Potential of allospecific gene-engineered T cells in transplantation gene therapy: specific T cell activation determines transgene expression in vitro and in vivo

T lymphocytes, regardless of their specificity, are considered key targets for genetic modification in the treatment of inherited or acquired human di seases. In this study, we generated Lewis T cell lines specific for Dark Ag outi rat alloantigens and tested the potential of allospecific T lymphocyte s as carriers of genes encoding therapeutic proteins in transplantation gen e therapy. These allospecific T lymphocytes were successfully, stably trans duced with enhanced green fluorescent protein (EGFP) by an Mo-MuLV-based re trovirus vector. A novel gene delivery protocol was utilized, resulting in nearly 100% EGFP-expressing T cells. This approach enabled tracking of allo specific transduced T cells in vivo and illustrates their transgene product ion by fluorometric determination after ex vivo isolation. Quantitation of EGFP transgene expression was used to determine the influence of T cell rec eptor-specific activation on transgene regulation. A strict positive correl ation between activation state and expression level was detected in vitro a nd in vivo. The activation-induced increase in transgene expression could b e blocked by interference with T cell activation signaling pathways by cycl osporin A, anti-CD4 MAb, or CTLA4-Ig. These data provide strong evidence th at direct or indirect effects caused by activation-induced transcription fa ctors are crucial in transgene upregulation. Allospecific activation in spl eens, lymph nodes, and transplanted grafts can be considered as antigen-spe cific targeting strategy. This activation might be useful in expressing the rapeutic proteins such as TGF-beta or IL-10 specific to these sites. T lymp hocyte priming and activation might be prevented or altered by modification of the local microenvironments, thereby exerting a therapeutic influence o n acute and chronic graft rejection processes.