Role of Micro-Chimerism in Inducing Immunological Tolerance by Intraportal Injection of Donor Spleen Cells in Rats

Abstract

Recently, we reported that intraportal (IP) injection of donor spleen cells (SPCs) prevented liver allograft rejection. Moreover, we developed a new method using polymerase chain reaction (PCR)-mediated restriction fragment length polymorphism (RFLP) analysis, and demonstrated micro-chimerism (MC) at the DNA level in the spleen 14 days after IP injection. In the present study, the long-term presence of injected allogeneic SPCs was investigated at the cellular level by immunofluorescence staining as well as the DNA level using RFLP analysis. Male ACI (RT1^a rats were used as the donors and Lewis (RT1¹) rats as the recipients. After DNA preparation from the lymphoid organs, RT1Bβ domain 1 region was amplified by PCR, and RFLP analysis was performed with PVuII restriction enzyme. In the immunofluorescence staining, the monoclonal antibody, MN4-91-6, was used to detect the injected donor ACI SPCs in a frozen specimen. We did not detect MC in Lewis rats intravenously injected with 5 x 10⁷ ACI SPCs on day 14. On the other hand, stable chimerism in the spleen was observed in intraportally injected rats up to 28 days after injection at not only the DNA level but also the cellular level. No chimerism was detected in other organs (including the thymus, lymph nodes, and liver). In conclusion, the long-term presence of injected allogeneic SPCs in the spleen was demonstrated after IP injection but not after IV injection, and this phenomenon may be one of the mechanisms involved in portal venous immunosuppression.