Development of an Enhanced VETO cells for the generation of Alloantigen-specific Tolerance

Abstract

Abstract Development of specific immunotherapies to prevent graft rejection and graft-versus-host disease offers the promise of selectively deleting only allospecific immune responses. Veto activity is the ability of a cell to specifically suppress/delete only T cells directed against antigens of the veto cells themselves, but not against third-party antigens. In the present study, we reasoned that providing TCR-like signaling to allogeneic major histocompatibility complex (MHC) molecules on the surface of CD8+CTLs might result in enhanced veto function. To generate the donor antigen specific veto cells, we have utilized the Chimeric Antigen Receptor (CAR) T cell approach, in which CD8+CTLs express an MHC alloantigen fused to signaling molecule apparatuses. We fused mouse MHC class I alloantigen (H-2Dd) to the hinge and trans-membrane domains of the mouse CD8 chain plus intracellular signaling sequences derived from mouse 4-1BB, and CD3-ζ chain creating various CAR constructs. These CARs were cloned into retroviral vectors. Ex-vivo transduced CD8+T cells (FVB-Dd) were adoptively transferred in the recipient parental mice strain (FVB-H-2q). 4–6 weeks post adoptive transfer, we detected low levels of Dd+ CD8+T cells in lymph node and spleen, ranging from 2–3% of total CD8+T cells. Dd expressing skin grafts from 3604 transgenic mice (H-2Dd transgenic mice on FVB-H-2q background) were transplanted on recipient mice and preliminary data suggests that skin graft survival was extended for 4 months (usual rejection in naïve mice is by 15–18 days).Veto activity of these Dd+CD8+T cells will be examined by in vitro Dd specific CTL assays, proliferation assays.