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Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-a and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-a and TNF in primary human pDCs. Induction of IFN-a in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the Nterminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus. [ABSTRACT FROM AUTHOR]

Background aims Monitoring cellular immune responses is one prerequisite for the rational development of cancer vaccines. Methods We describe an extensive effort to optimize and validate quantitatively an in vitro T-cell culture method by determining the phenotype and function of both CD4+ and CD8+ T cells, including measurement of the phenotype markers CCR7, CD45RA, CD28 and CD27 and the functional markers interferon (IFN)-γ, interleukin (IL)-2, macrophage inflammatory protein (MIP)-1β, tumor necrosis factor (TNF)-αand CD107a. Results Autologous peripheral blood mononuclear cells (PBMC) were potent stimulators that expanded antigen (Ag)-specific CD8+ T cells during short-term culture with the addition of IL-2 and IL-15 cytokines. Polyfunctional Ag-specific CD4+ and CD8+ T cells were detectable using this method. Conclusions Our culture system represents a robust human T-cell culture protocol that permits phenotypic, quantitative and qualitative evaluation of vaccine-induced CD4+ and CD8+ T-cell responses. [ABSTRACT FROM AUTHOR]

Fibroblast activation protein-a (FAP) is a cell surface serine protease expressed at sites of tissue remodeling in embryonic development. FAP is not expressed by mature somatic tissues except activated melanocytes and fibroblasts in wound healing or tumor stroma. FAP expression is specifically silenced in proliferating melanocytic cells during malignant transformation. To study the role of FAP as a tumor suppressor, the gene for mouse fap was cloned and mutated at the catalytic domain (FAP serine mutant, FSM). We found that expression of FAP or FSM at physiologic levels in mouse melanoma cells abrogated tumorigenicity. Remarkably, the mutant form FSM lacking specific serine protease activity was a more potent tumor suppressor. Tumor rejection was not due to adaptive immune responses because RAG1-/- mice challenged with melanoma cells expressing either FAP or FSM were not tumorigenic. In in vitro assays, FAP or FSM expression restored contact inhibition, led to cell cycle arrest at G0/G1 phase, and increased susceptibility to stress-induced apoptosis. Cell death in FAP+ or FSM+ melanoma cells was readily triggered by depletion of survival factors from the media, leading to subsequent activation of caspases via the intrinsic pathway. These results show that expression of FAP is a tumor suppressor that abrogates tumorigenicity through regulation of cell growth and survival.Oncogene (2004) 23, 5435-5446. doi:10.1038/sj.onc.1207730 Published online 10 May 2004 [ABSTRACT FROM AUTHOR]

Cancer cells express self-antigens that are weakly recognized by the immune system. Even though responses against autologous cells are difficult to induce, the immune system is still able to mount a response against cancer. The discovery of the molecular identity of antigens that are recognized by the immune system of melanoma patients has led to the elucidation of tumor immunity at a cellular and molecular level. Multiple pathways in both the priming and effector phases of melanoma rejection have been described. Animal models' active immunotherapies for melanoma show a requirement for the cellular compartment of the immune system in the priming phase, primarily CD4+T cells. More diverse elements are required for the effector phase, including components from the innate immune system (macrophages, complement and/or natural killer cells) and from the adaptive immune system (CD8+T cells and B cells). Minor differences in amino-acid sequences of antigens must determine the particular mechanisms involved in tumor rejection. Since the immune system contains T and B cells that recognize and reject autologous cells, a consequence of tumor immunity is potential autoimmunity. There are distinct pathways for tumor immunity and autoimmunity. The requirements for autoimmunity at the priming phase seem to be CD4+/IFN-? dependent while the effector mechanisms are alternative and redundant. Vitiligo (autoimmune hypopigmentation) can be mediated by T cells, Fc?R+macrophages and/or complement.Oncogene (2003) 22, 3180-3187. doi:10.1038/sj.onc.1206462 [ABSTRACT FROM AUTHOR]

Examines mechanisms underlying immunity to self antigens on cancer cells. Characteristics of tumor antigens; Analysis of tumor immunity in melanoma; Identifications of melanoma differentiation antigens; Description of the antibody-mediated tumor immunity; Clinical strategies for immunotherapy.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances immune responses by inducing the proliferation, maturation, and migration of dendritic cells, and the expansion and differentiation of B and T lymphocytes. Similar biological effects have been observed with the use of GM-CSF DNA in mouse models for therapy of cancer and infectious diseases, and its use is currently being investigated in clinical trials in combination with DNA vaccines. To further understand the adjuvant mechanisms of GM-CSF DNA, we examined early events following its administration. We found measurable levels of GM-CSF protein in the skin and muscle, as well as in serum. Measurements of other cytokine and chemokine levels revealed differential expression patterns over time. The early response was characterized by high levels of inflammatory molecules, including IL-1β, IL-6, TNFα, RANTES, MIP-1α and MCP-1, later followed by expression of precursor Th1 cytokines, IL-12 and IL-18, concomitant with IFNγ production. Local production of GM-CSF protein also resulted in the early recruitment of polymorphonuclear cells and later recruitment of mononuclear cells, including dendritic cells. These results have implications for understanding early events in the immune response to DNA vaccines, and provide a basis for development of new approaches to cancer vaccines, including the use of cytokine genes as adjuvants. [ABSTRACT FROM AUTHOR]

Determines the toxicity and immunogenicity of synthetic globo H-keyhole limpet hemocyanin conjugates, a carbohydrate antigen commonly found on breast cancer cells and its a potential target for vaccine therapy. Administration of the globo-H vaccine to metastatic breast cancer patients; Tabulation of observed toxicitiy of the vaccine.

Examines the adjuvant effects of inoculation of DNA encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) into skin. Delivery of DNA encoding GM-CSF by particle bombardment into skin that resulted in an increase of epidermal dendritic cells at the inoculation site.

Reveals that interdermal injection of granulocyte-macrophage colony-stimulating factor in patients with metastatic melanoma recruits dendritic cells. Staining of pretreatment and post-treatment skin and tumor biopsies; Quantification of positive cells; Method of preparing skin sites for vaccine delivery.