Influenza A Virus Negative Strand RNA Is Translated for CD8+ T Cell Immunosurveillance.

Probing the limits of CD8+ T cell immunosurveillance, we inserted the SIINFEKL peptide into influenza A virus (IAV)-negative strand gene segments. Although IAV genomic RNA is considered noncoding, there is a conserved, relatively long open reading frame present in segment 8, encoding a potential protein termed NEG8. The biosynthesis of NEG8 from IAV has yet to be demonstrated. Although we failed to detect NEG8 protein expression in IAV-infected mouse cells, cell surface Kb-SIINFEKL complexes are generated when SIINFEKL is genetically appended to the predicted C terminus of NEG8, as shown by activation of OT-I T cells in vitro and in vivo. Moreover, recombinant IAV encoding of SIINFEKL embedded in the negative strand of the neuraminidase-stalk coding sequence also activates OT-I T cells in mice. Together, our findings demonstrate both the translation of sequences on the negative strand of a single-stranded RNA virus and its relevance in antiviral immunosurveillance.

CXCR3 chemokine receptor enables local CD8(+) T cell migration for the destruction of virus-infected cells.

CD8(+) T cells play a critical role in 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 CXCL10 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.

Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation.

Detonation of an improvised nuclear device near a population center would cause significant casualties from the acute radiation syndrome (ARS) due to exposure to mixed neutron/gamma fields (MF). The pathophysiology of ARS involves inflammation, microvascular damage and alterations in immune function. Interactions between endothelial cells (EC) and hematopoietic cells are important not only for regulating immune cell traffic and function, but also for providing the microenvironment that controls survival, differentiation and migration of hematopoietic stem and progenitor cells in blood-forming tissues. Endothelial cells/leukocyte interactions also influence tumor progression and the results of anticancer therapies. In this study, we hypothesized that irradiation of endothelial cells would modulate their effects on hematopoietic cells and vice versa. Human umbilical vein endothelial cells (HUVEC) and immortalized T lymphocytes (Jurkat cells) were cultured individually and in co-culture after exposure to mixed fields. Effects of nonirradiated cells were compared to effects of irradiated cells and alterations in signaling pathways were determined. Mitogen-activated protein kinases (MAPKs) p38 and p44/42 (ERK1/2) in HUVEC exhibited higher levels of phosphorylated protein after exposure to mixed field radiation. IL-6, IL-8, G-CSF, platelet derived growth factor (PDGF) and angiopoietin 2 (ANG2) protein expression were upregulated in HUVEC by exposure to mixed field radiation. PCR arrays using HUVEC mRNA revealed alterations in gene expression after exposure to mixed fields and/or co-culture with Jurkat cells. The presence of HUVEC also influenced the function of Jurkat cells. Nonirradiated Jurkat cells showed an increase in proliferation when co-cultured with nonirradiated HUVEC, and a decrease in proliferation when co-cultured with irradiated HUVEC. Additionally, nonirradiated Jurkat cells incubated in media from irradiated HUVEC exhibited upregulation of activated caspase 3. Irradiation of Jurkat cells caused a G2/M arrest and increased adherence to HUVEC. When co-cultured with HUVEC, irradiated Jurkat cells exhibited G0/G1 arrest and increased apoptosis. The data indicate that gene expression and cell function of endothelial cells and hematopoietic cells are influenced by radiation and by interactions between the two cell types. These phenomena may affect the success of therapies for ARS and cancer.

The Gottingen minipig is a model of the hematopoietic acute radiation syndrome: G-colony stimulating factor stimulates hematopoiesis and enhances survival from lethal total-body γ-irradiation.

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 the discovery and development of novel radiation countermeasures. Among the advantages provided by this model, the similarities to human hematologic parameters and dynamics of cell loss/recovery after irradiation provide a convenient means to compare the efficacy of drugs known to affect bone marrow cellularity and hematopoiesis. METHODS AND MATERIALS: Male Gottingen minipigs, 4 to 5 months old and weighing 9 to 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 γ-irradiation (TBI) radiation dose for the hematopoietic syndrome, starting 24 hours after irradiation. RESULTS: The results indicated that granulocyte colony stimulating factor (G-CSF) enhanced survival, stimulated recovery from neutropenia, and induced mobilization of hematopoietic progenitor cells. In addition, the administration of G-CSF resulted in maturation of monocytes/macrophages. CONCLUSIONS: 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 they suggest that the efficacy of G-CSF in improving survival after total body irradiation may involve mechanisms other than increasing the numbers of circulating granulocytes.

Anatomically restricted synergistic antiviral activities of innate and adaptive immune cells in the skin.

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 that outlie 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.

Efficacy of radiation countermeasures depends on radiation quality.

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.