Antiviral immunity requires early and late mechanisms in which IFN-α and IL-12 play major roles. However, the initial events leading to their production remain largely unclear. Given the crucial role of TLR in innate recognition, we investigated their role in antiviral immunity in vivo. Upon murine CMV (MCMV) infection, both MyD88−/− and TLR9−/− mice were more susceptible and presented increased viral loads compared with C57BL/6, TLR2−/−, TLR3−/−, or TLR4−/− mice. However, in terms of resistance to infection, IFN-α production and in many other parameters of early inflammatory responses, the MyD88−/− mice showed a more defective response than TLR9−/− mice. In the absence of the TLR9/MyD88 signaling pathway, cytokine production was dramatically impaired with a complete abolition of bioactive IL-12p70 serum release contrasting with a high flexibility for IFN-α release, which is initially (36 h) plasmacytoid dendritic cell- and MyD88-dependent, and subsequently (44 h) PDC-, MyD88-independent and, most likely, TLR-independent. NK cells from MCMV-infected MyD88−/− and TLR9−/− mice displayed a severely impaired IFN-γ production, yet retained enhanced cytotoxic activity. In addition, dendritic cell activation and critical inflammatory cell trafficking toward the liver were still effective. In the long term, except for isotype switching to MCMV-specific IgG1, the establishment of Ab responses was not significantly altered. Thus, our results demonstrate a critical requirement of TLR9 in the process of MCMV sensing to assure rapid antiviral responses, coordinated with other TLR-dependent and -independent events that are sufficient to establish adaptive immunity.
Marrow stromal cells (MSCs) can be easily gene-modified and clonally expanded making them ideal candidates for transgenic cell therapy. However, recent reports suggest that MSCs possess immunosuppressive effects, which may limit their clinical applications. We investigated whether interleukin (IL)-2 gene-modified MSCs can be used to mount an effective immune response against the poorly immunogenic B16 melanoma model. We first show that primary MSCs mixed with B16 cells and injected subcutaneously in syngeneic recipients do not affect tumor growth. On the other hand, IL-2-producing MSCs mixed with B16 cells significantly delayed tumor growth in an IL-2 dose-dependent manner. Furthermore, we observed that matrix-embedded IL-2-producing MSCs injected in the vicinity of preestablished B16 tumors led to absence of tumor growth in 90% of treated mice (p < 0.001). We demonstrated that tumor-bearing mice treated with IL-2-producing MSCs developed CD8-mediated tumor-specific immunity and significantly delayed tumor growth of a B16 cell challenge (p < 0.05). In addition, treatment of cd8-/-, cd4-/- and beige mice revealed that CD8+ and natural killer (NK) cells, but not CD4+ cells, were required to achieve antitumor effect. In conclusion, MSCs can be exploited to deliver IL-2 and generate effective immune responses against melanoma in mice with normal immune systems.
Expression vectors encoding herpes simplex virus thymidine kinase (HSVTK) have been extensively used in cell and gene therapy applications either as anticancer "suicide" or as "self-destruct" transgenes in adoptive immunotherapy applications. In both gene therapy applications, reliable detection of HSVTK transgene expression is required in genetically engineered cells. Direct fluorescent labeling of the HSVTK protein may be the remedy. We designed a retrovector encoding a chimeric GFP-HSVTK fusion protein that can serve as a bifunctional suicide and reporter transgene. The fusion gene was incorporated in a VSV G-pseudotyped retrovector (vGFPTKfus) and high-titer stable retroviral producer was generated ( approximately 3 x 10(6) retroparticles/ml). Tumor cell lines transduced at an MOI of 8 for 3 days led to >90% gene transfer efficiency. Southern blot analysis confirmed that unrearranged proviral genomes integrated in chromosomal DNA. Protein extract immunoblot with HSVTK antisera revealed the presence of a 70-kDa protein consistent with the predicted size of an HSVTK-GFP fusion protein. Fluorescence microscopy and FACS analysis revealed that GFPTKfus-mediated fluorescence was nuclear localized and was 30-fold greater than that observed in a bicistronic HSVTK-GFP vector. Growth of cell lines expressing vGFPTKfus was significantly suppressed in the presence of ganciclovir. The DA3 mouse mammary carcinoma cell line was transduced with vGFPTKfus and implanted in syngeneic BALB/c mice. Preestablished tumors completely regressed in seven of nine mice treated with ganciclovir. Normal human peripheral blood T lymphocytes were transduced with vGFPTKfus and nucleus-restricted green fluorescence was observed. Sorting of green fluorescent lymphocytes allowed for selection of engineered cells. In conclusion, we demonstrate the utility of vGFPTKfus as a suicide/reporter transgene in tumor cells in vitro and in vivo. Furthermore, its potential use as an analytical and therapeutic tool targeting human T lymphocytes is shown.
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