With the approach, the target antigen is exclusively expressed by transduced fibroblasts; however, direct administration of retroviral vectors can lead to the transduction of cells near the injection site as well as those cells residing in tissues to which vector can be transported. We have recently demonstrated induction of immune responses after direct administration of retroviral vectors intramuscularly in mice, rhesus monkeys, and baboons (4). Direct injection of retroviral vector offers several advantages over the approach, primarily the elimination of the extensive efforts involved in generating autologous fibroblast cell lines for each patient. With the approach, the target antigen is exclusively expressed by transduced fibroblasts; however, direct administration of retroviral vectors can lead to the transduction of cells near the injection site as well as those cells residing in tissues to which vector can be transported. Consequently, it was not clear which cells were transduced by the retroviral vector, and which cells were responsible for presentation of retroviral vector-encoded antigens. In this paper, we have attempted RO4987655 to identify the vector-transduced cells and cells capable of presenting antigen to delineate mechanisms for induction of immune responses after direct injection of retroviral vectors. We have used retroviral vectors encoding HIV env/rev, -galactosidase (-gal), chicken ovalbumin, and firefly luciferase to identify the subsets of cells involved in antigen presentation and induction of immune responses. These four different antigen systems were used to verify that the mechanisms of transduction and induction of immune responses are comparable among retroviral vectors derived from the same packaging cell line and backbone construct regardless of the specific antigen they encode. Furthermore, each of these retroviral vectors offers unique advantages, such as the availability of extremely sensitive detection systems or antibodies suitable for immunohistochemistry. Using these assays, we not only detected cells belonging to each of these subsets, but have also estimated their relative figures. With this paper, we discuss the implications of these data in conjunction with the potential mechanisms of antigen RO4987655 demonstration following direct administration of retroviral vector. MATERIALS AND METHODS Retroviral Vectors. The nonreplicating, amphotropic murine retroviral vectors encoding HIV IIIB env/rev (N2-IIIBenv), bacterial -gal (N2–gal), firefly luciferase (N2-luci), and chicken ovalbumin (N2-ova) were used. Generation of retroviral vector backbone and production of high titer vectors [>1 107 colony-forming devices (cfu)/ml] were explained previously (4, 5). Purified, formulated high titer retroviral vector preparations were used in all experiments. Immunizations, Cytotoxic T Lymphocyte (CTL) Induction, and Cytotoxicity Assays. Six- to eight-week-old woman BALB/c (H-2d) or C57BL/6 (H-2b) mice from HarlanCSpragueCDawley were used in all experiments. On days 1, 4, and 7, mice were injected intramuscularly in both gastrocnemius muscle tissue with 100 l each of retroviral vector. For adoptive transfer experiments, BC-env fibroblasts stably transduced with N2-IIIBenv, or Transduction of the Dendritic Cell Portion and Adoptive Transfer. Naive mice were killed, and the dendritic cell portion was prepared as RO4987655 Cdc14B2 explained above and put into press comprising granulocyte/macrophage colony-stimulating element (500 devices/ml) and interleukin 4 (1000 devices/ml). DA/KT-1, the maker cell collection for N2-IIIBenv, was seeded in transwells (Corning Costar) having a pore size of 0.45 m, which does not allow any leakage of producer cells across the membrane. The dendritic cell portion was added to low adherence cells tradition wells (Corning Costar) and transwells comprising DA/KT-1 were placed above the dendritic cell portion. After 3 days of cocultivation, the recovered cells were washed three times with PBS before injection into naive recipient mice. Spleens from recipient mice were harvested 7 days later on and assayed for cytotoxicity as explained above. RESULTS Infiltrating Leukocytes in the Injection Sites Are Transduced with Retroviral Vector. We were interested in defining the types of cells that are transduced after direct administration RO4987655 of retroviral vectors in muscle mass and examined the injection sites by immunohistochemistry. Mice were immunized in the gastrocnemius muscle tissue having a retroviral vector encoding chicken ovalbumin (N2-ova, a total of 2.4 107 cfu per mouse). We selected ovalbumin as the primary system to analyze the injection sites because of the availability of antibodies suitable for immunohistochemistry. We also utilized HIV env/rev retroviral vector (N2-IIIBenv) and -gal retroviral vector (N2–gal) systems to verify the phenotypes of the infiltrating cells acquired using N2-ova (data not demonstrated). After three injections, mice were killed and injection site muscles were cryosectioned and analyzed by immunohistochemistry using antibodies against ovalbumin and a variety of leukocyte markers (Fig. ?(Fig.1).1). We found a large number of infiltrating cells in the endomesium near the injection site, and some of the infiltrating mononuclear cells indicated ovalbumin proteins (Fig. ?(Fig.11 (data not shown). Open RO4987655 in a separate window Number 1 Immunohistochemistry exam.
