Blockade Of Signal Transduction Prolongs Cardiac Allografts Survival In Mice

PART IProlongation of cardiac allografts survival in mice transfected withAAV-CTLA4IgObjective To evaluate the transfection rate and survival time of mouse cardiac allogrfts transfected with AAV-CTLA4Ig through coronary perfusion. Methods The animals were randomized divided into treated group, control group and untreated group. In treated group B10 (H2~b) heart allografts were transfected with AAV-CTLA4Ig through coronary perfusion before allogenic heart transplantation, the control group and untreated group were perfused with AAV-LacZ or phosphate buffered solution (PBS) respectively. AAV-LacZ was also used as a report gene, the expression of β-gal was determined by X-gal staining. The transfection efficiency and survival time of B10 (H2~b) heart allografts were evaluated after transplantd into C3H (H2~k) recipients. Results Thirty percent to forty percent of myocytes were X-gal staining strongly positive 4-6 weeks after perfusion of AAV-LacZ. Additional used veso-dilator reagents appeared no improvement of above transfer rate. Survival time of B10 (H2~b) heart allografts transfected with AAV-CTLA4-Ig was significantly prolonged in C3H (H2~k) recipients. Conclusion AAV vector can efficiently be transfected into mouse myocardium by coronary perfusion, which significantly inhibits graft rejection.PART IIInfluence of nuclear factor-kappa B antisense oligodeoxynucleotideson survival time of mouse cardiac allograftsObjective NF-kB has been shown to be a crucial transcription factor that regulates costimulatory molecules (CM) expression on the surface of antigen presenting cell (APC). We applied an antisense phosphorothioate oligodeoxynucleotides (ODN) to specifically inhibit mRNA of mouse NF-kB, resulting in a deficiency in CM expression on APC that may prolong organ allograft survival. Methods Anti-NF-kB ODN was delivered systemically by an osmotic pump implanted in the abdominal cavity of recipients following transplantation of vascularized heart allografts. The animals in control groups were given mismatched control ODN or were left untreated. Results Normal C3H (H2k) recipients rejected B10 (H2b) heart allografts at a mediansurvival time (MST) of 15 days. A 14-day administration of 12.5 mg·kg·day anti-NF-kB ODN prolonged the survival of cardiac allografts to an MST of 25 days (P <0.05). In contrast, a mismatched control ODN had no effective result (MST 17 days, P >0.05 compared to untreated controls). To investigate the underlying mechanisms of the immunosuppressive effect of anti-NF-kB ODN administration, graft infiltrating cells, spleen cells, and serum were collected from animals on day 7 post-transplantation. Freshly isolated graft infiltrating cells from anti-NF-kB ODN-treated recipients exhibited significantly decreased donor-specific CTL activity. Generation of CTL activity of spleen T cells from anti-NF-kB ODN-treated recipients was also impaired in comparison with untreated animals. Conclusion These data suggest that treatment with anti-NF-kB ODN markedly inhibits the cellular response of allograft recipients, resulting in significant prolongation of allograft survival.PART IIIDonor dendritic cells treated with CD80, CD86 antisenseoligonucleotide prolonged mouse cardiac allografts survival Objective The expression of costimulatory molecules on antigen-presenting cells (APC) is crucial in determining T-cell immune responses. We examined the effects of transduction with high-affinity antisense oligodeoxyribonucleotides (ODN) designed to target the mRNA of CD80 or CD86 on the phenotype and function of dendriticcells (DC). Methods DC were propagated from C57BL/10 (B10, H2~b) bone marrowcells after the stimulation of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4(IL-4), and then transduced with anti-CD80 or anti-CD86 antisense ODN (mismatched ODN as controls). The effect of antisense ODN on phenotype was examined by flow cytometry. The allostimulatory function of DC was assessed by mixed leukocyte reaction and cytotoxic activity in vitro, and the influence on allograft survival was assessed in vivo. Results ODN were effectively incorporated by DC, which were enhanced by the presence of lipofectamine. Antisense ODN targeting CD80 or CD86 mRNA specifically suppressed the expression of CD80 or CD86 in DC and inhibited their capacity to elicit theproliferative responses, donor-specific cytotoxic T-lymphocyte activity in C3H (H2~k )spleen T cells. It was associated with decreased IL-2, but elevated IL-4 production. Injection of DC transduced with anti-CD80 or CD86 antisense ODN significantly prolonged the survival of heart allografts. Conclusion Transduction with antisense ODN targeting CD80 or CD86mRNA is a feasible and effective method to modify DC that renders them tolerogenic by inducing T-cell hyporesponsiveness.