Intravenous administration of human embryonic stem cell-derived neural precursor cells attenuates cuprizone-induced central nervous system (CNS) demyelination.

AIMS: Previous studies have demonstrated the therapeutic potential for human embryonic stem cell-derived neural precursor cells (hES-NPCs) in autoimmune and genetic animal models of demyelinating diseases. Herein, we tested whether intravenous (i.v.) administration of hES-NPCs would impact central nervous system (CNS) demyelination in a cuprizone model of demyelination. METHODS: C57Bl/6 mice were fed cuprizone (0.2%) for 2 weeks and then separated into two groups that either received an i.v. injection of hES-NPCs or i.v. administration of media without these cells. After an additional 2 weeks of dietary cuprizone treatment, CNS tissues were analysed for detection of transplanted cells and differences in myelination in the region of the corpus callosum (CC). RESULTS: Cuprizone-induced demyelination in the CC was significantly reduced in mice treated with hES-NPCs compared with cuprizone-treated controls that did not receive stem cells. hES-NPCs were identified within the brain tissues of treated mice and revealed migration of transplanted cells into the CNS. A limited number of human cells were found to express the mature oligodendrocyte marker, O1, or the astrocyte marker, glial fibrillary acidic protein. Reduced apoptosis and attenuated microglial and astrocytic responses were also observed in the CC of hES-NPC-treated mice. CONCLUSIONS: These findings indicated that systemically administered hES-NPCs migrated from circulation into a demyelinated lesion within the CNS and effectively reduced demyelination. Observed reductions in astrocyte and microglial responses, and the benefit of hES-NPC treatment in this model of myelin injury was not obviously accountable to tissue replacement by exogenously administered cells.

Preferential coxsackievirus replication in proliferating/activated cells: implications for virus tropism, persistence, and pathogenesis.

Coxsackieviruses cause substantial human morbidity and mortality, but the underlying molecular mechanisms of disease remain obscure. Here, we review the effects that the cell status--both cellular activation, and the cell cycle--may have on the outcome of virus infection. We propose that these viruses have evolved to undergo productive infection in cells at the G1/S stage of the cell cycle, and to preferentially establish persistence/latent infection in quiescent cells, and we provide possible explanations for these outcomes. Finally, we consider the implications of these interactions for virus transmission and host pathology.

The cationic region from HIV tat enhances the cell-surface expression of epitope/MHC class I complexes.

The potential of genetic immunization has been acknowledged for almost a decade, but disappointing immunogenicity in humans has delayed its introduction into the clinical arena. To try to increase the potency of genetic immunization, we and others have evaluated 'translocatory' proteins, which are thought to exit living cells by an uncharacterized pathway, and enter neighboring cells in an energy-independent manner. Several laboratories, including our own, have begun to question these remarkable properties. Our previous studies showed that the ability of an epitope to induce major histocompatibility complex (MHC) class I restricted CD8(+) T cells was, indeed, enhanced by its being attached to the proposed translocatory sequence of the HIV-1 tat protein. However, we found little evidence that the increased immunogenicity resulted from transfer of the fusion peptide between living cells, and we proposed that it resulted instead from an increased epitope/MHC expression on the surface of transfected cells. Here, we directly test this hypothesis. We show that cells cotransfected with plasmids encoding an epitope, and the relevant MHC class I allele, can stimulate epitope-specific T cells, and that attachment of the epitope to a putative translocatory sequence - which we term herein an 'integral cationic region' (ICR) - leads to a marked increase in stimulatory activity. This elevated stimulatory capacity does not result from a nonspecific increase in MHC class I expression. We use a high-affinity T-cell receptor (TcR) specific for the epitope/MHC combination to quantitate directly the cell-surface expression of the immunogenic complex, and we show that the attachment of the tat ICR to an epitope results in a substantial enhancement of its cell-surface presentation. These data suggest an alternative explanation for the immune enhancement seen with ICRs.

Full-length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity.

Several proteins have been accorded the unusual ability to translocate across cell membranes in a receptor-independent and temperature-independent manner, and this activity has been mapped to a highly basic series of residues currently termed a 'protein transduction domain' (PTD). This translocatory attribute, if authentic, would be valuable for purposes of gene therapy and vaccination. We have evaluated the PTD from the human immunodeficiency virus type 1 (HIV) tat protein and we conclude that, when synthesized de novo, (1) the HIV tat PTD does not enhance the immunogenicity of a full-length protein to which it is tethered; and (2) the HIV tat PTD does not cause intercellular transfer of an attached marker protein, as judged by careful quantitative analyses. From our data, and from a review of published materials, we suggest that contrary to current dogma there is little evidence that these supposedly translocatory proteins can move between live cells. Furthermore, we suggest that PTDs do not act to enhance translocation, but instead merely to increase binding to the cell surface; in which case, the term 'protein transduction domain', and the related acronym, are misnomers which should be abandoned. Our conclusions explain why the most dramatic demonstrations of PTD efficacy have been obtained using fixed cells and/or denatured proteins, and have obvious implications for gene therapy and vaccination.

CD4(+) T cells induced by a DNA vaccine: immunological consequences of epitope-specific lysosomal targeting.

Our previous studies have shown that targeting DNA vaccine-encoded major histocompatibility complex class I epitopes to the proteasome enhanced CD8(+) T-cell induction and protection against lymphocytic choriomeningitis virus (LCMV) challenge. Here, we expand these studies to evaluate CD4(+) T-cell responses induced by DNA immunization and describe a system for targeting proteins and minigenes to lysosomes. Full-length proteins can be targeted to the lysosomal compartment by covalent attachment to the 20-amino-acid C-terminal tail of lysosomal integral membrane protein-II (LIMP-II). Using minigenes encoding defined T-helper epitopes from lymphocytic choriomeningitis virus, we show that the CD4(+) T-cell response induced by the NP(309-328) epitope of LCMV was greatly enhanced by addition of the LIMP-II tail. However, the immunological consequence of lysosomal targeting is not invariably positive; the CD4(+) T-cell response induced by the GP(61-80) epitope was almost abolished when attached to the LIMP-II tail. We identify the mechanism which underlies this marked difference in outcome. The GP(61-80) epitope is highly susceptible to cleavage by cathepsin D, an aspartic endopeptidase found almost exclusively in lysosomes. We show, using mass spectrometry, that the GP(61-80) peptide is cleaved between residues F(74) and K(75) and that this destroys its ability to stimulate virus-specific CD4(+) T cells. Thus, the immunological result of lysosomal targeting varies, depending upon the primary sequence of the encoded antigen. We analyze the effects of CD4(+) T-cell priming on the virus-specific antibody and CD8(+) T-cell responses which are mounted after virus infection and show that neither response appears to be accelerated or enhanced. Finally, we evaluate the protective benefits of CD4(+) T-cell vaccination in the LCMV model system; in contrast to DNA vaccine-induced CD8(+) T cells, which can confer solid protection against LCMV challenge, DNA vaccine-mediated priming of CD4(+) T cells does not appear to enhance the vaccinee's ability to combat viral challenge.

Enhancing T cell activation and antiviral protection by introducing the HIV-1 protein transduction domain into a DNA vaccine.

Protein transduction domains (PTD), which can transport proteins or peptides across biological membranes, have been identified in several proteins of viral, invertebrate, and vertebrate origin. Here, we evaluate the immunological and biological consequences of including PTD in synthetic peptides and in DNA vaccines that contain CD8(+) T cell epitopes from lymphocytic choriomeningitis virus (LCMV). Synthetic PTD-peptides did not induce detectable CD8(+) T cell responses. However, fusion of an open reading frame encoding a PTD to an epitope minigene caused transfected tissue culture cells to stimulate epitope-specific T cells much more effectively. Kinetic studies indicated that the epitope reached the surface of transfected cells more rapidly and that the number of transfected cells needed to stimulate T cell responses was reduced by 35- to 50-fold when compared to cells transfected with a standard minigene plasmid. The mechanism underlying the effect of PTD linkage is not clear, but transit of the PTD-attached epitope from transfected cells to nontransfected cells (cross presentation) seemed to play, at most, a minimal role. Mice immunized once with the plasmid encoding the PTD-linked epitope showed a markedly accelerated CD8(+) T cell response and, unlike mice immunized with a standard plasmid, were completely protected against a normally lethal LCMV challenge administered only 8 days post-immunization.

Viruses can silently prime for and trigger central nervous system autoimmune disease.

Although many viruses have been isolated from patients with multiple sclerosis (MS), as yet, no one agent has been demonstrated to cause MS. In contrast, epidemiological data indicate that viral infections are associated with exacerbations of MS. Here, we present data showing that virus infections can subclinically prime animals for central nervous system (CNS) autoimmune disease; long after the original infection has been eradicated, a nonspecific challenge/infection can trigger an exacerbation. The priming infectious agent must show molecular mimicry with self-CNS antigens such as glial fibrillary acidic protein (GFAP), myelin associated glycoprotein (MAG) or myelin proteolipid protein (PLP). The subsequent challenge, however, may be nonspecific; complete Freund's adjuvant (CFA), or infection with a recombinant vaccinia virus encoding an irrelevant protein, could trigger CNS disease. In the CNS, we could detect a mononuclear cell infiltration, but no demyelination was found. However, if the pathogenesis of MS is similar to that of this novel animal model for CNS autoimmune disease, our findings could help explain why exacerbations of MS are often associated with a variety of different viral infections.

Functional avidity maturation of CD8(+) T cells without selection of higher affinity TCR.

Unlike B cells, T cells lack the capacity to improve the affinity of their antigen receptors by somatic mutation. It is, therefore, believed that optimization of cellular immunity is mediated almost exclusively through selective expansion of T cells bearing receptors with the highest affinity for antigen. We show here that T cell responsiveness to peptide (termed "functional avidity") increased>50-fold during the early stages of viral infection. This indicated that T cells, like B cells, undergo extensive functional maturation in vivo. However, in contrast to B cells, maturation of the T cell response can occur without any appreciable change in T cell receptor affinity.

Two overlapping subdominant epitopes identified by DNA immunization induce protective CD8(+) T-cell populations with differing cytolytic activities.

Subdominant CD8(+) T-cell responses contribute to control of several viral infections and to vaccine-induced immunity. Here, using the lymphocytic choriomeningitis virus model, we demonstrate that subdominant epitopes can be more reliably identified by DNA immunization than by other methods, permitting the identification, in the virus nucleoprotein, of two overlapping subdominant epitopes: one presented by L(d) and the other presented by K(d). This subdominant sequence confers immunity as effective as that induced by the dominant epitope, against which >90% of the antiviral CD8(+) T cells are normally directed. We compare the kinetics of the dominant and subdominant responses after vaccination with those following subsequent viral infection. The dominant CD8(+) response expands more rapidly than the subdominant responses, but after virus infection is cleared, mice which had been immunized with the "dominant" vaccine have a pool of memory T cells focused almost entirely upon the dominant epitope. In contrast, after virus infection, mice which had been immunized with the "subdominant" vaccine retain both dominant and subdominant memory cells. During the acute phase of the immune response, the acquisition of cytokine responsiveness by subdominant CD8(+) T cells precedes their development of lytic activity. Furthermore, in both dominant and subdominant populations, lytic activity declines more rapidly than cytokine responsiveness. Thus, the lysis(low)-cytokine(competent) phenotype associated with most memory CD8(+) T cells appears to develop soon after antigen clearance. Finally, lytic activity differs among CD8(+) T-cell populations with different epitope specificities, suggesting that vaccines can be designed to selectively induce CD8(+) T cells with distinct functional attributes.

Using recombinant coxsackievirus B3 to evaluate the induction and protective efficacy of CD8+ T cells during picornavirus infection.

Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDpro cleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were approximately 1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+ T-cell response to the encoded epitope; this has implications for the proposed role of "cross-priming" during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+ memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in approximately 50-fold-lower virus titers in the heart and approximately 6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.

Animal models using lymphocytic choriomeningitis virus.

This unit includes protocols for inducing systemic infection and persistent infection of mice with lymphocytic choriomeningitis virus (LCMV). Methods used to measure T cell responses to LCMV are then described. A protocol to assess anti-LCMV immunity in vivo is also included. Support protocols for preparing LCMV stocks and measuring LCMV titers using a plaque assay are also included. Finally, a support protocol for detecting anti-LCMV antibodies by ELISA is presented.

Direct ex vivo kinetic and phenotypic analyses of CD8(+) T-cell responses induced by DNA immunization.

CD8(+) T-cell responses can be induced by DNA immunization, but little is known about the kinetics of these responses in vivo in the absence of restimulation or how soon protective immunity is conferred by a DNA vaccine. It is also unclear if CD8(+) T cells primed by DNA vaccines express the vigorous effector functions characteristic of cells primed by natural infection or by immunization with a recombinant live virus vaccine. To address these issues, we have used the sensitive technique of intracellular cytokine staining to carry out direct ex vivo kinetic and phenotypic analyses of antigen-specific CD8(+) T cells present in the spleens of mice at various times after (i) a single intramuscular administration of a plasmid expressing the nucleoprotein (NP) gene from lymphocytic choriomeningitis virus (LCMV), (ii) infection by a recombinant vaccinia virus carrying the same protein (vvNP), or (iii) LCMV infection. In addition, we have evaluated the rapidity with which protective immunity against both lethal and sublethal LCMV infections is achieved following DNA vaccination. The CD8(+) T-cell response in DNA-vaccinated mice was slightly delayed compared to LCMV or vvNP vaccinees, peaking at 15 days postimmunization. Interestingly, the percentage of antigen-specific CD8(+) T cells present in the spleen at day 15 and later time points was similar to that observed following vvNP infection. T cells primed by DNA vaccination or by infection exhibited similar cytokine expression profiles and had similar avidities for an immunodominant cytotoxic T lymphocyte epitope peptide, implying that the responses induced by DNA vaccination differ quantitatively but not qualitatively from those induced by live virus infection. Surprisingly, protection from both lethal and sublethal LCMV infections was conferred within 1 week of DNA vaccination, well before the peak of the CD8(+) T-cell response.

DNA vaccination to treat autoimmune diabetes.

In numerous animal models, DNA immunization has been shown to induce protective immunity against infectious diseases (viral, bacterial and protozoan) and cancers (1, 2). In these situations it is desirable to induce a strong immune response to the DNA-encoded antigen in order to generate an immune memory that enables the vaccine to respond more rapidly to subsequent challenge. The success of DNA vaccination in this regard has led to its rapid introduction into several human clinical trials (3, 4). However, in autoimmunity, undesirable immune responses to autoantigens are thought to lead to the destruction of target cells or organs, resulting in diseases such as myasthenia gravis, diabetes or multiple sclerosis. Thus, at first sight, it appears that immunization would more likely trigger autoimmunity than ameliorate it. Nevertheless, clinical experience has shown that certain immune-mediated diseases may be countered by low-dose antigen administration ('desensitization'), although the underlying mechanisms remain somewhat conjectural. Here, we will describe an intriguing approach to the prevention of autoimmune disease, in which we use a DNA vaccine encoding a self-antigen to abrogate autoimmune diabetes. The success of this strategy relies on the nature of the immune response induced by the DNA vaccine.

Coxsackievirus infection of the pancreas: evaluation of receptor expression, pathogenesis, and immunopathology.

Coxsackievirus type B (CVB) infection of the pancreas induces a massive cellular infiltrate composed of natural killer cells, T cells, and macrophages and leads to the destruction of exocrine tissue. The physiological manifestations of pancreatic CVB infection are correlated with viral tropism; the virus infects acinar cells but spares the islets of Langerhans. Here we evaluate the mechanisms underlying pancreatic inflammation and destruction and identify the determinants of viral tropism. T-cell-mediated immunopathology has been invoked, along with direct virus-mediated cytopathicity, to explain certain aspects of CVB-induced pancreatic disease. However, we show here that in the pancreas, the extent of inflammation and tissue destruction appears unaltered in the absence of the cytolytic protein perforin; these findings exclude any requirement for perforin-mediated lysis by natural killer cells or cytotoxic T cells in CVB3-induced pancreatic damage. Furthermore, perforin-mediated cytotoxic T-cell activity does not contribute to the control of CVB infection in this organ. In addition, we demonstrate that the recently identified coxsackie-adenovirus receptor is expressed at high levels in acinar cells but is barely detectable in islets, which is consistent with its being a major determinant of virus tropism and, therefore, of disease. However, further studies using various cell lines of pancreatic origin reveal secondary determinants of virus tropism.

Clinical implications of dysregulated cytokine production.

Cytokines are soluble proteins that are produced and secreted as part of the immune response to a variety of tissue insults including infection, cancer, and autoimmunity. Most cytokines are secreted by cells of the immune system, but some (for example, type I interferons) are released from "nonimmunological" cells such as fibroblasts and epithelial cells. Cytokines have pleiotropic effects, acting on many somatic cell types to modulate the host's immune response. For the most part, cytokines exert their antimicrobial actions locally---they are secreted by cells in the area of infection, and their effects are restricted to neighboring cells. While many of their local effects benefit the host, cytokines are soluble molecules that may act systemically and are often responsible for many of the symptoms of infection (e.g., headache, fever, myalgia). In high concentrations they can be toxic, or even lethal. Human clinical trials involving the systemic injection of purified cytokines such as interleukins 2 and 12 and tumor necrosis factor alpha provide compelling evidence for the toxicity of these molecules. Likewise, studies of septic shock syndrome demonstrate how overproduction/aberrant production of inflammatory cytokines can lead to rapid mortality. The host may attempt to counter high cytokine levels by releasing soluble cytokine receptors (sCR) or by synthesizing high-affinity anti-cytokine antibodies (acAb), and these natural responses have spawned great interest as potential therapeutic approaches for alleviating cytokine-mediated disease. However, recent studies indicate that these in vivo interactions are much more complex than previously realized; administration of sCR or acAb may either inhibit or (paradoxically) enhance cytokine activity. An alternative therapeutic approach is to intervene at the source of cytokine production. T cells initiate cytokine production only upon antigen contact and terminate synthesis almost immediately after this contact is broken. Thus T cells secrete cytokines specifically at sites of infection and do not continuously produce these potentially toxic molecules while migrating through uninfected tissues or the bloodstream. By learning more about the molecular mechanisms involved with on/off regulation of cytokine production we may be able to develop novel therapeutic drugs to protect against cytokine-mediated immunopathology. This review discusses the regulation of cytokine function by sCR and acAb and compares this to the regulatory mechanisms that are associated with antigen-specific cytokine release by T cells.

An autologous oral DNA vaccine protects against murine melanoma.

We demonstrated that peripheral T cell tolerance toward murine melanoma self-antigens gp100 and TRP-2 can be broken by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to minigenes encoding peptide epitopes gp100(25-33) and TRP-2(181-188). These epitopes contain dominant anchor residues for MHC class I antigen alleles H-2D(b) and H-2K(b), respectively. The DNA vaccine was delivered by oral gavage by using an attenuated strain of Salmonella typhimurium as carrier. Tumor-protective immunity was mediated by MHC class I antigen-restricted CD8(+) T cells that secreted T(H)1 cytokine IFN-gamma and induced tumor rejection and growth suppression after a lethal challenge with B16G3. 26 murine melanoma cells. Importantly, the protective immunity induced by this autologous DNA vaccine against murine melanoma cells was at least equal to that achieved through xenoimmunization with the human gp100(25-33) peptide, which differs in its three NH(2)-terminal amino acid residues from its murine counterpart and was previously reported to be clearly superior to an autologous vaccine in inducing protective immunity. The presence of ubiquitin upstream of the minigene proved to be essential for achieving this tumor-protective immunity, suggesting that effective antigen processing and presentation may make it possible to break peripheral T cell tolerance to a self-antigen. This vaccine design might prove useful for future rational designs of other recombinant DNA vaccines targeting tissue differentiation antigens expressed by tumors.

Virus-induced diabetes in a transgenic model: role of cross-reacting viruses and quantitation of effector T cells needed to cause disease.

Virus-specific cytotoxic T lymphocytes (CTL) at frequencies of >1/1, 000 are sufficient to cause insulin-dependent diabetes mellitus (IDDM) in transgenic mice whose pancreatic beta cells express as "self" antigen a protein from a virus later used to initiate infection. The inability to generate sufficient effector CTL for other cross-reacting viruses that fail to cause IDDM could be mapped to point mutations in the CTL epitope or its COO(-) flanking region. These data indicate that IDDM and likely other autoimmune diseases are caused by a quantifiable number of T cells, that neither standard epidemiologic markers nor molecular analysis with nucleic acid probes reliably distinguishes between viruses that do or do not cause diabetes, and that a single-amino-acid change flanking a CTL epitope can interfere with antigen presentation and development of autoimmune disease in vivo.