Presensitization and donor-specific immunity play a major role in acute and chronic rejection of kidney transplant patients. While assessment of humoral sensitization is well standardised by the measurement of donor-specific antibodies, analyses of donor-specific T-cells is not applied in clinical routine. Here, the major obstacle is either low quantity (limited amount of donor spleen cells) or quality (lack of sufficient matching between HLA-bank and donor HLA) of stimulator cells. To overcome this shortage we established a method utilizing exfoliated allograft cells from the urine as stimulator cells in a short term stimulation assay and analysed patients with/without acute rejection in post-transplant follow-up.

Donor-derived stimulator cells were generated from urine of kidney-transplant (KTx) patients with acute rejection (AR) and with an uncomplicated clinical course as controls (Co). PBMCs were obtained at days 0, 5-7, 11-17 and 60-81 in both groups and incubated with the urinary cells for 16h. Multi-parameter flow cytometry was used to assess the frequency, phenotype and functionality of donor-specific T-cells using activation markers, cytokines and effector molecules in patients with AR and Co.

Applying the novel assay we were able to demonstrate increased frequencies of donor-reactive CD4+ T-cells at the time point of AR. Even more, increased frequencies of donor-reactive CD4+ and CD8+ T-cells were observed already before transplantation in AR group. In contrast, donor-reactive T-cells were hardly detectable in Co patients before transplantation and during follow-up. In-depth functional and phenotypic characterization of the analysed cell populations revealed dominance of Th17 phenotype within donor-reactive CD4+ cells, whereas IFNg production was less pronounced.

In conclusion, we demonstrate the clinical utility of the newly established flow cytometry-based alloantigen-assay allowing quantification as well as functional and phenotypic characterization of donor-reactive T-cells. Our data might suggest an important role of Th17 cells for the pathogenesis of AR in KTx and allow for new diagnostic and therapeutic targets.

CITATION INFORMATION: Thieme C, Weist B, Müskes A, Reinke P, Westhoff T, Babel N. Assessment of Donor-Reactive T-Cell Immunity by the Novel Urine Cell-Derived Alloantigen Assay Allows Prediction of Acute Rejection in Renal Transplant Patients. Am J Transplant. 2017;17 (suppl 3).

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