Granzyme B is important for the ability of NK cells and CD8(+) T cells to kill their targets. However, we showed here that granzyme B-deficient mice clear both allogeneic and syngeneic tumor cell lines more efficiently than do wild-type (WT) mice. To determine whether regulatory T (Treg) cells utilize granzyme B to suppress immune responses against these tumors, we examined the expression and function of granzyme B in Treg cells. Granzyme B was not expressed in naive Treg cells but was highly expressed in 5%-30% of CD4(+)Foxp3(+) Treg cells in the tumor environment. Adoptive transfer of WT Treg cells, but not granzyme B- or perforin-deficient Treg cells, into granzyme B-deficient mice partially restored susceptibility to tumor growth; Treg cells derived from the tumor environment could induce NK and CD8(+) T cell death in a granzyme B- and perforin-dependent fashion. Granzyme B and perforin are therefore relevant for Treg cell-mediated suppression of tumor clearance in vivo.

The ubiquitin-proteasome pathway is the central mediator of regulated proteolysis in cells, and defects in this pathway are associated with cancer and neurodegenerative diseases. To assess 26S proteasome function in living animals, we developed a ubiquitin-luciferase reporter for bioluminescence imaging. The reporter was degraded rapidly under steady-state conditions and stabilized in a dose- and time-dependent manner in response to proteasome inhibitors. Using bioluminescence imaging after one dose of the chemo-therapeutic proteasome inhibitor bortezomib (PS-341), proteasome function in tumor xenografts was blocked within 30 min and returned to nearly baseline by 46 h. After a 2-week regimen of bortezomib, however, imaging of target tumors showed significantly enhanced proteasome inhibition that no longer returned to baseline. The ubiquitin-luciferase reporter enables repetitive tissue-specific analysis of 26S proteasome activity in vivo and should facilitate development and validation of proteasome inhibitors in mouse models, as well as investigations of the ubiquitin-proteasome pathway in disease pathogenesis.

Herpes simplex virus type 1 (HSV-1) can produce disseminated, systemic infection in neonates and patients with AIDS or other immunocompromising diseases, resulting in significant morbidity and mortality in spite of antiviral therapy. Components of host immunity that normally limit HSV-1 to localized epithelial and neuronal infection remain incompletely defined. We used in vivo bioluminescence imaging to determine effects of type I and II interferons (IFNs) on replication and tropism of HSV-1 infection in mice with genetic deficiency of type I, type II, or both type I and II IFN receptors. Following footpad or ocular infection of mice lacking type I IFN receptors, HSV-1 spread to parenchymal organs, including lung, liver, spleen, and regional lymph nodes, but mice survived. Deletion of type I and II IFN receptors produced quantitatively greatest and most widespread dissemination of virus to visceral organs and the nervous system, and these mice invariably died after ocular or footpad infection. Type II receptor knockout and wild-type mice had comparable viral replication and localization, with no systemic spread of HSV-1 or lethality. Therefore, while isolated deficiency of type II IFN receptors did not affect pathogenesis, loss of these receptors in combination with genetic deletion of type I receptors had a profound effect on susceptibility to HSV-1. These data demonstrate different effects of type I and II IFNs in limiting systemic dissemination of HSV-1 and further validate the use of bioluminescence imaging for studies of viral pathogenesis.