ENHANCEMENT OF DENDRITIC CELL TOLEROGENICITY BY GENETIC MODIFICATION USING ADENOVIRAL VECTORS ENCODING cDNA FOR TGFβ1

Abstract

77 Transforming growth factor β (TGFβ) inhibits the functional maturation of dendritic cells (DC). It has been shown that administration of mouse bone marrow (BM)-derived DC propagated in GM-CSF and TGFβ1(`TGFβ DC'), that are MHC class II+, but deficient in B7 family and CD40 molecule expression, prolongs donor-specific mouse cardiac allograft survival, associated with inhibition of anti-donor cytotoxic T lymphocyte(CTL) activity. The purpose of this study was to determine whether genetic engineering of these DC to overexpress TGFβ might enhance their tolerogenicity. Methods: genes encoding mouse TGFβ1 were transduced into B10 (H-2b) BM-derived TGFβ DC using replication-deficient adenoviral (Ad) vectors, and LacZ as reporter genes. Expression of co-stimulatory molecules on DC was monitored by flow cytometry.Results: the immune stimulatory effects of DC were determinedin vitro by MLR and CTL generation assays, and in vivo by their influence on B10 cardiac graft survival in C3H (H-2k) recipients. The transduction rate of Ad-LacZ into TGFβ DC was 86%, 83%, 78% and 76% at 100, 50, 20, and 10 MOI (multiplicity of infection), respectively. Ad-LacZ gene transfer did not affect the in vivo DC migration pattern, as detected by both donor MHC class II and X-gal staining of tissues following footpad injection into C3H mice. Transduction with Ad-LacZ at 50 MOI did not significantly affect TGFβ DC B7 or CD40 molecule expression or their allostimulatory activity in MLR, but enhanced the generation of CTL activity (51Cr release: 57.8 ± 7.5% vs 29.1±% in controls at E:T ratio of 100:1). TGFβ DC transduced with Ad-TGFβ1 producing 5 ng/106/d TGFβ1 did not show alterations, compared with Ad-LacZ controls, in MHC class II (40.5% vs 46.6%), CD80 (B7-1; 36.4% vs 39.6%), CD86 (B7-2: 28.1% vs 27.2%), or CD40 (14.5% vs 13.6%) expression, but showed reduced allostimulatory activity in MLR (5132 ± 139 vs 7154 ± 1074 cpm in Ad-LacZ controls) and generation of donor-specific CTL activity (51Cr release 20.4 ± 0.5% vs 57.8± 7.5% in Ad-LacZ controls). The life span of Ad-TGFβ1-transduced DC appeared prolonged in allogeneic recipients, as shown by histoimmunochemical staining of lymphoid tissue following footpad injection. Administration of B10 BM-derived Ad-TGFβ1 transduced TGFβ DC (2× 106) into C3H recipients of B10 cardiac grafts further prolonged graft survival (median graft survival time 21 days vs 16 days in Ad-LacZ-transduced DC injection controls, and 11 days in normal controls p<0.05). In conclusion, mouse BM-derived TGFβ DC can be efficiently transduced to express functional murine TGFβ1 genes using Ad vectors. This transduction does not appear to alter DC expression of MHC class II and co-stimulatory molecules, but increases their tolerogenicity in vivo as shown by further prolongation of cardiac graft survival in allogeneic recipients.