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 cellu lar alloresponse, a rat kidney transplantation model with culturing of graf t 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 In order to initiate acute cellular rejection, immunosuppression was withdrawn after 10 days. In filtrating lymphocytes were analysed using an in vitro culture system, allo wing cells to propagate from the biopsies to culture medium. The propagated cells were counted and analysed for subtype activation markers and donor-s pecificity using flow cytometry and a proliferation assay. Syngeneically tr ansplanted animals and animals given constant immunosuppression upon transp lantation were used as controls. During rejection, significantly more T lym phocytes 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 reject ion group were proliferating and showed donor-specific reactivity whereas m ononuclear spleen cells from animals in the same group did not show this do nor specificity. In conclusion, we have presented a rat kidney allotransplantation model wit h in vitro propagation of graft infiltrating, activated and donor-specific T lymphocytes. This technique offers a possibility to study cellular reacti vity in allotransplantation.