Unlike allografts or other solid organs or tissues, the availability of allogeneic keratinocytes is not the major problem or current limitation. Allogeneic keratinocytes are readily available from numerous tissue sources; moreover, these cells have tremendous growth potential. Estimates that are that a 1cm2 biopsy can be expanded to 1m2 of cultured epithelium within 30 days. The challenge is to develop strategies that facilitate the long term survival of allografts of cultured keratinocytes without the use of systemic immunosuppressive drugs, which depress the entire immune system, thereby increasing the risk of cancer and infections. Strategies are needed that achieve immunosuppression which is local and confined to the transplanted organ and/or immunosuppression, which induces donor-specific hypo-responsiveness or even donor-specific tolerance. These needs are especially relevant to the skin. Although the surgical procedure of skin transplantation is relatively easy, immunological barriers to the acceptance of skin allografts are formidable, thus making skin allografts perhaps the most difficult organ to transplant. It is our hypothesis that local immunosuppression and/or donor specific tolerance for grafts of cultured keratinocytes can be achieved through the over-expression of one or more immunosuppressive factors (FasL, CTLA-4, IL-10) by the keratinocytes of the graft. To test this hypothesis, we propose to genetically modify keratinocytes with recombinant retroviruses expressing these immunosuppressive factors and to evaluate the survival of these modified allogeneic cells in animal models of allograft rejection. Modified murine keratinocytes will be tested in 2 donor-recipient models with increasing levels of mismatch and severity of allograft response; mismatch of class I and mismatch of class I, and mismatch of class I, II, and minor antigens. Modified human keratinocytes will be tested in a model of human allograft rejection by grafting keratinocytes to immunodeficient mice which have been engrafted with human immune cells and are competent to reject an allograft. In each of these models, we will gain a better understanding of allograft rejection by determining if immunosuppressive factor expression alters the kinetics and cell populations involved in the response. We will also determine if this regimen induces donor-specific tolerance. In addition to providing new information on the pathways of allograft rejection, these studies may lead to the development of a """"""""universal"""""""" epidermal allograft for numerous applications in gene therapy.
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