469. Induction of Donor Specific Tolerance Using Gene Therapy and Sublethal Conditioning

Abstract

Top of pageAbstract Introduction: Induction of molecular chimerism following genetic engineering of autologous hematopoietic stem cells can be used to induce transplantation tolerance following reconstitution of myeloablated hosts. In this study we set out to determine the minimum conditioning requirements to induce molecular chimerism and achieve donor-specific tolerance. Methods: B10.AKM (H-2Kk, Ik, Dq) mice received either 3, 6 or 10 Gy of whole body irradiation and were reconstituted the next day with either 4|[times]|106 mock or VSV-Kb (VSV-G enveloped retroviruses carrying the gene encoding H-2Kb (Kb)) transduced syngeneic bone marrow. In addition, recipient mice received 0.5 mg of anti-CD154 mAb (MR1) on the day of reconstitution and a second dose 3 days later. Reconstituted mice were then examined to determine expression of Kb on bone marrow derived cells and whether donor specific tolerance was induced by analyzing Kb-specific T cell responses using cytokine ELISPOT and CTL assays and by grafting with both donor-type and third party skin. Results: sixty percent of mice treated with 6 Gy of irradiation alone became chimeric and expressed Kb on 1.8 |[plusmn]| 1.7% of their peripheral blood leukocytes (PBL). The addition of anti-CD154 mAb allowed stable, multi-lineage chimerism to develop with as little as 3 Gy of irradiation, and increased both the percentage of mice that became chimeric (100%) as well as the percentage of Kb expression in the PBL (5.3 |[plusmn]| 2.6 %). T cells from mice conditioned with 3 Gy, anti-CD154 and VSV-Kb transduced bone marrow were unable to lyse targets expressing Kb, and contained substantially fewer Kb-reactive IL-2 and IFN-|[gamma]| producing CD4 T cells. Furthermore, this sublethal conditioning regimen induced donor specific tolerance in chimeric recipients as evidenced by the permanent survival of MHC class I disparate B10.MBR (H-2Kb, Ik, Dq) skin grafts. All mice promptly rejected third party BALB/c (H-2Kd) skin grafts. Conclusions: Sublethal conditioning and costimulation molecule blockade combined with genetic manipulation of autologous bone marrow is a new and potent way to induce donor specific tolerance. The use of genetically modified autologous bone marrow eliminates the possibility of inducing graft versus host disease which is encountered in allogeneic bone marrow transplants.