Retroviral delivery of viral interleukin-10 into myeloid dendritic cells markedly inhibits their allostimulatory activity and promotes the induction of T-cell hyporesponsiveness.

Abstract

Dendritic cells (DC) play critical roles in the initiation and modulation of immune responses and may determine the balance between tolerance and immunity. Viral interleukin-10 (vIL-10), encoded by the Epstein-Barr virus, is highly homologous to the "immunosuppressive" cytokine, mammalian IL-10. It impairs antigen-presenting cell function but lacks certain immunostimulatory properties of mammalian IL-10. We accomplished the following: (1) evaluated the effects of vIL-10 protein on DC phenotype and function, (2) transduced mouse bone marrow-derived DC to express vIL-10, and (3) assessed the impact of transgene expression on DC allostimulatory activity. DC progenitors propagated from bone marrow of B10 (H2b) mice in granulocyte-macrophage colony-stimulating factor plus IL-4 were repeatedly transduced by centrifugation, using retroviral supernatant obtained from the BOSC 23 ecotropic packaging cell line. To evaluate transduction efficiency, DC were transduced with the retroviral vector MFG-enhanced green fluorescence protein as a marker gene. Transgene and key cell surface molecule expression were examined by flow cytometry. The level of vIL-10 gene product in the culture supernatant was quantitated by ELISA. DC function was assessed by evaluation of the ability of DC to induce allogeneic (C3H;H2k) T-cell proliferation and cytotoxic T lymphocytes in primary mixed leukocyte reactions. Secondary mixed leukocyte reactions were used to test for T-cell hyporesponsiveness. The early addition of vIL-10 protein to cultures inhibited DC maturation and function. vIL-10 gene transfer was achieved with an approximate transduction efficiency of 35 to 40%. Transduced DC expressed vIL-10 at a level of 40 ng/10(6) cells/48 hr. In comparison with controls, vIL-10-transduced cells showed decreased surface expression of major histocompatibility complex class II and costimulatory molecules, reduced ability to stimulate T-cell proliferation and cytotoxic T lymphocyte generation, and potential to induce alloantigen-specific hyporesponsiveness. DC can be effectively transduced to express vIL-10 and limit their ability to stimulate in vitro. These genetically engineered antigen-presenting cells may have therapeutic potential to inhibit undesired immune responses to allo- or autoantigens.