Lymphocytic Choriomeningitis Virus Glycoprotein Research Articles

Endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase SKI-1/S1P.

The envelope glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is posttranslationally cleaved into two subunits. We show here that this endoproteolytic processing is not required for transport to the cell surface but is essential for LCMV GP to mediate infectivity of pseudotyped retroviral vectors. By systematic mutational analysis of the LCMV GP cleavage site, we determined that the consensus motif R-(R/K/H)-L-(A/L/S/T/F)(265) is essential for the endoproteolytic processing. In agreement with the identified consensus motif, we show that the cellular subtilase SKI-1/S1P cleaves LCMV GP.

Transduction of dendritic cells by antigen-encoding lentiviral vectors permits antigen processing and MHC class I–dependent presentation

Because antigen-presenting dendritic cells (DCs) play a major role in the polarization of T cells, including T(H)2 cells involved in allergy, strategies to modify DCs genetically are required. The purpose of this investigation was to transduce murine bone marrow-derived DCs with lentiviral vectors encoding antigen to demonstrate antigen processing and MHC class I-dependent presentation. Bone marrow leukocytes were incubated with antigen-encoding lentiviral constructs and cultured with GM-CSF, IL-4, and Flt-3 ligand. The capacity of the resulting DCs to express, process, and present antigen was tested in vitro. An average of 40% of DCs expressed antigen after 1 week of culture when antigen encoded by the lentiviral vector construct was green fluorescent protein. To demonstrate that transduced antigen can be presented by DCs on MHC class I, we chose the lymphocytic choriomeningitis virus glycoprotein (gp) as a model antigen, inasmuch as it is recognized by CD8 T cells from transgenic mice expressing an MHC class I-restricted T-cell receptor specific for the epitope of positions 33 through 41 of gp. DCs transduced with lentiviral construct encoding gp and matured with LPS activated transgenic T cells in an antigen-specific fashion. Using transporter associated with antigen presentation (TAP)-deficient mice, we show that presentation of the gp33-41 epitope is TAP-dependent, confirming processing of gp by the endogenous pathway. These results demonstrate that CD8 T cells can recognize MHC class I epitopes processed from antigen in DCs transduced with lentiviral vectors. Lentiviral transduction of DCs and antigen presentation to CD8 T cells could be exploited for immunotherapy, because allergen-specific CD8 T cells have been shown to be suppressive in IgE-dependent allergy models.

Calcineurin Aalpha plays an exclusive role in TCR signaling in mature but not in immature T cells.

Calcineurin has been demonstrated as one of the key enzymes in TCR-mediated signaling cascades that lead to the transcription of a variety of cytokines including IL-2. In this study, we addressed the role of calcineurin in lymphocyte development and peripheral T cell responses using the lymphocytic choriomeningitis virus glycoprotein peptide p33-specific, TCR (P14)-transgenic T cells that were deficient in calcineurin subunit A alpha-isoform (CNAalpha(-/-)). Fetal thymic organ culture of P14/CNAalpha(-/-) lobes showed no defect in positive or negative selection of thymocytes. In addition, peptide-induced peripheral T cell deletion was also normal in CNAalpha-deficient T cells. In terms of mature T cell function, a reduction in proliferation, and IL-2 and IFN-gamma production was observed upon stimulation of P14/CNAalpha(-/-) T cells with the antigenic peptide. Impaired NF-AT nuclear localization was also observed. These results suggest that CNAalphais important for mature T cell function, but has a limited role in thymocyte development.

Apoptosis of autoreactive CD8 lymphocytes as a potential mechanism for the abrogation of type 1 diabetes by islet-specific TNF-alpha expression at a time when the autoimmune process is already ongoing.

The role of TNF-alpha in type 1 diabetes pathogenesis is controversial. Using double transgenic mice expressing (i) the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) as an islet self-antigen and (ii) TNF-alpha under control of a tetracycline-regulated promotor system (tTA) in the pancreatic beta cells, we could previously demonstrate a differential effect of TNF-alpha on the incidence of type 1 diabetes. Most interestingly, late expression of TNF-alpha resulted in a reversion of mice that were already diabetic to a nondiabetic state. Here we provide a model of how experienced autoaggressive CD8 lymphocytes are dying by apoptosis as a result of beta cell-specific TNF-alpha expression at a time when the autoimmune process is already ongoing.

Enhanced T cell responses contribute to the genetic predisposition of CD8-mediated spontaneous autoimmunity.

A number of factors have been demonstrated to influence the induction of pathogenic autoimmune responses, including the loss of regulatory T cells. To assess the contribution of regulatory T cells in CD8(+) T cell-mediated autoimmunity, RIP-gp/P14 double-transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) on pancreatic beta-islet cells, together with T cells expressing an LCMV-gp-specific T cell receptor (TCR), were crossed to RAG 2-deficient mice. The loss of potentially regulatory T cells, however, did not contribute to diabetes induction. Surprisingly, both RIP-gp/P14-RAG(+/-) and RIP-gp/P14-RAG(-/-) developed spontaneous disease, suggesting an influence of the 129 genetic background on disease susceptibility. Further studies demonstrated that disease susceptibility was not due to nonspecific T cell activation, nor to enhanced cross-presentation of LCMV-gp, nor to decreased expression levels of the negative regulatory molecule CD5. Disease susceptibility did associate, however, with enhanced T cell responses. Thus, T cell hyperactivity combined with various genetic factors may predispose an individual to autoimmunity.

Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range.

Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.

Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes.

The lineage relationship between short-lived effector T cells and long-lived memory cells is not fully understood. We have described T-GFP mice previously, in which naive and early activated T cells express GFP uniformly, whereas cells that have differentiated into effector cytotoxic T cells selectively lose GFP expression. Here we studied antigen-specific CD8 T cell differentiation using T-GFP mice crossed to the TCR transgenic (Tg) mice P14 (specific for the lymphocytic choriomeningitis virus glycoprotein peptide, gp33-41). After activation with antigenic peptide, P14XT-GFP CD8(+) T cells cultured in high-dose IL-2 developed into cells with effector phenotype and function: they were blastoid, lost GFP expression, expressed high levels of activation and effector markers, and were capable of immediate cytotoxic function. In contrast, cells cultured in IL-15 or low-dose IL-2 never developed into full-fledged effector cells. Rather, they resembled memory cells: they were smaller, were GFP(+), did not express effector markers, and were incapable of immediate cytotoxicity. However, they mediated rapid-recall responses in vitro. After adoptive transfer, they survived in vivo for at least 10 weeks and mounted a secondary immune response after antigen rechallenge that was as potent as endogenously generated memory cells. In addition to providing a simple means to generate memory cells in virtually unlimited numbers, our results suggest that effector differentiation is not a prerequisite for memory cell generation.

Perforin-independent regulation of dendritic cell homeostasis by CD8(+) T cells in vivo: implications for adaptive immunotherapy.

We investigated here the effects of perforin on CTL responses during interaction of dendritic cells (DC) with cytotoxic T lymphocytes in vivo. Using MHC class I tetramers complexed with the immunodominant CTL epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP33), we followed the kinetics of DC-induced CTL responses. GP33-presenting DC induced rapid primary expansion of both perforin-competent and -deficient CTL with similar kinetics. Secondary CTL responses in perforin-deficient and normal control mice after DC-booster immunization were more rapid than the primary responses, but never reached the high initial levels, suggesting that reactivated memory CTL eliminated the antigen-presenting DC and thereby limited the booster effect. Whereas killingof DC in vitro was strictly dependent on perforin, elimination of GP33-presenting DC by CTL in vivo was largely independent of perforin and Fas. Taken together, these results suggest that control of DC homeostasis by CTL, i. e. elimination of DC by the effector cells they had elicited, is controlled via multiple and probably redundant signals and represents an important fail-safe mechanism to avoid exaggerated CTL responses.

Perforin-independent regulation of dendritic cell homeostasis by CD8+ T cellsin vivo: implications for adaptive immunotherapy

We investigated here the effects of perforin on CTL responses during interaction of dendritic cells (DC) with cytotoxic T lymphocytes in vivo. Using MHC class I tetramers complexed with the immunodominant CTL epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP33), we followed the kinetics of DC-induced CTL responses. GP33-presenting DC induced rapid primary expansion of both perforin-competent and -deficient CTL with similar kinetics. Secondary CTL responses in perforin-deficient and normal control mice after DC-booster immunization were more rapid than the primary responses, but never reached the high initial levels, suggesting that reactivated memory CTL eliminated the antigen-presenting DC and thereby limited the booster effect. Whereas killingof DC in vitro was strictly dependent on perforin, elimination of GP33-presenting DC by CTL in vivo was largely independent of perforin and Fas. Taken together, these results suggest that control of DC homeostasis by CTL, i. e. elimination of DC by the effector cells they had elicited, is controlled via multiple and probably redundant signals and represents an important fail-safe mechanism to avoid exaggerated CTL responses.

Rapid peptide turnover and inefficient presentation of exogenous antigen critically limit the activation of self-reactive CTL by dendritic cells.

This study evaluated to what extent presentation of exogenously acquired self-Ags via MHC class I molecules on DC might contribute to the activation of self-reactive CTL and subsequent development of autoimmune disease. We show here by using the rat insulin promotor lymphocytic choriomeningitis virus glycoprotein model of autoimmune diabetes that the activation of self-reactive CTL by DC after uptake of exogenous Ag is very limited, first by the short half-life of MHC class I-associated peptides on DC in vitro and in vivo, and second by the rather inefficient MHC class I presentation of cell-associated self-Ags by DC. These two mechanisms are probably crucial in establishing high thresholds for the induction of self-reactive CTL that prevent autoimmune sequelae after release of sequestered and previously immunologically ignored tissue Ags.

Recombinant expression of lymphocytic choriomeningitis virus strain WE glycoproteins: a single amino acid makes the difference.

Cytoplasmic vector systems are generally used for expression of lymphocytic choriomeningitis virus (LCMV) proteins. However, we achieved high levels of cell surface glycoproteins using a standard nuclear expression plasmid. Expression was independent of other LCMV proteins but was blocked by a missense mutation within the original LCMV(WE) glycoprotein cDNA.

Break of T cell ignorance to a viral antigen in the liver induces hepatitis.

To study peripheral tolerance of CD8 T cells to a classically MHC-restricted peptide Ag expressed in hepatocytes, ALB1 transgenic (tg) mice expressing the CTL epitope GP33 of the lymphocytic choriomeningitis virus glycoprotein under control of the mouse albumin promoter were generated. ALB1 mice exclusively expressed the GP33 transgene in the liver and, at a 100- to 1000-fold lower level, in the thymus. TCR-tg mice specific for the GP33 epitope were used to directly follow GP33-specific T cells in vivo. These experiments revealed that 1) thymic expression of the GP33 transgene led to incomplete central deletion of TCR-tg cells; and 2) peripheral TCR-tg cells in ALB1 mice ignored the GP33 transgene expressed in hepatocytes. Ignorance of adoptively transferred TCR-tg cells in ALB1 mice was broken by infection with lymphocytic choriomeningitis virus, leading to induction of hepatitis in ALB1, but not in control, mice. Taken together, we have established a novel model of virus-induced CD8 T cell-mediated autoimmune hepatitis in mice and demonstrate that naive CD8 T cells may ignore Ags expressed in the liver.

CD8+ T Cells Secreting Type 2 Lymphokines Are Defective in Protection against Viral Infection

Effector T cells secreting type 1 and/or type 2 lymphokines (Tc1, Tc0, Tc2) were generated in vitro from CD8+ T cells of mice with a transgenic TCR recognizing lymphocytic choriomeningitis virus (LCMV) glycoprotein to compare their effector function in vitro and in vivo. Tc1, Tc2, and Tc0 showed similar Fas- and perforin-mediated cytotoxicity in vitro. Upon adoptive transfer, Tc2 and Tc0 effectors were less efficient than Tc1 at controlling LCMV or recombinant vaccinia virus expressing the LCMV glycoprotein in vivo. Tc2 and Tc0 had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro. Therefore, the reduced antiviral activity in vivo of Tc2 and Tc0 compared with Tc1 is not due to reduced cytotoxic activity or IFN-γ secretion but may be explained by defective homing to the target organ due to decreased expression and/or lower activity of adhesion molecules.

CpG-DNA activates in vivo T cell epitope presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses.

MHC class I-restricted T cell epitopes lack immunogenicity unless aided by IFA or CFA. In an attempt to circumvent the known inflammatory side effects of IFA and CFA, we analyzed the ability of immunostimulatory CpG-DNA to act as an adjuvant for MHC class I-restricted peptide epitopes. Using the immunodominant CD8 T cell epitopes, SIINFEKL from OVA or KAVYNFATM (gp33) from lymphocytic choriomeningitis virus glycoprotein, we observed that CpG-DNA conveyed immunogenicity to these epitopes leading to primary induction of peptide-specific CTL. Furthermore, vaccination with the lymphocytic choriomeningitis virus gp33 peptide triggered not only CTL but also protective antiviral defense. We also showed that MHC class I-restricted peptides are constitutively presented by immature dendritic cells (DC) within the draining lymph nodes but failed to induce CTL responses. The use of CpG-DNA as an adjuvant, however, initiated peptide presenting immature DC progression to professional licensed APC. Activated DC induced cytolytic CD8 T cells in wild-type mice and also mice deficient of Th cells or CD40 ligand. CpG-DNA thus incites CTL responses toward MHC class I-restricted T cell epitopes in a Th cell-independent manner. Overall, these results provide new insights into CpG-DNA-mediated adjuvanticity and may influence future vaccination strategies for infectious and perhaps tumor diseases.

Antiviral protection after DNA vaccination is short lived and not enhanced by CpG DNA.

In this study, we investigated the potential of a DNA vaccine expressing the minimal cytotoxic T lymphocyte (CTL) epitope gp33 of the lymphocytic choriomeningitis virus glycoprotein to protect against infection of a non-lymphoid organ and compared this to protection against a systemic infection. Furthermore, since immune stimulatory sequences have been shown to augment CTL responses, we examined the capacity of CpG DNA to enhance CTL memory. The data show that DNA vaccination with a gp33-based gene construct induced short-lived gp33-specific CTL which protected against a systemic infection but not against a peripheral infection. Immune stimulatory sequences were incapable of either prolonging CTL memory or promoting protection against infection of a peripheral organ.

Virus-induced non-specific signals cause cell cycle progression of primed CD8(+) T cells but do not induce cell differentiation.

In this report the significance of virus-induced non-specific T cell activation was re-evaluated using transgenic mice in which about half of the CD8(+) T cells expressed a TCR specific for amino acids 33-41 of lymphocytic choriomeningitis virus glycoprotein I. This allowed tracing of cells with known specificity and priming history in an environment also containing a normal heterogeneous CD8(+) population which served as an intrinsic control. Three parameters of T cell activation were analyzed: cell cycle progression, phenotypic conversion and cytolytic activity. Following injection of the IFN inducer poly(I:C), proliferation of memory (CD44(hi)) CD8(+) T cells but no phenotypic or functional activation was observed. Following injection of an unrelated virus [vesicular stomatitis virus (VSV)], naive TCR transgenic cells did not become significantly activated with respect to any of the parameters investigated. In contrast, memory TCR transgenic cells were found to proliferate extensively early after VSV infection (day 0-3), whereas limited proliferation was observed later (day 3-6) when proliferation of non-transgenic CD8(+) T cells is maximal. This aborted response did not result from anergy to TCR stimulation, as memory TCR transgenic cells proliferated vigorously upon stimulation with their nominal peptide. Despite the massive proliferation of memory cells observed early after VSV infection, no phenotypic or functional activation was observed. Together these findings indicate that both non-specific and antigen-specific signals contribute to the initial virus-induced proliferation of CD8(+) T cells, but for further proliferation and differentiation to take place, TCR-ligand interaction is required. The implications for maintenance of T cell memory is discussed.

Identification of a cross-reactive self ligand in virus-mediated autoimmunity.

Molecular mimicry has been considered to be one of the potential mechanisms underlying the induction of autoimmune diseases. Using a TCR-transgenic model specific for lymphocytic choriomeningitis virus (LCMV) we have examined the potential for cross-reactive recognition of tissue-restricted self peptides. Several peptides were identified that were able to cross-react with the TCR-transgenic virus-specific T cells in vitro. One peptide was derived from dopamine beta-mono-oxygenase, an enzyme expressed in the adrenal medulla. Interestingly, after activation of the transgenic T cells with LCMV glycoprotein peptides or viruses, infiltration of the adrenal medulla was detected in conjunction with alterations in dopamine metabolism. However, complete destruction of the adrenal medulla was not observed. This suggests that molecular mimicry may be sufficient for self recognition and infiltration, but other factors clearly contribute to chronic autoimmune disease.

Retroviral vectors pseudotyped with lymphocytic choriomeningitis virus.

Pseudotyping can improve retroviral vector stability and transduction efficiency. Here, we describe a novel pseudotype of murine leukemia virus packaged with lymphocytic choriomeningitis virus (LCMV). This pseudotype was stable during ultracentrifugation and infected several cell lines from different species. Moreover, LCMV glycoproteins were not cell toxic.

Role of antigen-presenting cells in mediating tolerance and autoimmunity.

The mechanisms that determine whether receptor stimulation leads to lymphocyte tolerance versus activation remain poorly understood. We have used rat insulin promoter (RIP)-gp/P14 double-transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) on pancreatic beta-islet cells together with T cells expressing an LCMV-gp-specific T cell receptor to assess the requirements for the induction of autoimmunity. Our studies have shown that administration of the gp peptide gp33 leads to the activation of P14-transgenic T cells, as measured by the upregulation of activation markers and the induction of effector cytotoxic activity. This treatment also leads to expansion and deletion of P14 T cells. Despite the induction of cytotoxic T lymphocyte activity, peptide administration is not sufficient to induce diabetes. However, the administration of gp peptide together with an activating anti-CD40 antibody rapidly induces diabetes. These findings suggest that the induction of tolerance versus autoimmunity is determined by resting versus activated antigen-presenting cells.

CpG-containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune responses with T-cell peptide vaccines.

Synthetic nonmethylated oligonucleotides containing CpG dinucleotides (CpG-ODNs) have been shown to exhibit immunostimulatory activity. CpG-ODNs have the capacity to directly activate B cells, macrophages, and dendritic cells, and we show here that this is reflected by cell surface binding of oligonucleotides to these cell subsets. However, T cells are not directly activated by CpG-ODNs, which correlates with the failure to bind to the T-cell surface. Efficient competition for CpG-induced B-cell activation by non-CpG-containing oligonucleotides suggests that oligonucleotides might bind to an as yet undefined sequence-nonspecific receptor prior to cellular activation. Induction of protective T-cell responses against challenge infection with lymphocytic choriomeningitis virus (LCMV) or with recombinant vaccinia virus expressing the LCMV glycoprotein was achieved by immunizing mice with the immunodominant major histocompatibility complex class I-binding LCMV glycoprotein-derived peptide gp33 together with CpG-ODNs. In these experiments, B cells, potentially serving as CpG-ODN-activated antigen-presenting cells (APCs), were not required for induction of protective immunity since CpG-ODN-gp33-immunized B-cell-deficient mice were equally protected against challenge infection with both viruses. This finding suggested that macrophages and/or dendritic cells were sufficiently activated in vivo by CpG-ODNs to serve as potent APCs for the induction of naive T cells. Furthermore, treatment with CpG-ODN alone induced protection against infection with Listeria monocytogenes via antigen-independent activation of macrophages. These data suggest that CpG activation of macrophages and dendritic cells may provide a critical step in CpG-ODN adjuvant activity.