Ole Marker

Role of CD40 ligand and CD28 in induction and maintenance of antiviral CD8+ effector T cell responses.

The primary aim of this report was to evaluate the immune responses of CD40 ligand-deficient (CD40L−/−) mice infected with two viruses known to differ markedly in their capacity to replicate in the host. Lymphocytic choriomeningitis virus (LCMV) is a natural mouse pathogen that replicates widely and extensively, whereas vesicular stomatitis virus (VSV) spreads poorly. We found that the primary response of CD40L−/− mice toward VSV is significantly impaired; proliferation of both CD4+ and CD8+ cells is reduced 2- to 3-fold, few CD8+ cells acquire an activated phenotype, and little functional activity is induced. Very similar results were obtained in VSV-infected, CD28-deficient mice. In contrast, neither CD40L nor CD28 was required for induction of a primary CD8+ response toward LCMV. Surprisingly, lack of CD4+ T cells had no impact on the primary immune response toward any of the viruses, even though the CD40 ligand dependence demonstrated for VSV would be expected to be associated with CD4 dependence. Upon coinfection of VSV-infected mice with LCMV, the requirement for CD40 ligand (but not CD28) could be partially bypassed, as evidenced by a 3-fold increase in the frequency of VSV-specific CD8+ T cells on day 6 postinfection. Finally, despite the fact that the primary LCMV-specific CD8+ response is virtually unimpaired in CD40L−/− mice, their capacity to maintain CD8+ effector activity and to permanently control the infection is significantly reduced. Thus, our results demonstrate that the importance of CD40/CD40L interaction for activation of CD8+ T cells varies between viruses and over time.

CCR2+ and CCR5+ CD8+ T cells increase during viral infection and migrate to sites of infection

Chemokines and their receptors play a critical role in the selective recruitment of various leukocyte subsets. In this study, we correlated the expression of multiple chemokine and CC chemokine receptor (CCR) genes during the course of intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV), which are prototypic of a noncytopathic and a cytopathic virus, respectively. Infection of mice with either virus resulted in rapid activation and overlapping cerebral expression of a number of chemokine genes. Infection with VSV i.c. causes a rapidly lethal, T cell‐independent encephalitis, and infection resulted in a dramatic early up‐regulation of chemokine gene expression. Similar marked up‐regulation of chemokine expression was not seen until late after LCMV infection and required the presence of activated T cells. Cerebral CCR gene expression was dominated by CCR1, CCR2 and CCR5. However, despite a stronger initial chemokine signal in VSV‐infected mice, only LCMV‐induced T cell‐dependent inflammation was found to be associated with substantially increased expression of CCR genes. Virus‐activated CD8+ T cells were found to express CCR2 and CCR5, whereas activated monocytes/macrophages expressed CCR1 in addition to CCR2 and CCR5. Together, these CCR profiles readily account for the CCR profile prominent during CD8+‐dependent CNS inflammation.

THE ROLE OF CD4+ T CELLS IN CELL-MEDIATED IMMUNITY TO LCMV : STUDIES IN MHC CLASS I AND CLASS II DEFICIENT MICE

Parameters of the virus‐specific T‐cell response were analysed in order to dissect the contribution of CD4+ and CDS+ T cells to cell‐mediated immunity to lymphocytic choriomeningitis virus. In MHC class II deficient mice, initial T‐cell responsiveness was not impaired, but virus clearance was delayed, and virus‐specific Td activity declined more rapidly. Furthermore, class I restricted Tc memory appeared to be impaired in these mice. To directly evaluate the role of CD4+ cells in virus clearance and T‐cell mediated inflammation, MHC class I deficient mice were also studied. No virus‐specific delayed‐type hypersensitivity reaction was detected following infection of the footpad, and only a few mice died from intracerebral challenge. However analysis of markers of T‐cell activation as well as direct evaluation of CSF infiammation unveiled a low degree of T‐cell activation and a chronic cellular exudate. This low‐grade response was associated with some degree of virus control as organ titres were lower in these animals than in matched T‐cell deficient nu/nu mice or class I deficient mice treated with anti‐CD4 monoclonal antibody. This confirms that CD4+ cells are not needed to induce a virus‐specific CD8+ T‐cell response, but our findings strongly suggest that CD4+ T cells are critical for maintaining full antiviral immunity. Furthermore, CD4+ T cells per se have a low potential for mediating virus‐specific infiammation that is associated with a low degree of virus control.

Lymphocytic Choriomeningitis Virus Infection is Associated with Long-Standing Perturbation of LFA-1 Expression on CD8+ T Cells

Flow cytometric analysis of splenocytes from mice infected with lymphocytic Choriomeningitis virus revealed marked and long‐standing up‐regulation of LFA‐1 expression on CD8+, but not on CD4+ T cells. Appearance of CD8+ T cells with a changed expression of adhesion molecules reflected polyclonal activation and expansion which was demonstrated not to depend on CD4+ T cells or their products. Cell sorting experiments defined virus‐specific CTL to be included in this population (LFA‐1hiMEL‐14lo), but since about 80% of splenic CD8+ T cells have a changed phenotype, extensive bystander activation must take place; this is indicated also by the finding that CD8+LFA‐lhi cells transiently express several markers of cellular activation, e. g. transferrin receptor, IL‐2Rα and β. Analysis of cells from the cerebrospinal fluid of mice infected intracerebrally showed that virtually all T cells present belonged to the CD8LFA‐lhi subset and, correspondingly, the ligand ICAM‐1 was found to be up‐regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV‐primed donor splenocytes with anti‐LFA‐1 markedly inhibited the transfer of virus‐specific delayed‐type hypersensitivity to naive recipients. Together, these findings indicate that up‐regulation of LFA‐1 expression is a critical factor involved in directing activated CD8+ T cells to sites of viral infection.

Sensitization to Lipopolysaccharide in Mice with Asymptomatic Viral Infection: Role of T Cell-Dependent Production of Interferon-γ

The interplay between viral infection and lipopolysaccharide (LPS) was studied. Infection with a noncytopathogenic virus, lymphocytic choriomeningitis virus (LCMV), was found to sensitize mice to low doses of LPS. In vivo, this hypersensitivity correlated with hyperproduction of tumor necrosis factor-alpha (TNF-alpha), and in vitro, LPS-stimulated splenic adherent cells produced increased amounts of TNF-alpha. Hyperproduction of TNF-alpha was temporally correlated with virus-induced production of interferon-gamma (IFN-gamma); only marginally increased IFN-gamma and TNF-alpha production was observed in LCMV-infected, T cell-deficient mice and in mice infected with vesicular stomatitis virus, a virus that induces much less T cell activation than does LCMV. Finally, LCMV infection was much less efficient in priming IFN-gamma knockout mice for hyperproduction of TNF-alpha. These findings indicate that clinically silent viral infections may induce hypersensitivity to LPS through T cell activation and subsequent production of IFN-gamma; this sensitizes monocytes/macrophages for hyperproduction of TNF-alpha.

Virus-activated T cells regulate expression of adhesion molecules on endothelial cells in sites of infection

To study the role of cell adhesion molecules in the fatal CD8+ T-cell mediated meningitis which is induced by intracerebral infection with lymphocytic choriomeningitis virus, the expression of relevant molecules on inflammatory cells and local endothelium was analyzed immunohistochemically. Most inflammatory cells were strongly positive for LFA-1, VLA-4, Pgp-1 and ICAM-1. Expression of ICAM-1 and VCAM-1 was upregulated on the endothelial cells in immunocompetent mice, but not in T-cell deficient nude mice. Analysis of mice deficient in either CD4+ or CD8+ T cells, revealed that not only was the inflammatory reaction dependent on the presence of CD8+ cells, but these cells also appeared to be required for maximal upregulation of ICAM-1 and VCAM-1 on the endothelial cells. These results indicate that virus-specific CD8+ T cells are crucially involved in regulating the inflammatory reaction through effects on endothelial expression of adhesion molecules.

Breakdown of blood-brain barrier function in the murine lymphocytic choriomeningitis virus infection mediated by virus-specific CD8+ T cells.

Intracerebral inoculation of lymphocytic choriomeningitis virus (LCMV) generally results in a fatal T cell-mediated meningitis. In a previous study we have demonstrated a compromised blood-brain barrier (BBB) under such conditions. Using semi-quantitative radiography and the low molecular tracer 2-amino-[1-14C]isobutyric acid we now demonstrate an uncompromised BBB in i.c. infected T cell-deficient nu/nu mice, but serious dysfunction in heterozygous littermates. Transfer experiments were used to characterize and compare the cell subset(s) involved in inducing BBB dysfunction and fatal disease. It was demonstrated that Thy-1+, CD8+ class I-restricted T cells were mandatory for the increase in BBB permeability as well as for mortality. In addition, depletion of class II-restricted CD4+ cells significantly weakened both effects of cell transfer. These results support the idea of a causal relationship between virus-specific T cell activity, BBB dysfunction, and fatal disease. Furthermore, the data indicate that T helper cells augment the response of LCMV-specific CD8+ effector cells.

Role of interferon-γ in the pathogenesis of LCMV-induced meningitis: unimpaired leucocyte recruitment, but deficient macrophage activation in interferon-γ knock-out mice

Abstract Generally, interferon- γ (IFN- γ ) is considered a critical regulator of T cell mediated inflammation. For this reason, we investigated the pathogenesis of lymphocytic choriomeningits in mice with a targeted defect of the gene encoding this cytokine. Our results revealed that IFN- γ is redundant in the afferent phase of the antiviral T cell response as well as a local mediator of this T cell mediated inflammatory disease. However, IFN- γ may play an indirect role as it is involved in reducing extraneural infection that may compete with CNS for available effector cells. Analysis of the inflammatory exudate disclosed that leucocyte recruitment was unimpaired in the absence of IFN- γ as was the upregulation of ICAM-1 and VCAM-1 on endothelium at the inflammatory site. However, local macrophage activation (production of tumor necrosis- α and NO) was significantly impaired. Notably, a viral peptide could also elicit a T cell mediated inflammatory response in virus-primed IFN- γ knock-out mice, indicating that redundancy of this cytokine as a proinflammatory mediator is not restricted to inflammatory reactions triggered by an active infection. Thus, T cell mediated inflammation may be induced in the absence of IFN- γ and local macrophage activation.

High-dose survival in the lymphocytic choriomeningitis virus infection is accompanied by suppressed DTH but unaffected T-cell cytotoxicity

Provided that intracerebral inoculation is applied, an increase in the virus dose from 102to 104 LD50 of lymphocytic choriomeningitis virus (LCMV) leads to strikingly reduced mortality. To analyse the background for this autointerferencc, we measured several virologic and immunologic variables in mice infected with these doses of virus. In the high‐dose mice we found generally higher organ virus titres and serum interferon titres than in the low‐dose mice. Since we could demonstrate that virus‐specific T‐cell cytotoxicity in spleen, peripheral blood, and meningeal exudate was similar after intracerebral infection with large and small virus doses, and since the LCMV infection in the brain qualitatively and quantitatively was independent of the size of virus inoculum, the explanation for the survival of the high‐dose animals is obviously not lack of possibilities for interaction between cytotoxic T cells and infected sensitive targets in the central nervous system. On the other hand, high doses of virus caused a clear suppression of the LCMV‐specific delayed‐type hypersensitivity(DTH). In addition, when splenocytes from high‐dose animals were transferred either intravenously or locally into the footpad of newly virus‐challenged mice. DTH was markedly suppressed as compared with the response after transfer of spleen cells from low‐dose mice. We therefore conclude that autointerferencc in the LCMV infection is due to a selective suppression of Td function. Large amounts of persistent virus late after infection with high doses of virus suggest a central role for Td function also in virus clearance. Finally, our results indicate the existence of two subsets of K, D region‐restricted T cells, one mediating cytotoxicity and the other mediating DTH. This possibility is discussed.

Inducible nitric-oxide synthase plays a minimal role in lymphocytic choriomeningitis virus-induced, T cell-mediated protective immunity and immunopathology

By using mice with a targetted disruption in the gene encoding inducible nitric-oxide synthase (iNOS), we have studied the role of nitric oxide (NO) in lymphocytic choriomeningitis virus (LCMV)-induced, T cell-mediated protective immunity and immunopathology. The afferent phase of the T cell-mediated immune response was found to be unaltered in iNOS-deficient mice compared with wild-type C57BL/6 mice, and LCMV- induced general immunosuppression was equally pronounced in both strains. In vivo analysis revealed identical kinetics of virus clearance, as well as unaltered clinical severity of systemic LCMV infection in both strains. Concerning the outcome of intracerebral infection, no significant differences were found between iNOS-deficient and wild-type mice in the number or composition of mononuclear cells found in the cerebrospinal fluid on day 6 post-infection. Likewise, NO did not influence the up-regulation of proinflammatory cytokine/chemokine genes significantly, nor did it influence the development of fatal meningitis. However, a reduced virus-specific delayed-type hypersensitivity reaction was observed in iNOS-deficient mice compared with both IFN-gamma-deficient and wild-type mice. This might suggest a role of NO in regulating vascular reactivity in the context of T cell-mediated inflammation. In conclusion, these findings indicate a minimal role for iNOS/NO in the host response to LCMV. Except for a reduced local oedema in the knockout mice, iNOS/NO seems to be redundant in controlling both the afferent and efferent phases of the T cell-mediated immune response to LCMV infection.