Ex vivo propagation and characterization of lymphocytes from rejecting rat-kidney allografts

Today, most clinically used methods for analysis of alloreactivity in organ transplantation are based on humoral immunity. In order to study the cellular alloresponse, a rat kidney transplantation model with culturing of graft infiltrating lymphocytes was developed. Kidney transplantations between inbred rat strains were performed with the animals initially immunosuppressed with cyclosporine. In order to initiate acute cellular rejection, immunosuppression was withdrawn after 10 days. Infiltrating lymphocytes were analysed using an in vitro culture system, allowing cells to propagate from the biopsies to culture medium. The propagated cells were counted and analysed for subtype activation markers and donor-specificity using flow cytometry and a proliferation assay. Syngeneically transplanted animals and animals given constant immunosuppression upon transplantation were used as controls. During rejection, significantly more T lymphocytes were propagating from the biopsies compared to controls. A higher percentage of the propagated T lymphocytes in the rejection group expressed activation markers [CD25 and major histocompatibility complex (MHC) class II antigen] compared to spleen- and peripheral blood T lymphocytes from the same individuals. Propagated mononuclear cells from biopsies in the rejection group were proliferating and showed donor-specific reactivity whereas mononuclear spleen cells from animals in the same group did not show this donor specificity. In conclusion, we have presented a rat kidney allotransplantation model with in vitro propagation of graft-infiltrating, activated and donor-specific T lymphocytes. This technique offers a possibility to study cellular reactivity in allotransplantation.