SUMMARY CD8+ T cells play a critical role limiting peripheral virus replication, yet how they locate virus-infected cells within tissues is unknown. Here, we have examined the environmental signals that CD8+ T cells use to localize and eliminate virus-infected skin cells. Epicutaneous vaccinia virus (VV) infection, mimicking human smallpox vaccination, greatly increased expression of the CXCR3 chemokine receptor ligands CXCL9 and -10 in VV-infected skin. Despite normal T cell numbers in the skin, Cxcr3−/− mice exhibited dramatically impaired CD8+ T cell-dependent virus clearance. Intravital microscopy revealed that Cxcr3−/− T cells were markedly deficient in locating, engaging, and killing virus-infected cells. Further, transfer of wild-type CD8+ T cells restored viral clearance in Cxcr3−/−animals. These findings demonstrate a function for CXCR3 in enhancing the ability of tissue-localized CD8+ T cells to locate virus-infected cells and thereby exert anti-viral effector functions.

SUMMARY Despite extensive ex vivo investigation, the spatiotemporal organization of immune cells interacting with virus-infected cells in tissues remains uncertain. To address this, we used intravital multiphoton microscopy to visualize immune cell interactions with virus-infected cells following epicutaneous vaccinia virus (VV) infection of mice. VV infects keratinocytes in epidermal foci, and numerous migratory dermal inflammatory monocytes outlying the foci. We observed Ly6G+ innate immune cells infiltrating and controlling foci, while CD8+ T cells remained on the periphery killing infected monocytes. Most antigen-specific CD8+ T cells in the skin did not interact with virus-infected cells. Blocking the generation of reactive nitrogen species relocated CD8+ T cells into foci, modestly reducing viral titers. Depletion of Ly6G+ and CD8+ cells dramatically increased viral titers, consistent with their synergistic but spatially segregated viral clearance activities. These findings highlight previously unappreciated differences in the anatomic specialization of antiviral immune cell subsets.

Purpose We are characterizing the Gottingen minipig as an additional large animal model for advanced drug testing for the Acute Radiation Syndrome (ARS), to enhance discovery and development of novel radiation countermeasures. Among the advantages provided by this model, the similarities to human hematological parameters and dynamics of cell loss/recovery following irradiation provide a convenient means to compare efficacy of drugs known to affect bone marrow cellularity and hematopoiesis. Methods and Materials Male Gottingen minipigs, 4–5 months old and weighing 9–11 kg were used for this study. We tested the standard off-label treatment for ARS, rhG-CSF (Neupogen®, 10 μg/kg/day for 17 days), at the estimated LD70/30 total-body gamma-irradiation (TBI) radiation dose for the hematopoietic syndrome, starting 24 hours after irradiation. Results Results indicate G-CSF enhanced survival, stimulated recovery from neutropenia, and induced mobilization of hematopoietic progenitor cells. In addition, administration of G-CSF resulted in maturation of monocytes/macrophages. Conclusion These results support continuing efforts toward validation of the minipig as a large animal model for advanced testing of radiation countermeasures and characterization of the pathophysiology of ARS, and suggest that the efficacy of G-CSF in improving survival after total body irradiation may involve mechanisms other than increasing numbers of circulating granulocytes.

The detonation of a nuclear weapon or a nuclear accident represent possible events with significant exposure to mixed neutron/γ-radiation fields. Although radiation countermeasures generally have been studied in subjects exposed to pure photons (γ or X rays), the mechanisms of injury of these low linear energy transfer (LET) radiations are different from those of high-LET radiation such as neutrons, and these differences may affect countermeasure efficacy. We compared 30-day survival in mice after varying doses of pure γ and mixed neutron/γ (mixed field) radiation (MF, Dn/Dt = 0.65), and also examined peripheral blood cells, bone marrow cell reconstitution, and cytokine expression. Mixed-field-irradiated mice displayed prolonged defects in T-cell populations compared to mice irradiated with pure γ photons. In mouse survival assays, the growth factor granulocyte colony-stimulating factor (G-CSF) was effective as a (post-irradiation) mitigator against both γ-photons and mixed-field radiation, while the thrombopoietin (TPO) mimetic ALXN4100TPO was effective only against γ irradiation. The results indicate that radiation countermeasures should be tested against radiation qualities appropriate for specific scenarios before inclusion in response plans.