Patrick M. Stuart

Inducible Nonlymphoid Expression of Fas Ligand Is Responsible for Superantigen-Induced Peripheral Deletion of T Cells

Fas (CD95) and Fas ligand (FasL) play major roles in staphylococcal enterotoxin B (SEB)-induced peripheral deletion of Vbeta8+ T cells. We found that peripheral deletion was defective in radiation chimeras with non-functional tissue FasL, regardless of the FasL status of the bone marrow-derived cells. SEB induced a dramatic upregulation of FasL expression and function in nonlymphoid cells of liver and small intestine. This effect was resistant to inhibition by cyclosporin A, which also failed to inhibit peripheral deletion. In SCID animals nonlymphoid tissues did not express FasL in response to SEB unless transplanted lymphocytes were present. Thus, some immune responses induce FasL in nonlymphoid tissues, which in turn kills activated lymphocytes, leading to peripheral T cell deletion.

Apoptosis, tolerance, and regulatory T cells--old wine, new wineskins.

Summary:  Antigen‐specific unresponsiveness (or tolerance) has always been an important area of research. Interest in the fate of apoptotic cells and their ability to tolerize has revived interest in some of the older models involving hapten‐modified self. Recently, we have examined the mechanisms by which intravenous injection of trinitrophenol‐coupled spleen cells leads to systemic tolerance. These studies have revealed an important role for Fas/Fas ligand interactions, caspases, CD40/CD40L, and regulatory CD4+ and CD8+ T cells. Extension of these studies to peripheral deletion of T‐cell antigen receptor transgenic T cells has shown that deletion and active regulation of immune responses may be important mechanisms for the control of potentially damaging autoimmune responses.

Peripheral Deletion of Antigen-Specific T Cells Leads to Long-Term Tolerance Mediated by CD8+ Cytotoxic Cells

Peripheral deletion is one mechanism by which potentially self-reactive clones are removed whether they escape thymic deletion. We have examined the consequences of deleting Ag-specific T cells by i.v. injection of soluble Ag. Deletion of DO11.10 T cells by peptide was mediated predominately via a Fas/FasL mechanism. Animals that underwent deletion were tolerant to subsequent immunization with Ag, even when tolerant mice were given fresh Ag-specific DO11.10 T cells before immunization. Tolerance was mediated by CD8+ T cells that killed the DO11.10-transgenic T cells in vivo. These data demonstrate that the programmed cell death of large numbers of T cells leads to peripheral tolerance mediated by CD8+ CTLs.

Therapeutic vaccination with vhs− herpes simplex virus reduces the severity of recurrent herpetic stromal keratitis in mice

Virion host shutoff (vhs)-deficient herpes simplex virus (HSV) was tested as a therapeutic vaccine in a mouse model of UV light-induced recurrent herpetic stromal keratitis. Four weeks after primary corneal infection, mice were vaccinated intraperitoneally with vhs(-) vaccine or control. Four weeks after vaccination, the eyes of latently infected mice were UV-B irradiated to induce recurrent virus shedding and disease. Post-irradiation corneal opacity in latently infected, vhs(-)-vaccinated mice was significantly reduced compared to control-vaccinated mice (P=0.007 to 0.035). The incidence and duration of recurrent virus shedding were the same in both groups. Antibody titres were increased (P=0.05) and delayed type hypersensitive responses were unaffected by vhs(-) vaccination. Combined with studies using different vaccination timing and vhs(-) genotypes, these data suggest that deletion of vhs is a useful strategy in the development of a therapeutic HSV vaccine, and that temporal and genetic factors influence vaccination outcome.

Mice with Mutations in Fas and Fas Ligand Demonstrate Increased Herpetic Stromal Keratitis following Corneal Infection with HSV-1

HSV-1 infection of the cornea leads to a potentially blinding immunoinflammatory lesion of the cornea, termed herpetic stromal keratitis. It has also been shown that one of the factors limiting inflammation of the cornea is the presence of Fas ligand (FasL) on corneal epithelium and endothelium. In this study, the role played by FasL expression in the cornea following acute infection with HSV-1 was determined. Both BALB/c and C57BL/6 (B6) mice with HSV-1 infection were compared with their lpr and gld counterparts. Results indicated that mice bearing mutations in the Fas Ag (lpr) displayed the most severe disease, whereas the FasL-defective gld mouse displayed an intermediate phenotype. It was further demonstrated that increased disease was due to lack of Fas expression on bone marrow-derived cells. Of interest, although virus persisted slightly longer in the corneas of mice bearing lpr and gld mutations, the persistence of infectious virus in the trigeminal ganglia was the same for all strains infected. Further, B6 mice bearing lpr and gld mutations were also more resistant to virus-induced mortality than were wild-type B6 mice. Thus, neither disease nor mortality correlated with viral replication in these mice. Collectively, the findings indicate that the presence of FasL on the cornea restricts the entry of Fas+ bone marrow-derived inflammatory cells and thus reduces the severity of HSK.

Delivery of Interferon-γ by an adenovirus vector blocks herpes simplex virus Type 1 reactivation in vitro and in vivo independent of RNase L and double-stranded RNA-dependent protein kinase pathways

HSV-1 is a significant human pathogen that can result in the loss of sight as a result of episodic reactivation of latent virus from sensory ganglion neurons. In this study the potential efficacy of anti-viral cytokine expression in preventing latent virus reactivation was investigated. Both type I (IFN-beta) and type II (IFN-gamma) IFN transgene expression following transduction of trigeminal ganglion explant cultures significantly reduced the incident of HSV-1 reactivation that in the case of IFN-beta was dependent on the presence of double stranded RNA-dependent protein kinase and RNase L. In vivo, expression of the IFN-gamma but not IFN-beta transgene significantly delayed and reduced the frequency of reactivation of latent mice exposed to UV light without discernable inflammation. This result is the first report that demonstrates the ability to block reactivation using an ectopic cytokine expression system and warrants further exploration as a means to prevent HSV-1 reactivation.

Interferon gamma is not required for recurrent herpetic stromal keratitis

The role that interferon-gamma (IFNgamma) plays during herpetic stromal keratitis (HSK) has not been definitively determined. In primary HSK most reports suggest that IFNgamma may help control viral replication and contribute to corneal pathology. However, its role in recurrent HSK has not been directly addressed. The present study addresses its role in recurrent HSK by comparing HSK in latently infected normal and IFNgamma gene knockout (GKO) on the C57BL/6 background. We initially evaluated HSK following primary infection and observed that GKO mice had higher tear film virus titers, but virtually identical ocular disease as normal mice. In contrast, following reactivation of latent virus, GKO mice had a greater incidence and severity of opacity, neovascularization, and blepharitis. Interestingly, the incidence of reactivation after UV-B exposure was equivalent in GKO and normal mice, but virus shedding was increased in the GKO groups. We also observed diminished delayed-type hypersensitivity responses in GKO mice, as expected. These data indicate that IFNgamma is important for the control of virus replication in both primary and recurrent ocular HSV infection in C57BL/6 mice. The enhanced recurrent disease seen in GKO mice may be the result of increased viral titers and persistence in these mice which act to prolong the stimulation of an inflammatory response.

FasL, leukocytes and vascular modeling

The recent paper by Ishida et al.1 examines the role of leukocytes, specifically T lymphocytes, in vascular remodeling and vaso-obliteration in the rat eye. The authors conclude that T lymphocytes bind to the vasculature and model it by using Fas ligand (FasL) to prune the developing vessels. The authors also conclude from their examination of oxygen-induced vaso-obliteration that FasL on T lymphocytes induces apoptosis of hyperoxygenated endothelial cells. For these observations to have biological relevance, they should transcend species specificity. Because similar models of retinal angiogenesis exist in the mouse, one should be able to use the mouse to test predictions arising from these important observations. First, if Fas-FasL interactions are involved in retinal vessel development, Fas-deficient (lpr) and FasL-deficient (gld) mice should show vascular defects. Second, vessels in mice without these proteins should not undergo vaso-obliteration to the same degree as do wild-type animals. Third, rodents without T lymphocytes (Rag-/- or severe combined immunodeficient mice and T-cell-deficient rats) and/or cytotoxic lymphocytes (MHC class I− or CD8-deficient) should show aberrant retinal vessel development. Fourth, if the retinal vessels in these mice do not develop normally, this should severely impair normal development of the retina. Fifth, examination of these strains for vaso-obliteration should reveal that this process is severely impaired compared with wild-type animals.

Human cytomegalovirus infection in a retinoblastoma cell line in vitro

Abstract · Background: The focus of these studies was to determine whether the Y79 human retinoblastoma cell line could function as a good in vitro model system for studying human cytomegalovirus (HCMV) infection. · Methods: Y79 cells were exposed to an HCMV mutant carrying a LacZ gene, and the resulting β-galactosidase expression in infected cells was assessed by flow cytometry. The extent to which the three classes of viral gene products – immediate early, early, and late proteins – were expressed was analyzed by immunohistochemical staining and Western blotting. Infected Y79 cells were also co-cultivated on human foreskin fibroblast (SF cell) cultures to recover virus. · Results: Infection of Y79 cells with the virus resulted in β-galactosidase expression as detected by flow-cytometric analysis. Immunohistochemical staining revealed that a portion of Y79 cells expressed antigens reactive to monoclonal antibodies against immediate early, early, and late HCMV proteins. The 43-kDa early gene product was also detected by Western blotting. Infected Y79 cells co-cultivated on SF cell cultures yielded infectious foci, which turned blue following X-gal staining, demonstrating productive HCMV infection in the Y79 cells. · Conclusion: These results demonstrate that while HCMV can productively infect Y79 cultures, it does so in a highly inefficient manner, leading these authors to conclude that this cell line does not provide a particularly good model system to study HCMV infection.