Quantification of lymphocytes propagating from rat-kidney allografts — a tool to monitor anti-rejection treatment

Abstract

Background: Morphological evaluation of kidney biopsies applying the Banff Classification is closely correlated to clinical parameters of rejection. However, this classification is insufficient for monitoring the effect of instituted anti-rejection therapy. The objective of the present study was to develop a diagnostic tool enabling rapid assessment of the effects of an attempted anti-rejection treatment. Materials and methods: A kidney allotransplantation model in the rat was applied. All animals were initially maintained on cyclosporine A (CsA). In order to induce an episode of acute cellular rejection, CsA was withdrawn for 5 days. Thereafter, the immunosuppressive treatment was restarted. Core biopsies were taken before, and at various times after, the introduction of anti-rejection therapy. Infiltrating cells were isolated using an in vitro culture system, allowing cells to propagate from the biopsies to culture medium. Propagated cells were counted and analysed for subtype and activation markers using flow cytometry. The results were compared with immunohistochemical and morphological analyses of the grafts. Results: By applying the in vitro culture system it was possible to demonstrate a reduction in outgrowth of mononuclear cells from core biopsies as early as 2 days after the start of anti-rejection therapy. At this time-point, as well as 2 days later, morphological and immunohistochemical analyses of biopsies showed ongoing acute cellular rejection, with no differences between biopsies taken before and after restart of CsA-therapy. The percentage of propagating T lymphocytes expressing the activation markers CD25 or MHC class II did not differ between grafts with ongoing rejection and grafts obtained from CsA-treated rats, neither did the ratio of CD4/CD8-positive cells. Conclusion: Our findings indicate that the ex vivo propagation method could function as a complement to routine histology, not only in the diagnosis of cellular rejection but also in order to rapidly reveal a failure of an anti-rejection therapy to halt the rejection process.