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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Takayama
T
T
Department of Surgery, Thomas E. Starzl Transplantation Institute and University of Pittsburgh, Pennsylvania 15213, USA.
Nishioka
Y
Y
Lu
L
L
Lotze
M T
MT
Tahara
H
H
Thomson
A W
AW
D000818:Animals; D003713:Dendritic Cells / Q000502:physiology*; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D016753:Interleukin-10 / Q000494:pharmacology*; D015847:Interleukin-4 / Q000494:pharmacology; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D012190:Retroviridae / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000502:physiology; D014764:Viral Proteins / Q000494:pharmacology*
D007166:Immunosuppressive Agents; D014764:Viral Proteins; D016753:Interleukin-10; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor