Jejunal Administration of Donor Splenocytes in Vascularised Composite Tissue Allotransplant Recipient Rats---The Effect and Role of CD4+CD25+ Regulatory T Cell

Composite tissue allotransplantation (CTA), which refers to the en bloc transplantation of neurovascularised functional anatomical units containing heterogenous tissues, such as a hand or the larynx, holds great potential for reconstructive surgery. However CTA recipients currently require lifelong immunosuppressive therapy and may suffer from the associated wide-ranging and sometimes severe side effects. Even in the presence of immunosuppressive therapy, chronic rejection of allotransplants still occurs. The induction of immunologic unresponsiveness (or tolerance) in recipients to donor tissues is one approach that may overcome these obstacles in transplantation. Immune tolerance to donor tissues can be achieved through inducing a state of chimerism by using bone marrow transplantation (BMT), but it may not be an acceptable tolerance protocol for CTA because of total body irradiation-induced toxic effects. It has recently been increasingly demonstrated that oral administration of antigens or donor splenocytes can modulate the immune response in experimental autoimmune diseases in animals and humans, and prolong the survival of allografts such as heart and kidney. Combination therapy using delayed low-dose cyclosporine A (CsA) or donor hepatocytes appears more capable of prolonging allograft survival. Several investigators suggest that CD4+CD25+ T cells, which release suppressive cytokines such as interleukin (IL)-4, IL-10, and transforming growth factor beta (TGFβ), are involved in the induction of immune tolerance through oral administration of donor splenocytes. The route of administration of donor splenocytes may be critical for its efficacy. Ishido et al demonstrated that intrajejunal administration of donor splenocytes significantly increased heart allograft survival in comparison to oral administration. Although the administration of donor splenocytes appears to prolong solid organ allograft survival, the effect of intrajejunal administration of donor splenocytes on CTA survival and the possible underlying mechanisms have not been elucidated. In order to answer these questions, we will use the well-characterised major histocompatibility complex (MHC)-mismatched rat model in which Brown-Norway (BN) rats are used as donors and Lewis (LEW) rats are used as recipients. Firstly, a gastrojejunostomy will be sited, using biomedical silicone tubing, and subcutaneously tunneled to reside on the dorsum of the neck of recipient Lewis rats. After a one-week recovery period, these rats will receive a groin flap containing skin and subcutaneous tissue from BN rats. Recipient rats will be treated with delayed, low-dose, subtherapeutic CsA (2.5 PROBABLY 4 mg/kg/day) from day 3 after the transplantation. In the first-year of study, we will establish four different experimental groups (Table I) to investigate that whether the intrajejunal administration of a mixture of donor splenocytes and hepatocytes is more effective in prolonging groin flap survival in comparison to monocellular (splenocytes or hepatocytes) administration. If successful, the donor specificity of tolerance will be assessed using third party groin skin flap from an irrelevant inbred strain such as ACI rats, a cross between the August and Copenhagen-Irish strain. Table I. Allotransplant LEW rats with or without intrajejunal feeding of splenocytes and/or hepatocytes In the second-year of study we will divided animals into two groups (Table II) to investigate whether CD4+CD25+ T cells play an important role in prolonging CTA survival when using the intrajejunal route to administer ※donor cells. (The donor cells can be splenocyte alone, hepatocyte alone or mixture of splenocyte and hepatocyte depending on the result of the first year study.) Table II. Allotransplant LEW rats with or without intrajejunal feeding of donor cells If there is a positive correlation between the generation of CD4+CD25+ T cells and the prolongation of CTAs, in the third-year of study we will establish three more groups (Table III) to investigate whether this primed population of CD4+CD25+ T cells can transfer donor specific immune tolerance from intrajejunally treated Lewis rats (※using donor BN splenocytes and/or hepatocytes depending on the result of the first year study) to naive LEW rats. Table III. Allotransplant LEW rats with intravenous administration of CD4+CD25+ T cells isolated from naive or treated LEW rats Our preliminary results using the in vitro mixed lymphocyte reaction assay have shown that the responsiveness of the recipient T cell population to alloantigen is reduced in LEW rats which have been treated by intrajejunal donor BN splenocytes for 6 consecutive days. Since T cell activation to alloantigen is critical to allograft rejection, our preliminary results encourage further studies. The results from the above studies should provide valuable information regarding the effects of intrajejunal administration of donor splenocytes (and/or hepatocytes) in combination with delayed low-dose CsA on the prolongation of CTA survival, as well as the role of CD4+CD25+ T cells in the establishment of tolerance by intrajejunal administration of donor cells.

Project ID:PC9508-0870
External Project ID:NSC95-2314-B182-019-MY3