Functional significance of CD4+ and CD8+ T lymphocytes in the immune response to murine gammaherpesvirus 68

Abstract

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of murid rodents and is closely related to Human Herpesvirus 8, Herpesvirus saimiri and Epstein Barr virus. Intranasal infection of inbred mouse strains with MHV-68 results in the lungs of these animals becoming productively infected with virus. In immunocompetent mice, MHV-68 is cleared from the lungs by day 10 after infection. By this time the virus has reached the spleen and has adopted a latent form of infection in B lymphocytes. Depletion of CD8+ T cells from mice, prior to infection, results in uncontrolled MHV-68 replication in the lungs and death of the animal by day 12 post-infection. Such evidence indicates that CD8+ T cells play an important role in the immune response of mice to primary infection with MHV-68.In this study T lymphocyte responses to MHV-68 were examined in vitro and in vivo.In vitro work focused on developing a conventional assay to measure MHV-68-specific CTL activity. It was found that splenocytes from MHV-68-infected mice consistently lysed 'S11' cells, a B cell lymphoma line originally isolated from an MHV-68-infected mouse. All S11 cells are latently infected with MHV-68 and around 5% of these cells support viral replication. Treatment of S11 cells with the anti-viral drug, 4'- S-EtdU, is known to prevent lytic MHV-68 protein expression. 4'-S-EtdU-treated S11 target cells were killed by lymphocytes from infected mice. This indicates that the T lymphocytes responsible for killing S11 cells were specific for a latent antigen of MHV-68. No measurable cytolytic activity against any other target cell line, infected in vitro with MHV-68, was detected.S11 has been shown to divide and expand when implanted subcutaneously into nude (T cell-deficient) mice. S11 does not expand when implanted into normal, immunocompetent mice, implying that T lymphocytes play a key role in inhibiting tumour formation. To investigate this theory, S11 cells were injected subcutaneously into nude mice. This was followed by transfer of re-stimulated lymphocyte populations, enriched for either CD4+ or CD8+ T cells, into the animals. This protocol consistently resulted in regression of S11 tumours. Splenocyte populations depleted of CD8+ T lymphocytes were most effective in preventing tumour formation. This suggests that CD4+ T cells play a major role in preventing B cell lymphoma outgrowth. Immunohistochemical analyses highlighted populations of macrophages and CD4+ T cells in regressing tumours. It may be hypothesised that CD4+ T cells elicit tumour regression via the initiation of a delayed-type hypersensitivity (DTH) response.Further in vivo work involved depleting BALB/c mice T lymphocyte subsets (CD4 and CD8) at a range of times before or after MHV-68 infection. Subsequently, virus titre in the lungs was assessed by plaque assay. Latently-infected B cells in the spleen were quantified by infectious centre assay and visualised by in situ hybridisation. Using these techniques it was demonstrated that both CD4+ and CD8+ T lymphocytes contribute to clearance of MHV-68 from the lungs. CD8+ T cells also appear to limit the extent of B lymphocyte infection in the spleen. However, CD8+ T cells do not appear to be responsible for the decline in latently infected B cells which occurs in MHV-68 infected mice 2 to 3 weeks after infection.In conclusion, MHV-68 infection of inbred laboratory mouse strains has potential to model immunological responses to primary gammaherpesvirus infection in humans. In addition, histopathology induced by the S11 cell line implanted into nude mice could provide insight into gammaherpesvirus-associated B lymphoproliferative disease in immunocompromised individuals.