Supplementary MaterialsSupplementary material 41598_2019_55442_MOESM1_ESM. from your urine of kidney transplant patients and used them as stimulators for donor-reactive T-cells, which we analyzed by circulation cytometry. We could demonstrate that using the TreaT-assay the quantification and characterization of alloreactive T-cells is usually superior to other stimulators. In a pilot study, the number of pre-transplant alloreactive T-cells negatively correlated with the post-transplant eGFR. Frequencies of pre-transplant CD161+ alloreactive CD4+ T-cells and granzyme B generating alloreactive CD8+ T-cells were substantially higher in patients with early acute rejection compared to patients without complications. In conclusion, we established a novel assay for the assessment of donor-reactive memory T-cells based on kidney cells with the potential to predict early acute rejection and post-transplant eGFR. in a short-term activation approach32. Previously, we as well as others could show that the number of pre-transplant donor-reactive IFN-producing cells measured by ELISPOT correlates with post-transplant glomerular filtration rate23C25 and predicts early AR13,26,33,34. However, stimulator cells applied in these assays present several limitations. They are either of restricted availability (splenocytes) or lack sufficient matching (HLA-bank cells). Furthermore, functional and phenotypic analysis of alloreactive cells with ELISPOT applied in previous studies is very limited3. Here, we present the Transplant reactive T cells (TreaT)-Assay, a novel multi-parameter circulation cytometry-based diagnostic tool using an easily accessible and renewable urine-derived donor-specific source of stimulator cells for monitoring allograft-specific T cells. Compared to the currently used sources our model has the advantages of high quantity, availability, and quality. The cultivation process is easy to perform. The outgrowth of cells from your urine worked for all those patients included in our study. As we could show, the majority of cells in the cultures are TEC. Since the urine was collected from a pigtail catheter, the allograft origin of TEC can be ensured. Therefore, this method offers a non-invasive way to procure kidney allograft cells. Regarding the quality, urinary cells have been shown earlier to be fully functional renal tubular cells35. Most important in our setting is usually their stimulatory capacity. We could show that urine-derived TEC, like TEC from other sources21,36, up-regulate both HLA-ABC and CDR molecules in a pro-inflammatory environment. These cells can therefore act as atypical antigen presenting cells and activate memory T cells37,38. The specificity of alloreactive T cells and ML367 the influence of pro-inflammatory conditions around the stimulatory capacity of TEC is usually displayed by the differing reactivity of CD4+ and CD8+ T cells. Homeostatic HLA-ABC expression was sufficient ML367 to trigger a CD8+ T cell response, while CD4+ T cells only reacted on inflammatory treated TEC with upregulated HLA-DR molecules. Further, comparing autologous with allogenic activation, we could demonstrate that this activation of T cells followed by TEC activation was due to allogenic capacity of TEC and not due to unspecific cytokine pre-treatment of TEC. Thus, T ML367 cells of healthy volunteers show little to no reaction towards autologous TEC, while allogenic TEC could elicit a measurable reactivity. Taken together, these experiments show that TEC have the ability to induce a specific T cell alloreaction without provoking significant unspecific reactivity. Knowing that we can specifically monitor TEC-induced donor-reactive T cells, we assessed the sensitivity of our assay in comparison to splenocytes, currently Rabbit Polyclonal to OPN3 the most commonly used stimulator source. Previously, some authors stated the presence of tissue-specific alloreactivity by HLA-molecules presenting kidney cell specific peptides39. Accordingly, splenocytes would only monitor a portion of the alloreactive T cells as their HLA-molecules do not bind the peptides present in the kidney-allograft. The presence of tissue-specific T cells in the kidney-transplantation setting was already shown more than two decades ago by demonstrating that some clones of graft-infiltrating T cells lyse TEC, but not splenocytes isolated from your corresponding donor39C45. In ML367 line with these results, we observed a significantly lower reactivity upon activation with donor-splenocytes as compared to the donor-derived TEC, despite a higher expression of HLA-molecules around the splenocytes. This underscores the superiority of our TEC-based alloreactivity-assay and suggests that it ML367 may reflect donor- and tissue-specific reactivity as well as the intragraft situation more accurately than currently used sources for stimulator cells. To show the clinical power of the established assay, we performed a proof-of-principle study.