It is widely believed that viral clearance is mediated principally by the destruction of infected cells by CTLs. In this report, we use a transgenic mouse model of HBV replication to demonstrate that this assumption may not be true for all viruses. We find that adoptively transferred virus-specific CTLs can abolish HBV gene expression and replication in the liver without killing the hepatocytes. This antiviral function is mediated by IFN gamma and TNF alpha secreted by the CTL or by the antigen-nonspecific macrophages and T cells that they activate following antigen recognition. These cytokines activate two independent virocidal pathways: the first pathway eliminates HBV nucleocapsid particles and their cargo of replicating viral genomes, while the second pathway destabilizes the viral RNA. Intracellular viral inactivation mechanisms such as these could greatly amplify the protective effects of the immune response, while failure of such mechanisms could lead to viral persistence or to the death of the host.

During hepatitis B virus (HBV) infection, distinct host-virus interactions may establish the patterns of viral clearance and persistence and the extent of virusassoiated pathology. It is generally thought that HBV-speciflc class I-restricted cytotoxic T lymphocytes (CTLs) play a critical role in this process by destroying infected hepatocytes. This cytopathic m nism, however, could be lethal if most of the hepatocytes are Infected. In the current study, we demonstrate that class I-retricted HBV-speclfic CTLs profoundly suppress hepatoceliular HBV gene expression in HBV transgenic mice by a noncytolytic process, the strength ofwhich greatly exceeds the cytopathic effect of the CTLs in magnitude and duration. We also show that the regulatory effect of the CTLs is initially mediated by interferon y and tumor necrosis factor a, is delayed in onset, and becomes independent of these cytokines shortly after it begins. The data indicate that the anti-viral CTL response activates a complex regulatory cascade that inhibits hepatoceflular HBV gene expression without kifling the cell.The extent to which this mechanism contributes to viral clearance or viral persistence during HBV infection rem to be determined.The control of hepatitis B virus (HBV) infection is thought to be mediated by the class I-restricted cytotoxic T-lymphocyte (CTL) response. Patients with acute viral hepatitis, who successfully clear the virus, mount a multispecific polyclonal CTL response to several HBV-encoded antigens, whereas persistently infected patients with chronic hepatitis do not (1). Hepatitis B surface antigen (HBsAg)-specific murine CTL clones cause a necroinflammatory liver disease when they are injected into HBsAg-positive transgenic mice, and the cytopathic effect of the CTLs is largely mediated by the inflammatory cytokines that they release when they are activated by antigen recognition (2, 3). While the data indicate that the CTL response to HBV plays a critical role in viral clearance and disease pathogenesis, the extent to which viral clearance depends on the destruction of infected cells is not known at this time.We have shown that hepatocellular HBV gene expression in transgenic mice is negatively regulated, noncytopathically, by the pharmacological administration of recombinant interleukin (IL) 2 and tumor necrosis factor (TNF) a and that the IL-2 effect is mediated by TNF-a, which inhibits HBV gene expression by a posttranscriptional mechanism in these animals (4-6). We now report that hepatocellular HBV gene expression is profoundly inhibited, noncytopathically, by HBsAg-specific class I-restricted CTLs activated physiologically by antigen recognition in vivo, and we show that the inhibitory effect of the CTLs, which is mediated by TNF-a and interferon y (IFN-y), greatly exceeds their cytopathic effect in magnitude and duration. MATERIALS AND METHODSHBV Transgenic Mice. The two lineages studied were selected because they do not develop evidence of spontaneous liver disease and they display lineage-specific differences...

Hepatitis B virus (HBV) infection is thought to be controlled by virus-specific cytotoxic T lymphocytes (CTL). We have recently shown that HBV-specific CTL can abolish HBV replication noncytopathically in the liver of transgenic mice by secreting tumor necrosis factor a (TNF-a) and interferon y (IFN-,y) Because the effector functions of these antiviral cytokines are independent of their source, HBV gene expression and replication should also be suppressed if the appropriate cytokines are induced in the liver by HBV-nonspecific stimuli. To examine this hypothesis, we monitored HBV gene expression and replication in serum and liver of HBV transgenic mice during lymphocytic choriomeningitis virus (LCMV) infection.LCMV, an arenavirus that is a natural pathogen of mice, causes a noncytopathic infection of many tissues, including the liver (5). Inoculation of adult immunocompetent mice with moderate doses of most LCMV isolates produces an acute infection, with virus clearance occurring in 7-14 days. LCMV clearance is mediated by CD8+, major histocompatability complex class I-restricted CTL (6). If an effective CTL response is not generated, a persistent infection is established (7). Therefore, mice infected with LCMV at birth or in utero develop a lifelong persistent infection because they are unable to mount an effective CTL response, primarily because of thymic deletion of LCMV-reactive T cells (8,9).We now report that hepatocellular HBV gene expression and replication are profoundly and noncytopathically suppressed during acute and persistent LCMV infection in HBV transgenic mice, and we demonstrate that these effects are mediated principally by TNF-a and IFN-a/P produced by LCMV-infected intrahepatic macrophages.MATERIALS AND METHODS HBV Transgenic Mice. The HBV transgenic mice used in this study (lineage 1.3.32) have been previously described (10). The hepatocytes from these animals replicate HBV from an integrated greater-than-genome-length HBV transcriptional template at levels comparable with that seen in the infected livers of patients with chronic hepatitis, without any evidence of cytopathology (10). Lineage 1.3.32, originally produced in a C57BL/6/SJL hybrid, was expanded by repetitive backcrossing against C57BL/6 and routinely backcrossed one generation against B10.D2 to produce H-2bxd F1 hybrids before LCMV infection.LCMV Isolates and Infection of Mice. Two different clones of the WE isolate of LCMV were used in these studies: clones 2.2 and 54 (M. N. Teng, P.B., M.B.A.O., and J. C. de la Torre, unpublished results). The titers of the LCMV stocks, and also infectious virus titers in murine tissues, were determined by plaque assay on Vero cells (11). Adult male HBV transgenic mice (10 weeks old) were infected by i.v. inoculation of 2 x 106 plaque-forming units (pfu) of LCMV WE clone 2.2. Newborn transgenic mice were infected within 24 hr of birth by i.c. inoculation of 103 pfu of LCMV WE clone 54.LCMV-Specific CTL Assay. LCMV-specific CTL activity was quantitated using a standard 5tCr release a...

Characterization of Brn-3.0 and identification of a highly related member (Brn-3.1) of the class IV POUdomain family suggest potential roles of Brn-3.0 in the development of retinal ganglion cells and sensory neurons, as well as potential roles in the pituitary gland and the immune system. Brn-3.0 is expressed in the pituitary gland and in a corticotroph cell line. A functional DNA response element has been identified in the proopiomelanocortin promoter. In contrast to previously described mammalian POU-domain proteins, Brn-3.0 binds relatively ineffectively to known octamer DNA motifs, but instead binds with high affinity to a distinct set ofDNA elements, functioning as a transcriptional activator. Brn-3.0, Brn-3

To investigate the mechanism by which viruses are cleared from neurons in the central nervous system, we have utilized a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (OBLV60). After intranasal inoculation, OBLV60 grew preferentially in the olfactory bulbs of BALB/c mice. Using in situ hybridization, we found that viral RNA localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. Virus was cleared rapidly from the olfactory bulb between 5 and 11 days. Athymic nude mice failed to eliminate the virus, demonstrating a requirement for T lymphocytes. Immunosuppression of normal mice with cyclophosphamide also prevented clearance. Both CD4+ and CD8+ T-cell subsets were important, as depletion of either of these subsets delayed viral clearance. Gliosis and infiltrates of CD4+ and CD8+ cells were detected by immunohistochemical analysis at 6 days. The role of cytokines in clearance was investigated by using an RNase protection assay for interleukin-la (IL-la), IL-10i, IL-2, IL-3, IL-4, IL-5, IL-6, tumor necrosis factor alpha (TNF-o), TNF-I, and gamma interferon (IFN-,y). In immunocompetent mice there was upregulation of RNA for IL-la, IL-11", IL-6, TNF-a, and IFN-y at the time of clearance. Nude mice had comparable increases in these cytokine messages, with the exception of IFN-y. Induction of major histocompatibility complex class I (MHC-I) molecules on cells in infected brains was demonstrated by immunohistochemical analyses in normal and nude mice, suggesting that IFN-y may not be necessary for induction of MHC-I on neural cells in vivo.

One hypothesis for the etiology of central nervous system (CNS) autoimmune disease is that infection by a virus sharing antigenic epitopes with CNS antigens (molecular mimicry) elicits a virus-specific immune response that also recognizes self-epitopes. To address this hypothesis, transgenic mice were generated that express the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus (LCMV) as self in oligodendrocytes. Intraperitoneal infection with LCMV strain Armstrong led to infection of tissues in the periphery but not the CNS, and the virus was cleared within 7–14 d. After clearance, a chronic inflammation of the CNS resulted, accompanied by upregulation of CNS expression of MHC class I and II molecules. A second LCMV infection led to enhanced CNS pathology, characterized by loss of myelin and clinical motor dysfunction. Disease enhancement also occurred after a second infection with unrelated viruses that cross-activated LCMV-specific memory T cells. These findings indicate that chronic CNS autoimmune disease may be induced by infection with a virus sharing epitopes with a protein expressed in oligodendrocytes and this disease may be enhanced by a second infection with the same or an unrelated virus. These results may explain the association of several different viruses with some human autoimmune diseases.

The Epstein-Barr virus (EBV)-associated Although phenotypic analysis of LPDs has suggested similarities with LCLs, based on positive immunohistology for CD23, CD58 (LFA-3), and CD54 (ICAM-1) in three lesions (12), other studies found positive CD23 expression in only five of nine LPD lesions (13), and three lesions were positive for the expression of CD38, a surface antigen associated with plasma-cell formation (13). Furthermore, histopathologic analysis characterizes LPD as polymorphic B-cell immunoblasts with variable degrees ofplasmacytoid features (14)(15)(16).In this study, we have addressed the question of whether EBV-associated LPDs are mature LCL-like activated B cells or represent a more differentiated plasmacytoid phenotype, by examining patterns of cellular and viral gene expression in EBV-induced LPDs of severe combined immunodeficiency/ human (SCID/hu) chimeric mice, a model that has been shown to be remarkably similar to human posttransplant . Recent studies on EBV-induced tumor formation in SCID/hu chimeric mice have shown that CD23 and CD20 expression by the tumor cells is greatly reduced relative to donor-matched LCLs (20,21) and that tumor cells have a plasmacytoid morphology (17, 18). We injected LCLs into SCID mice and compared the resultant tumor cells to the input LCL. Analysis of LCL-derived tumors, rather than tumors arising in SCID mice engrafted with human peripheral blood (17, 18, 20), ensures that all tumor cells are EBVinfected and avoids the possible presence of reactive (i.e., uninfected) B-cell populations. We present data to show that phenotypic changes in the tumor cells consistent with a terminally differentiated plasma cell were observed and that the tumor cells have significantly reduced EBNA2 and LMP1 gene expression relative to the input LCLs, indicating that

SummaryB cell knockout mice p, MT/p, MT were used to examine the requirement for B cell antigen (Ag) presentation in the establishment of CD4 + T cell tolerance. CD4 + T cells from p, MT mice injected with exogenous protein Ag in adjuvant responded to in vitro challenge by transcription of cytokine mKNA, cytokine secretion, and proliferation. Peripheral tolerance could be established in p~MT mice with a single dose of deaggragated protein. This tolerance was manifested by a loss of T cell proliferation and cytokine production (including both T helper cell type 1 [Thl]-and Th2-related cytokines), indicating that B cells are not required for the induction of peripheral T cell tolerance and suggesting that the dual zone tolerance theory is not applicable to all protein Ags and is not mediated through Ag presentation by B cells.T he role of B cells in Ag presentation to peripheral CD4 + T cell responses is controversial. Although B cells are the most abundant MHC class II-positive cell and have been shown in some studies to be involved in the activation of peripheral T cells, other investigators have argued against the notion that B cells are necessary as APCs for induction of T cell responses. Thus, results from studies have ranged from, first, the B cell as the crucial initiating APC in the lymph node (1), through second, the B cell as a necessary mediator of T cell clonal expansion and secondary proliferation (2-5), to third, normal priming and proliferation of T cells in the absence of B cells (6, 7). These studies all examined Ag-specific T cell activation in mice that had been rendered deficient in B cells, either by continuous injection of anti-p, Ab, by reconstitution of SCID mice with naive T cells, or, most recently, by targeted deletion of either the IgM heavy chain gene (8) or the JH gene segment (5, 9). The role of B cells in the induction of peripheral T cell tolerance is less controversial. B cells can serve as APCs in the induction of Ag-specific tolerance in naive CD4 + and CD8 + T cells (10, 11) and in Ag-specific T cell clones (12). These reports led to speculation by investigators that tolerance induction in virgin T cells is a primary function of the B cell.To reevaluate the role of B cells as APCs for activation and tolerization ofCD4 + T cells, we used the B cell knockout mouse ~MT/p~MT (hereafter p~MT), which was generated by introduction of a nonsense mutation into the transmembrane exon of the IgM heavy chain gene, resulting in a total deletion of B cells (13). The Ag used to investigate CD4 + T cell responses in these mice was human ~/-globulin (HGG), 1 which is a T-dependent Ag able to induce either tolerance or activation ofT cells, depending on the injection procedure. HGG injected subcutaneously in adjuvant induces T cell proliferation and cytokine secretion as well as a significant anti-HGG Ag response, whereas a single injection of deaggregated HGG (DHGG) induces a long-lasting peripheral tolerance that affects both B cells and T cells (reviewed in 14 and 15). In the case ofT cells, ...