express CCR5. In this study, we investigated whether TN could be efficiently HIV-1 trans infected by professional antigen-presenting B lymphocytes and myeloid dendritic cells (DC) in the absence of global T cell activation. We found that B cells, but not DC, have a unique ability to efficiently trans infect TNin vitro In contrast, both B cells and DC mediated HIV-1 trans infection of memory and activated CD4+ T cells. Moreover, we found that TN isolated from HIV-1-infected nonprogressors (NP) harbor significantly disproportionately lower levels of HIV-1 DNA than TN isolated from progressors. This is consistent with our previous finding that antigen-presenting cells (APC) derived from NP do not efficiently trans infect CD4+ T cells due to alterations in APC cholesterol metabolism and cell membrane lipid raft organization. These findings support that B cell-mediated trans infection of TN with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression.IMPORTANCE The latent human immunodeficiency virus type 1 (HIV-1) reservoir in persons on antiretroviral therapy (ART) represents a major barrier to a cure. Although most studies have focused on the HIV-1 reservoir in the memory T cell subset, replication-competent HIV-1 has been isolated from TN, and CCR5-tropic HIV-1 has been recovered from CCR5neg TN from ART-suppressed HIV-1-infected individuals. In this study, we showed that CCR5neg TN are efficiently trans infected with R5-tropic HIV-1 by B lymphocytes, but not by myeloid dendritic cells. Furthermore, we found that TN isolated from NP harbor no or significantly fewer copies of HIV-1 DNA than those from ART-suppressed progressors. These findings support that B cell-mediated trans infection of TN with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression. Understanding the establishment and maintenance of the HIV-1 latent reservoir is fundamental for the design of effective treatments for viral eradication.

Effect of Statin Use on Inflammation and Immune Activation Biomarkers in HIV-Infected Persons on Effective Antiretroviral Therapy.

Immune activation and inflammation are hallmarks of chronic HIV infection and are etiologically linked to major causes of morbidity and mortality among HIV-infected persons, including coronary artery disease and cancer. Systemic immune activation is dampened, but not resolved, with use of combination antiretroviral therapy (cART). Statins are cardioprotective drugs that also appear to have immunomodulatory and anti-inflammatory properties. We sought to understand the association between statin use, cART, and levels of circulating immune markers in a longitudinal cohort study. From 2004 to 2009, statin use was ascertained in male participants of the Multicenter AIDS Cohort Study (MACS) using interviewer-administered questionnaires. Twenty-four circulating markers of immune activation and inflammation were measured in archived serial samples from a subset of cohort members using multiplex assays. Propensity-adjusted generalized gamma models were used to compare biomarkers' distributions by statin use, and multivariable linear regression models were used to assess the effect of initiating statin on these biomarkers. Overall, 1,031 cART-exposed individuals with HIV infection were included in this study. Statin use was reported by 31.5% of cART-exposed participants. Compared to nonstatin users on cART, statin users on cART had lower levels of IP-10, IL-10, and IL-12p70, and the effect of statin use was decreased in participants using lipophilic statins (atorvastatin, simvastatin, fluvastatin, or lovastatin); these results were statistically significant (p < .05). Among cART users not on aspirin, starting statins decreased levels of high sensitivity c-reactive protein (hsCRP), IL-12p70, and IL-6. Statin therapy is associated with reduced levels of certain biomarkers of immune activation and inflammation in cART users, which may contribute to a lower burden of disease.

Efficient HIV-1 Trans Infection of CD4+ T Cells Occurs in the Presence of Antiretroviral Therapy.

BACKGROUND: Antiretroviral therapy (ART) has dramatically improved the quality of life of people with HIV-1 infection (PWH). However, it is not curative, and interruption of ART results in rapid viral rebound. Cell-to-cell transfer of HIV-1, or trans infection, is a highly efficient mechanism of virus infection of CD4+ T cells by professional antigen-presenting cells (APCs), that is, dendritic cells (DCs), macrophages, and B lymphocytes. METHODS: APC from HIV seronegative donors treated with ART in vitro (CCR5 agonist, NRTI, PI and NNRTI, alone or in combination), were loaded with HIV R5-tropic HIVBal and mixed with autologous or heterologous CD4+ T lymphocytes to assess trans infection. Ex vivo APC from chronic HIV-infected MACS participants before and after initiation of ART, were also loaded with HIV R5-tropic HIVBal and tested for trans infection against autologous or heterologous CD4+ T lymphocytes. Virus replication was measured by p24 ELISA. RESULTS: Here we show in vitro that antiretroviral drugs did not block the ability of DCs and B cells to trans-infect CD4+ T cells, although they were effective in blocking direct cis infection of CD4+ T cells. Moreover, ex vivo DCs and B cells from ART-suppressed PWH mediated efficient HIV-1 trans infection of CD4+ T cells, which were resistant to direct cis infection. CONCLUSIONS: Our study supports a role for HIV-1 trans infection in maintenance of the HIV-1 reservoir during ART.

Inefficient HIV-1 trans Infection of CD4+ T Cells by Macrophages from HIV-1 Nonprogressors Is Associated with Altered Membrane Cholesterol and DC-SIGN.

Professional antigen-presenting cells (APC; myeloid dendritic cells [DC] and macrophages [MΦ]; B lymphocytes) mediate highly efficient HIV-1 infection of CD4+ T cells, termed trans infection, that could contribute to HIV-1 pathogenesis. We have previously shown that lower cholesterol content in DC and B lymphocytes is associated with a lack of HIV-1 trans infection in HIV-1-infected nonprogressors (NP). Here, we assessed whether HIV-1 trans infection mediated by another major APC, MΦ, is deficient in NP due to altered cholesterol metabolism. When comparing healthy HIV-1 seronegatives (SN), rapid progressors (PR), and NP, we found that monocyte-derived MΦ from NP did not mediate HIV-1 trans infection of autologous CD4+ T cells, in contrast to efficient trans infection mediated by SN and PR MΦ. MΦ trans infection efficiency was directly associated with the number of DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-expressing MΦ. Significantly fewer NP MΦ expressed DC-SIGN. Unesterified (free) cholesterol in MΦ cell membranes and lipid rafting was significantly lower in NP than PR, as was virus internalization in early endosomes. Furthermore, simvastatin (SIMV) decreased the subpopulation of DC-SIGN+ MΦ as well as cis and trans infection. Notably, SIMV decreased cell membrane cholesterol and led to lipid raft dissociation, effectively mimicking the incompetent APC trans infection environment characteristic of NP. Our data support that DC-SIGN and membrane cholesterol are central to MΦ trans infection, and a lack of these limits HIV-1 disease progression. Targeting the ability of MΦ to drive HIV-1 dissemination in trans could enhance HIV-1 therapeutic strategies.IMPORTANCE Despite the success of combination antiretroviral therapy, neither a vaccine nor a cure for HIV infection has been developed, demonstrating a need for novel prophylactic and therapeutic strategies. Here, we show that efficiency of MΦ-mediated HIV trans infection of CD4+ T cells is a unique characteristic associated with control of disease progression, and it is impaired in HIV-infected NP. In vitro treatment of MΦ from healthy donors with SIMV lowers their cholesterol content, which results in a strongly reduced trans infection ability, similar to the levels of MΦ from NP. Taken together, our data support the hypothesis that MΦ-mediated HIV-1 trans infection plays a role in HIV infection and disease progression and demonstrate that the use of SIMV to decrease this mechanism of virus transfer should be considered for future HIV therapeutic development.

Human Herpesvirus 8 Infects and Replicates in Langerhans Cells and Interstitial Dermal Dendritic Cells and Impairs Their Function.

The predominant types of dendritic cells (DC) in the skin and mucosa are Langerhans cells (LC) and interstitial dermal DC (iDDC). LC and iDDC process cutaneous antigens and migrate out of the skin and mucosa to the draining lymph nodes to present antigens to T and B cells. Because of the strategic location of LC and iDDC and the ability of these cells to capture and process pathogens, we hypothesized that they could be infected with human herpesvirus 8 (HHV-8) (Kaposi's sarcoma [KS]-associated herpesvirus) and have an important role in the development of KS. We have previously shown that HHV-8 enters monocyte-derived dendritic cells (MDDC) through DC-SIGN, resulting in nonproductive infection. Here we show that LC and iDDC generated from pluripotent cord blood CD34+ cell precursors support productive infection with HHV-8. Anti-DC-SIGN monoclonal antibody (MAb) inhibited HHV-8 infection of iDDC, as shown by low expression levels of viral proteins and DNA. In contrast, blocking of both langerin and the receptor protein tyrosine kinase ephrin A2 was required to inhibit HHV-8 infection of LC. Infection with HHV-8 did not alter the cell surface expression of langerin on LC but downregulated the expression of DC-SIGN on iDDC, as we previously reported for MDDC. HHV-8-infected LC and iDDC had a reduced ability to stimulate allogeneic CD4+ T cells in the mixed-lymphocyte reaction. These results indicate that HHV-8 can target both LC and iDDC for productive infection via different receptors and alter their function, supporting their potential role in HHV-8 pathogenesis and KS.IMPORTANCE Here we show that HHV-8, a DNA tumor virus that causes Kaposi's sarcoma, infects three types of dendritic cells: monocyte-derived dendritic cells, Langerhans cells, and interstitial dermal dendritic cells. We show that different receptors are used by this virus to infect these cells. DC-SIGN is a major receptor for infection of both monocyte-derived dendritic cells and interstitial dermal dendritic cells, yet the virus fully replicates only in the latter. HHV-8 uses langerin and the ephrin A2 receptor to infect Langerhans cells, which support full HHV-8 lytic replication. This infection of Langerhans cells and interstitial dermal dendritic cells results in an impaired ability to stimulate CD4+ helper T cell responses. Taken together, our data show that HHV-8 utilizes alternate receptors to differentially infect and replicate in these tissue-resident DC and support the hypothesis that these cells play an important role in HHV-8 infection and pathogenesis.

Regulatory T Cell Effect on CD8+ T Cell Responses to Human Herpesvirus 8 Infection and Development of Kaposi's Sarcoma.

We assessed CD8+ T cell reactivity to human herpesvirus 8 (HHV-8; Kaposi's sarcoma [KS]-associated herpesvirus) and the role of CD4+CD25hiFoxP3+ regulatory T cells (Treg) in HHV-8- and HIV-coinfected participants of the Multicenter AIDS Cohort Study who did or did not develop KS. There were similarly low CD8+ T cell interferon-γ responses to MHC class I-restricted epitopes of HHV-8 lytic and latent proteins over 5.7 years before KS in participants who developed KS compared to those who did not. T cell reactivity to HHV-8 antigens was low relative to responses to a combination of cytomegalovirus, Epstein-Barr virus and influenza A virus (CEF) peptide epitopes, and dominant HIV peptide epitopes. There was no change in %Treg in the HHV-8- and HIV-coinfected participants who did not develop KS, whereas there was a significant increase in %Treg in HHV-8- and HIV-coinfected participants who developed KS beginning 1.8 years before development of KS. Removal of Treg enhanced HHV-8-specific T cell responses in HHV-8- and HIV-coinfected participants who did or did not develop KS, with a similar pattern observed in response to CEF and HIV peptides. Thus, long-term, low levels of anti-HHV-8 CD8+ T cell reactivity were present in both HHV-8- and HIV-coinfected men who did and did not develop KS. This was related to moderately enhanced Treg function.

Multiplex assay reliability and long-term intra-individual variation of serologic inflammatory biomarkers.

BACKGROUND: Circulating cytokines, chemokines, and soluble cytokine receptors can serve as biomarkers of inflammation and immune dysregulation. Good reliability of multiplex platforms, which allow for simultaneous, comprehensive biomarker assessment, is critical for their utility in epidemiologic studies. We examined the reliability of the Meso-Scale Discovery (MSD) platform to simultaneously quantitate 15 cytokines and chemokines and the Luminex platform (R&D Systems) to quantitate 5 soluble receptors and 2 chemokines and cytokines and evaluated long-term within-person correlation of these biomarkers. METHODS: The detectability and reliability of these assay systems were assessed using the same external controls across plates and archived sera from 250 HIV- men in the Multicenter AIDS Cohort Study. Using up to four visits per person from 1984 to 2009, age-adjusted intraclass correlation coefficients (ICC) of biomarkers with >80% detectability (CCL11, CXCL8, CXCL10, CCL2, CCL4, CCL13, CCL17, CXCL13, IL-10, IL-12p70, IL-6, TNF-α, BAFF, sCD14, sCD27, sgp130, sIL-2Rα, and sTNF-R2) were obtained using linear mixed models. RESULTS: Most biomarkers were detectable in 80% of control samples; IFN-γ, GM-CSF, and IL-2 were undetectable in >20% of samples. Among the HIV-uninfected men, most biomarkers showed fair to strong within-person correlation (ICC>0.40) up to 15years. The ICC for CXCL8 was good in the short term but decreased with increasing time between visits, becoming lower (ICC<0.40) after 8years. CONCLUSIONS: These multiplexed assays showed acceptable reliability for use in epidemiologic research, despite some technical variability and limitations in cytokine quantitation. Most biomarkers displayed moderate-to-excellent intra-individual variability over the long term, suggesting their utility in prospective studies investigating etiologic associations with diverse chronic conditions.

Human herpesvirus 8 induces polyfunctional B lymphocytes that drive Kaposi's sarcoma.

UNLABELLED: Kaposi's sarcoma (KS) is an unusual neoplasia wherein the tumor consists primarily of endothelial cells infected with human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus) that are not fully transformed but are instead driven to excess proliferation by inflammatory and angiogenic factors. This oncogenic process has been postulated but unproven to depend on a paracrine effect of an abnormal excess of host cytokines and chemokines produced by HHV-8-infected B lymphocytes. Using newly developed measures for intracellular detection of lytic cycle proteins and expression of cytokines and chemokines, we show that HHV-8 targets a range of naive B cell, IgM memory B cell, and plasma cell-like populations for infection and induction of interleukin-6, tumor necrosis factor alpha, macrophage inhibitory protein 1α, macrophage inhibitory protein 1ß, and interleukin-8 in vitro and in the blood of HHV-8/HIV-1-coinfected subjects with KS. These B cell lineage subsets that support HHV-8 infection are highly polyfunctional, producing combinations of 2 to 5 of these cytokines and chemokines, with greater numbers in the blood of subjects with KS than in those without KS. Our study provides a new paradigm of B cell polyfunctionality and supports a key role for B cell-derived cytokines and chemokines produced during HHV-8 infection in the development of KS. IMPORTANCE: Kaposi's sarcoma (KS) is the most common cancer in HIV-1-infected persons and is caused by one of only 7 human cancer viruses, i.e., human herpesvirus 8 (HHV-8). It is unclear how this virus causes neoplastic transformation. Development and outgrowth of endothelial cell lesions characteristic of KS are hypothesized to be dependent on virus replication and multiple immune mediators produced by the KS cells and inflammatory cells, yet the roles of these viral and cell factors have not been defined. The present study advances our understanding of KS in that it supports a central role for HHV-8 infection of B cells inducing multiple cytokines and chemokines that can drive development of the cancer. Notably, HIV-1-infected individuals who developed KS had greater numbers of such HHV-8-infected, polyfunctional B cells across a range of B cell phenotypic lineages than did HHV-8-infected persons without KS. This intriguing production of polyfunctional immune mediators by B cells serves as a new paradigm for B cell function and classification.

Dendritic cells: key players in human herpesvirus 8 infection and pathogenesis.

Human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus) is an oncogenic gammaherpesvirus that primarily infects cells of the immune and vascular systems. HHV-8 interacts with and targets professional antigen presenting cells and influences their function. Infection alters the maturation, antigen presentation, and immune activation capabilities of certain dendritic cells (DC) despite non-robust lytic replication in these cells. DC sustains a low level of antiviral functionality during HHV-8 infection in vitro. This may explain the ability of healthy individuals to effectively control this virus without disease. Following an immune compromising event, such as organ transplantation or human immunodeficiency virus type 1 infection, a reduced cellular antiviral response against HHV-8 compounded with skewed DC cytokine production and antigen presentation likely contributes to the development of HHV-8 associated diseases, i.e., Kaposi's sarcoma and certain B cell lymphomas. In this review we focus on the role of DC in the establishment of HHV-8 primary and latent infection, the functional state of DC during HHV-8 infection, and the current understanding of the factors influencing virus-DC interactions in the context of HHV-8-associated disease.

Human herpesvirus 8 glycoprotein B binds the entry receptor DC-SIGN.

We have previously shown that human herpesvirus 8 (HHV-8) uses DC-SIGN as an entry receptor for dendritic cells, macrophages and B cells. The viral attachment protein for DC-SIGN is unknown. HHV-8 virions contain five conserved herpesvirus glycoproteins, a single unique glycoprotein, and two predicted glycoproteins. Previous studies have shown that DC-SIGN binds highly mannosylated glycoproteins. The HHV-8 glycoprotein B (gB) has been reported to be highly mannosylated, and therefore we hypothesized that gB will bind to DC-SIGN. In this report we confirm that gB has a high mannose carbohydrate structure and demonstrate for the first time that it binds DC-SIGN in a dose-dependent manner. We also identify key amino acids in the DC-SIGN carbohydrate recognition domain that are required for HHV-8 infection and compare these results with published binding regions for ICAM-2/3 and HIV-1 gp120. These results clarify some of the initial events in HHV-8 entry and can be used for the design of targeted preventive therapies.

Alterations in cholesterol metabolism restrict HIV-1 trans infection in nonprogressors.

ABSTRACT HIV-1-infected nonprogressors (NP) inhibit disease progression for years without antiretroviral therapy. Defining the mechanisms for this resistance to disease progression could be important in determining strategies for controlling HIV-1 infection. Here we show that two types of professional antigen-presenting cells (APC), i.e., dendritic cells (DC) and B lymphocytes, from NP lacked the ability to mediate HIV-1 trans infection of CD4(+) T cells. In contrast, APC from HIV-1-infected progressors (PR) and HIV-1-seronegative donors (SN) were highly effective in mediating HIV-1 trans infection. Direct cis infection of T cells with HIV-1 was comparably efficient among NP, PR, and SN. Lack of HIV-1 trans infection in NP was linked to lower cholesterol levels and an increase in the levels of the reverse cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in APC but not in T cells. Moreover, trans infection mediated by APC from NP could be restored by reconstitution of cholesterol and by inhibiting ABCA1 by mRNA interference. Importantly, this appears to be an inherited trait, as it was evident in APC obtained from NP prior to their primary HIV-1 infection. The present study demonstrates a new mechanism wherein enhanced lipid metabolism in APC results in remarkable control of HIV-1 trans infection that directly relates to lack of HIV-1 disease progression. IMPORTANCE HIV-1 can be captured by antigen-presenting cells (APC) such as dendritic cells and transferred to CD4 helper T cells, which results in greatly enhanced viral replication by a mechanism termed trans infection. A small percentage of HIV-1-infected persons are able to control disease progression for many years without antiretroviral therapy. In our study, we linked this lack of disease progression to a profound inability of APC from these individuals to trans infect T cells. This effect was due to altered lipid metabolism in their APC, which appears to be an inherited trait. These results provide a basis for therapeutic interventions to control of HIV-1 infection through modulation of cholesterol metabolism.

Selective induction of CTL helper rather than killer activity by natural epitope variants promotes dendritic cell-mediated HIV-1 dissemination.

The ability of HIV-1 to rapidly accumulate mutations provides the virus with an effective means of escaping CD8(+) CTL responses. In this study, we describe how subtle alterations in CTL epitopes expressed by naturally occurring HIV-1 variants can result in an incomplete escape from CTL recognition, providing the virus with a selective advantage. Rather than paralyzing the CTL response, these epitope modifications selectively induce the CTL to produce proinflammatory cytokines in the absence of target killing. Importantly, instead of dampening the immune response through CTL elimination of variant Ag-expressing immature dendritic cells (DC), a positive CTL-to-DC immune feedback loop dominates whereby the immature DC differentiate into mature proinflammatory DC. Moreover, these CTL-programmed DC exhibit a superior capacity to mediate HIV-1 trans-infection of T cells. This discordant induction of CTL helper activity in the absence of killing most likely contributes to the chronic immune activation associated with HIV-1 infection, and can be used by HIV-1 to promote viral dissemination and persistence. Our findings highlight the need to address the detrimental potential of eliciting dysfunctional cross-reactive memory CTL responses when designing and implementing anti-HIV-1 immunotherapies.

Fifty percent tissue culture infective dose assay for determining the titer of infectious human herpesvirus 8.

We have developed a human herpesvirus 8 (HHV-8) 50% tissue culture infective dose (TCID50) assay using the T1H6-DC-SIGN cell line. Infection of T1H6-DC-SIGN cells with HHV-8 induces expression of ß-galactosidase, which was used to determine TCID50 levels. Validation of TCID50 values was performed by immunofluorescence assay of HHV-8 infection of immature dendritic cells at various TCID50 doses.

Professional antigen presenting cells in human herpesvirus 8 infection.

Professional antigen presenting cells (APC), i.e., dendritic cells (DC), monocytes/macrophages, and B lymphocytes, are critically important in the recognition of an invading pathogen and presentation of antigens to the T cell-mediated arm of immunity. Human herpesvirus 8 (HHV-8) is one of the few human viruses that primarily targets these APC for infection, altering their cytokine profiles, manipulating their surface expression of MHC molecules, and altering their ability to activate HHV-8-specific T cells. This could be why T cell responses to HHV-8 antigens are not very robust. Of these APC, only B cells support complete, lytic HHV-8 infection. However, both complete and abortive virus replication cycles in APC could directly affect viral pathogenesis and progression to Kaposi's sarcoma (KS) and HHV-8-associated B cell cancers. In this review, we discuss the effects of HHV-8 infection on professional APC and their relationship to the development of KS and B cell lymphomas.

Monofunctional and polyfunctional CD8+ T cell responses to human herpesvirus 8 lytic and latency proteins.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. It is postulated that CD8(+) T cell responses play an important role in controlling HHV-8 infection and preventing development of disease. In this study, we investigated monofunctional and polyfunctional CD8(+) T cell responses to HHV-8 lytic proteins gB (glycoprotein B) and K8.1 and latency proteins LANA-1 (latency-associated nuclear antigen-1) and K12. On the basis of our previous findings that dendritic cells (DC) reveal major histocompatibility complex (MHC) class I epitopes in gB, we used a DC-based system to identify 2 novel epitopes in gB, 2 in K8.1, 5 in LANA-1, and 1 in K12. These new HHV-8 epitopes activated monofunctional and polyfunctional CD8(+) T cells that produced various combinations of gamma interferon, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1ß, and cytotoxic degranulation marker CD107a in healthy HHV-8-seropositive individuals. We were also able to detect HHV-8-specific CD8(+) T cells in peripheral blood samples using HLA A*0201 pentamer complexes for one gB epitope, one K8.1 epitope, two LANA-1 epitopes, and one K12 epitope. These immunogenic regions of viral lytic and latency proteins could be important in T cell control of HHV-8 infection.

Primary human immunodeficiency virus type 1-specific CD8+ T-cell responses induced by myeloid dendritic cells.

Induction of an antigenically broad and vigorous primary T-cell immune response by myeloid dendritic cells (DC) in blood and tissues could be important for an effective prophylactic or therapeutic vaccine to human immunodeficiency virus type 1 (HIV-1). Here we show that a primary CD8(+) T-cell response can be induced by HIV-1 peptide-loaded DC derived from blood monocytes of HIV-1-negative adults and neonates (moDC) and by Langerhans cells (LC) and interstitial, dermal-intestinal DC (idDC) derived from CD34(+) stem cells of neonatal cord blood. Optimal priming of single-cell gamma interferon (IFN-gamma) production by CD8(+) T cells required CD4(+) T cells and was broadly directed to multiple regions of Gag, Env, and Nef that corresponded to known and predicted major histocompatibility complex class I epitopes. Polyfunctional CD8(+) T-cell responses, defined as single-cell production of more than one cytokine (IFN-gamma, interleukin 2, or tumor necrosis factor alpha), chemokine (macrophage inhibitory factor 1beta), or cytotoxic degranulation marker CD107a, were primed by moDC, LC, and idDC to HIV-1 Gag and reverse transcriptase epitopes, as well as to Epstein-Barr virus and influenza A virus epitopes. Thus, three major types of blood and tissue myeloid DC targeted by HIV-1, i.e., moDC, LC, and idDC, can prime multispecific, polyfunctional CD8(+) T-cell responses to HIV-1 and other viral antigens.

Cytokine production by human herpesvirus 8-infected dendritic cells.

We have shown previously that human herpesvirus 8 (HHV-8)-infected dendritic cells (DCs) undergo incomplete maturation and have a defective antigen-presenting function. Here, we examined the effects of HHV-8 infection on cytokine production, which is critical to the function of DCs. We detected expression of interleukin (IL)-6, tumour necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES and IL-12p40 from 2 to 6 h post-infection, and these peaked by 15-24 h. Expression of these factors decreased 24-48 h post-infection, with the exception of TNF-alpha which remained high throughout the entire 72 h. Interestingly, while IL-12p40 expression increased post-infection, bioactive IL-12p70 was not detected in the supernatants. These results suggest an intentional skewing of cytokine production in HHV-8-infected DCs towards induction of a Th2 response.

Human herpesvirus 8 infects and replicates in primary cultures of activated B lymphocytes through DC-SIGN.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. Although latent HHV-8 DNA can be detected in B cells from persons with these cancers, there is little information on the replication of HHV-8 in B cells. Indeed, B cells are relatively resistant to HHV-8 infection in vitro. We have recently shown that DC-SIGN, a C-type lectin first identified on dendritic cells (DC), is an entry receptor for HHV-8 on DC and macrophages. We have also demonstrated previously that B lymphocytes from peripheral blood and tonsils express DC-SIGN and that this expression increases after B-cell activation. Here we show that activated blood and tonsillar B cells can be productively infected with HHV-8, as measured by an increase in viral DNA, the expression of viral lytic and latency proteins, and the production of infectious virus. The infection of B cells with HHV-8 was blocked by the pretreatment of the cells with antibody specific for DC-SIGN or with mannan but not antibody specific for xCT, a cystine/glutamate exchange transporter that has been implicated in HHV-8 fusion to cells. The infection of B cells with HHV-8 resulted in increased expression of DC-SIGN and a decrease in the expression of CD20 and major histocompatibility complex class I. HHV-8 could also infect and replicate in B-cell lines transduced to express full-length DC-SIGN but not in B-cell lines transduced to express DC-SIGN lacking the transmembrane domain, demonstrating that the entry of HHV-8 into B cells is related to DC-SIGN-mediated endocytosis. The role of endocytosis in viral entry into activated B cells was confirmed by blocking HHV-8 infection with endocytic pathway inhibitors. Thus, the expression of DC-SIGN is essential for productive HHV-8 infection of and replication in B cells.