Dendritic Cells Loaded with Tumor B Cells Elicit Broad Immunity against Murine Gammaherpesvirus 68 but Fail To Prevent Long-Term Latency

It is still unknown whether a noninfectious gammaherpesvirus vaccine is able to prevent or reduce virus persistence. This led us to use dendritic cells loaded with tumor B cells as a vaccine approach for the murine gammaherpesvirus 68 (γHV68) model of infection. Dendritic cells loaded with UV-irradiated latently infected tumor B cells induce broad, strong, and long-lasting immunity against γHV68. Dendritic cell vaccination prevents the enlargement of lymph nodes and severely limits acute infection and early latency but does not prevent γHV68 from establishing long-term latency. Our findings support the concept that attenuated viruses may be the best vaccine option for preventing gammaherpesvirus persistence.Gammaherpesviruses have very high prevalence, infecting 95% of the world population. Natural infection does not induce sterilizing immunity (21, 30). Murine gammaherpesvirus 68 (γHV68) has important biological similarities to its human counterparts and is a good model for characterizing the immune response and for testing vaccine strategies (11, 33). Gammaherpesvirus vaccines designed to induce neutralizing antibodies reduce the incidence and symptoms of infectious mononucleosis (26) but are only minimally protective (1, 7, 22, 28). Peptide- or epitope-based vaccines that induce T-cell responses affect the early phase of infection but do not alter long-term latency (9, 17, 19, 29, 32). Infection with latency-attenuated viruses induces protection against a challenge with wild-type γHV68, although the vaccine virus persists in the host (6, 25, 30) except in the case of γHV68 AC-RTA (16). These findings with live-attenuated viruses reflect the ability of latency-defective viruses to elicit a wide range of humoral and cell-mediated immune responses and suggest that optimal broad immunity may achieve protection. Dendritic cells (DC) are at the core of the immune response, and they are also the main target of adjuvants. Ex vivo-loaded dendritic cells can induce humoral immunity and strong T-cell immunity (3) and accelerated generation of memory T cells (2). Dendritic cells loaded with multiple antigens could circumvent the narrow antigen specificity of peptide- or epitope-based vaccines and lack the safety concerns associated with live-attenuated herpesviruses. Thus, dendritic cell vaccination can be attractive where other approaches have failed or as a tool for elucidating mechanisms of immune protection. Here, we wanted to test whether dendritic cells loaded ex vivo with a broad range of viral antigens would ameliorate disease and confer protection to gammaherpesvirus infection by inducing strong and broad cellular and humoral immunity.     

Gamma Interferon Signaling in Macrophage Lineage Cells Regulates Central Nervous System Inflammation and Chemokine Production

Intracranial (i.c.) infection of mice with lymphocytic choriomeningitis virus (LCMV) results in anorexic weight loss, mediated by T cells and gamma interferon (IFN-γ). Here, we assessed the role of CD4⁺ T cells and IFN-γ on immune cell recruitment and proinflammatory cytokine/chemokine production in the central nervous system (CNS) after i.c. LCMV infection. We found that T-cell-depleted mice had decreased recruitment of hematopoietic cells to the CNS and diminished levels of IFN-γ, CCL2 (MCP-1), CCL3 (MIP-1α), and CCL5 (RANTES) in the cerebrospinal fluid (CSF). Mice deficient in IFN-γ had decreased CSF levels of CCL3, CCL5, and CXCL10 (IP-10), and decreased activation of both resident CNS and infiltrating antigen-presenting cells (APCs). The effects of IFN-γ signaling on macrophage lineage cells was assessed using transgenic mice, called “macrophages insensitive to interferon gamma” (MIIG) mice, that express a dominant-negative IFN-γ receptor under the control of the CD68 promoter. MIIG mice had decreased levels of CCL2, CCL3, CCL5, and CXCL10 compared to controls despite having normal numbers of LCMV-specific CD4⁺ T cells in the CNS. MIIG mice also had decreased recruitment of infiltrating macrophages and decreased activation of both resident CNS and infiltrating APCs. Finally, MIIG mice were significantly protected from LCMV-induced anorexia and weight loss. Thus, these data suggest that CD4⁺ T-cell production of IFN-γ promotes signaling in macrophage lineage cells, which control (i) the production of proinflammatory cytokines and chemokines, (ii) the recruitment of macrophages to the CNS, (iii) the activation of resident CNS and infiltrating APC populations, and (iv) anorexic weight loss.Immune cell recruitment to and infiltration of the central nervous system (CNS) is central to the pathology of a variety of inflammatory neurological diseases, including infectious meningoencephalitis, multiple sclerosis, and cerebral ischemia (59, 60). Chemokines have been shown to be highly upregulated in both human diseases and animal models of neuroinflammation and are thought to be important mediators of immune cell entry into the CNS (59, 60). For example, during experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS), the chemokines CCL2 (monocyte chemoattractant protein 1 [MCP-1α]), CCL3 (macrophage inflammatory protein 1α [MIP-1α]), CCL5 (regulated upon activation, T-cell expressed and secreted [RANTES]), and CXCL10 (gamma interferon [IFN-γ]-inducible protein 10 [IP-10]) are produced by either resident CNS cells or infiltrating cells (27) and serve to amplify the ongoing inflammatory response (25, 28). However, in some EAE studies, neither CCL3 nor CXCL10 were required for disease (72, 73). During CNS viral infection, CXCL10 and CCL5 are highly produced in several models (2, 41, 48, 82). In addition, mice deficient in CCR5, which binds (among others) CCL3 and CCL5, do not display impaired CNS inflammation after certain viral infections (13). Thus, the role of chemokines in CNS inflammation is likely complex and dissimilar between autoimmune and viral infection models.IFN-γ is present in the CNS during autoimmunity and infection (7, 54, 69). Several studies suggest that IFN-γ can be a potent inducer of CNS chemokine expression. Adenoviral expression of IFN-γ in the CNS strongly induced CCL5 and CXCL10 mRNA and protein, and this induction was dependent on the presence of the IFN-γ receptor (50). In EAE and Toxoplasma infection, mice deficient in IFN-γ or the IFN-γ receptor demonstrated reduced expression of several chemokines, including CCL2, CCL3, CCL5, and CXCL10 (26, 69). However, given the near-ubiquitous expression of the IFN-γ receptor (44), the mechanisms by which IFN-γ regulates CNS chemokine production remain to be elucidated.We studied neuroinflammation and immune-mediated disease using a well-studied mouse model of infection with lymphocytic choriomeningitis virus (LCMV). Intracranial (i.c.) injection of mice with LCMV results in seizures and death 6 to 8 days after inoculation. The onset of symptoms is associated with a massive influx of mononuclear cells into the cerebrospinal fluid (CSF), meninges, choroid plexus, and ependymal membranes (6, 8, 18), as well as the presence of proinflammatory cytokines (7, 38). The immune response is critical for disease, since infection of irradiated or T-cell-depleted mice leads to persistent infection with very high levels of virus in multiple tissues without the development of lethal meningitis (18, 34, 64). i.c. LCMV infection of β₂-microglobulin-deficient mice (β₂m^−/− mice) also results in meningitis and production of proinflammatory cytokines and chemokines; however, meningitis occurs with a later onset and lower severity compared to wild-type mice (17, 24, 53, 57). Interestingly, i.c. LCMV infection of these mice also causes severe anorexia and weight loss (33, 38, 46, 52, 57) that is mediated by major histocompatibility complex (MHC) class II-restricted, CD4⁺ T cells (17, 46, 53, 57). Anorexia and weight loss are also observed in wild-type mice, but they succumb to lethal meningitis shortly thereafter (33), making study of this particular aspect of disease difficult. LCMV-induced weight loss, similar to what we have observed in β₂m^−/− mice also occurs in perforin-deficient mice, which possess CD8⁺ T cells (37). Although some reports have observed weight loss after peripheral LCMV infection (11, 45), we note that these studies used high doses of the clone 13 strain of LCMV, in contrast to our studies which have used the Armstrong strain of LCMV and orders of magnitude less virus (33, 38, 46, 52, 57). Although we cannot exclude a contribution of peripheral cells to weight loss in our i.c. Armstrong infection model, we previously showed that this weight loss does not occur with peripheral infection with LCMV Armstrong (33, 38), indicating that interactions between the CNS and the immune system are contribute substantially to disease.During LCMV infection, there is biphasic production of IFN-γ: a small, early peak of IFN-γ (most likely produced by NK or NKT cells), followed by T-cell-mediated production of IFN-γ (23, 75). Further, both CD4⁺ T cells and CD8⁺ T cells produce large amounts of IFN-γ after LCMV infection and T-cell production of IFN-γ is critical for LCMV-induced weight loss (35). Chemokines, especially CXCL10, CCL5, and CCL2, and their receptors, are upregulated in the brain after i.c. LCMV infection (2, 13). Brain chemokine mRNA expression after i.c. LCMV infection is reduced in IFN-γ-deficient mice and relatively absent in athymic mice (2). However, the mechanism(s) by which T cells and IFN-γ mediate the effects on CNS chemokine expression, cellular infiltration into the CNS, and LCMV-induced anorexic weight loss remain unclear.In the present study, we focused on two major questions. The first question concerned the role of IFN-γ on immune cell recruitment to and chemokine/cytokine production within the CNS? We found that macrophages and myeloid dendritic cells (DCs) require IFN-γ for their accumulation within the CNS. Second, since macrophages and myeloid DCs are the predominant cellular infiltrate, we sought to determine whether IFN-γ signaling on these cells was direct with regard to their recruitment and to chemokine/cytokine production. We found that IFN-γ signaling in macrophage lineage cells contributes significantly to their recruitment, to chemokine production in the CNS, and to anorexic weight loss. Together, these data suggest that much of the proinflammatory effects of IFN-γ in the CNS are mediated by the effects of IFN-γ on CD68-bearing cells.     

Interferons Accelerate Decay of Replication-Competent Nucleocapsids of Hepatitis B Virus

Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host antiviral immunity against hepatitis B virus (HBV) infection in vivo. However, the molecular mechanism by which these antiviral cytokines suppress HBV replication remains elusive. Using an immortalized murine hepatocyte (AML12)-derived cell line supporting tetracycline-inducible HBV replication, we show in this report that both IFN-α and IFN-γ efficiently reduce the amount of intracellular HBV nucleocapsids. Furthermore, we provide evidence suggesting that the IFN-induced cellular antiviral response is able to distinguish and selectively accelerate the decay of HBV replication-competent nucleocapsids but not empty capsids in a proteasome-dependent manner. Our findings thus reveal a novel antiviral mechanism of IFNs and provide a basis for a better understanding of HBV pathobiology.Hepatitis B virus (HBV) is a noncytopathic hepatotropic DNA virus which belongs to the family Hepadnaviridae (11, 44). Despite the fact that most adulthood HBV infections are transient, approximately 5 to 10% of infected adults and more than 90% of infected neonates fail to clear the virus and develop a lifelong persistent infection, which may progress to chronic hepatitis, cirrhosis, and primary hepatocellular carcinoma (4, 33, 34). It has been shown by several research groups that resolution of HBV and other animal hepadnavirus infection in vivo depends on both killing of infected hepatocytes by viral antigen-specific cytotoxic T lymphocytes and noncytolytic suppression of viral replication, which is most likely mediated by inflammatory cytokines, such as gamma interferon (IFN-γ) and tumor necrosis factor α (TNF-α) (10, 12, 15, 20, 26, 27, 48). Moreover, together with five nucleoside or nucleotide analogs that inhibit HBV DNA polymerase, alpha IFN (IFN-α) and pegylated IFN-α are currently available antiviral medications for the management of chronic hepatitis B. Compared to the viral DNA polymerase inhibitors, the advantages of IFN-α therapy include a lack of drug resistance, a finite and defined treatment course, and an increased likelihood for hepatitis B virus surface antigen (HBsAg) clearance (8, 39). However, only approximately 30% of treated patients achieve a sustained virological response to a standard 48-month pegylated IFN-α therapy (6, 32). Thus far, the antiviral mechanism of IFN-α and IFN-γ and the parameters determining the success or failure of IFN-α therapy in chronic hepatitis B remain elusive. Elucidation of the mechanism by which the cytokines suppress HBV replication represents an important step toward understanding the pathobiology of HBV infection and the molecular basis of IFN-α therapy of chronic hepatitis B.Considering the mechanism by which IFNs noncytolytically control HBV infection in vivo, it is possible that the cytokines either induce an antiviral response in hepatocytes to directly limit HBV replication or modulate the host antiviral immune response to indirectly inhibit the virus infection. However, due to the fact that IFN-α and -γ do not inhibit or only modestly inhibit HBV replication in human hepatoma-derived cell lines (5, 22, 23, 30), the direct antiviral effects of the cytokines and their antiviral mechanism against HBV have been studied with either an immortalized hepatocyte cell line derived from HBV transgenic mice or duck hepatitis B virus (DHBV) infection of primary duck hepatocytes (37, 53). While these studies revealed that IFN treatment significantly reduced the amount of encapsidated viral pregenomic RNA (pgRNA) in both mouse and duck hepatocytes, further mechanistic analyses suggested that IFN-α inhibited the formation of pgRNA-containing nucleocapsids in murine hepatocytes (52) but shortened the half-life of encapsidated pgRNA in DHBV-replicating chicken hepatoma cells (21). Moreover, the fate of viral DNA replication intermediates or nucleocapsids in the IFN-treated hepatocytes was not investigated in the previous studies.To further define the target(s) of IFN-α and -γ in the HBV life cycle and to create a robust cell culture system for the identification of IFN-stimulated genes (ISGs) that mediate the antiviral response of the cytokines (25), we established an immortalized murine hepatocyte (AML-12)-derived stable cell line that supported a high level of HBV replication in a tetracycline-inducible manner. Consistent with previous reports, we show that both IFN-α and IFN-γ potently inhibited HBV replication in murine hepatocytes (37, 40). With the help of small molecules that inhibit HBV capsid assembly (Bay-4109) (7, 47) and prevent the incorporation of pgRNA into nucleocapsids (AT-61) (9, 29), we obtained evidence suggesting that the IFN-induced cellular antiviral response is able to distinguish and selectively accelerate the decay of HBV replication-competent nucleocapsids but not empty capsids in a proteasome-dependent manner. Our findings provide a basis for further studies toward better understanding of IFN′s antiviral mechanism, which might ultimately lead to the development of strategies to improve the efficacy of IFN therapy of chronic hepatitis B.     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.