TRANSDUCTION OF DENDRITIC CELLS WITH ADENOVIRAL VECTORS ENCODING CTLA4-Ig MARKEDLY REDUCES THEIR ALLOSTIMULATORY ACTIVITY

Abstract

109 Background: Dendritic cells (DC) lacking sufficient expression of B7-1(CD80) and B7-2(CD86) can induce alloantigen-specific T cell anergyin vitro and prolong cardiac allograft survival. The failure of these cells to induce allograft tolerance may due to the “late” upregulation of costimulatory molecules on the DC following their interaction with host T cells. Aim: In this study, we examined whether genetic modification of donor DC with an adenoviral vector encoding murine CTLA4-Ig(Ad-CTLA4-Ig) could potentiate their tolerogenity. Methods: DC were propagated from bone marrow (BM) of B10 (H-2b) mice with GM-CSF+IL-4(“IL-4 DC”) or GM-CSF+TGFβ1 (“TGFβ DC”). Compared with IL-4 DC, TGFβ DC were 1) DEC 205+, MHC class II+, CD40low and B71/2low, 2) poor stimulators of allogeneic T cell proliferation and CTL production, 3) capable of phenotypic and functional maturation following addition of IL-4, or withdrawal of TGFβ1. DC were infected with Ad LacZ at different moi, and intracellular LacZ activity was determined by X Gal staining 24 hr after infection. The capacity of the Adtransduced DC to induce T cell proliferation was analyzed by three-day primary MLR using C3H T cells as responders. Cytotoxic T cell (CTL) generation was determined by 4 hr 51Cr release assay, using EL4(H-2b) cells as targels. In vivo migration and survival of Ad-LacZ transduced B10 DC were examined by immunohistochemical staining with α IAb mAb. Results. Both IL-4 DC and TGFβ DC were efficiently transduced by Ad LacZ. More than 85% of the DC were X Gal positive at moi 100-50, 60-70% at moi 20, 40-50% at moi 10. Ad-LacZ transduction enhanced costimulatory molecule expression on TGFβ DC, but not on IL-4 DC, in dose dependent manner. The allostimulatory function of the DC in MLR and induction of CTL responses was significantly enhanced by Ad LacZ transduction at 100 moi. However, both IL-4 DC and TGFβ DC transduced with Ad-CTLA4-Ig at moi 50 exhibited 60-70% reduction in their capacity to induce allogeneic T cell proliferation and CTL responses. The supernatant from Ad-CTLA4-Ig transduced IL-4 DC dramatically inhibited C3H T cell proliferation stimulated by unmodified B10 IL-4 DC. The poor allostimulatory function of TGFβ DC in MLR was further inhibited by Ad-CTLA4-Ig transduction. The migration pattern of B10 Ad-LacZ transduced DC and non-transduced DC, determined by donor MHC class II (IAb) staining, was very similar after their local injection into C3H recipients. Conclusions: BM-derived DC can be efficiently transduced and functionally modified with an adenoviral vector encoding the CTLA4-Ig gene. Ad-CTLA4-Ig transduction shows potential for maximizing the tolerogenic activity of DC.