The TreaT-Assay: A Novel Urine-Derived Donor Kidney Cell-Based Assay for Prediction of Kidney Transplantation Outcome

Donor-reactive immunity plays a major role in rejection after kidney transplantation, but analysis of donor-reactive T-cells is not applied routinely. However, it has been shown that this could help to identify patients at risk of acute rejection. A major obstacle is the limited quantity or quality of the required allogenic stimulator cells, including a limited availability of donor-splenocytes or an insufficient HLA-matching with HLA-bank cells. To overcome these limitations, we developed a novel assay, termed the TreaT (Transplant reactive T-cells)-assay. We cultivated renal tubular epithelial cells from the urine of kidney transplant patients and used them as stimulators for donor-reactive T-cells, which we analyzed by flow cytometry. We could demonstrate that using the TreaT-assay the quantification and characterization of alloreactive T-cells is 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 producing 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

Immune Response in Moderate to Critical Breakthrough COVID-19 Infection After mRNA Vaccination

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines

High incidence and viral load of HHV-6A in a multi-centre kidney transplant cohort

Human herpesvirus 6 (HHV-6) is a common opportunistic pathogen in kidney transplant recipients. Two distinct species of HHV-6, HHV-6A and HHV-6B, have been identified, of which the latter seems to be dominant. However, it is unclear whether they increase the likelihood of other viral reactivations. We characterized a multi-centre cohort of 93 patients along nine study visits for viral load. We tested for the following viruses: HHV-6A and HHV-6B, the herpesviruses cytomegalovirus (CMV) and Epstein-Barr virus (EBV) and the polyomavirus BK (BKV). We detected HHV-6A viral load in 48 (51.6%) patients, while the incidence of HHV-6B was much lower, being detected in 6 (6.5%) patients. The incidence of HHV-6A was higher than of BKV, CMV and EBV. HHV-6A also demonstrated higher viral loads than the rest of viruses. There was a non-significant trend of association between HHV-6A and HHV-6B as co-infection, whereas no increased incidence of other viruses among patients with HHV-6A reactivation was observed. There was no negative effect of high HHV-6A (>10,000 copies/ml) load on markers of renal graft and hepatic function or blood count twelve months post-transplant. In contrast to previously published data, our results show a clear dominance of HHV-6A in peripheral blood when compared to HHV-6B, with higher incidence and viral load levels. Despite the high HHV-6A loads observed, we did not identify any negative effects on posttransplant outcome

Das TreaT-Assay – Bestimmung des immunologischen Risikos nach Nierentransplantation durch Messung der T-Zell-Alloreaktivität gegen Transplantatzellen aus dem Urin

In the past decades, the prognosis of kidney transplant patients improved significantly. This can be attributed to a prolonged graft survival due to the introduction of very effective immunosuppressive drugs and a strong reduction of rejection episodes. On the other hand, the immunosuppressive medication increases the risk of infectious, cardiovascular and malignant diseases, which are the main reason for death with a functioning graft. Personalized immunosuppressive therapy could minimize the risk of complications due to the therapy, while still providing long-term protection of the allograft. For the introduction of personalized medicine, however, a marker that reliably mirrors the risk of cellular rejection is needed to guide immunosuppressive therapy. The key players in cellular rejection are alloreactive T cells. Especially memory T cells targeting proteins of the transplant cells can limit the short- and long-term functionality of the graft. The aim of this thesis was therefore to investigate how the reactivity of T cells towards the allograft can be measured, and how these methods can be used in a test system for the risk of rejection episodes. Cells of the organ donor are needed to activate alloreactive T cells and to determine their frequency. We were the first to describe the application of transplant cells derived from the urine of the transplant recipient for this matter. Therefore, we cultivated tubular epithelial cells from the urine and showed that these cells present HLA-proteins, the target molecules of alloreactive T cells. Alloreactive helper as well as cytotoxic T cells of transplant recipients reacted to the tubular epithelial cells of the kidney allograft donor. This reaction was even stronger than to splenocytes of the donor, an alternative source of stimulator cells. In a proof of concept study patients with a higher number of pre-transplant alloreactive T cells had a worse kidney function in follow-up after transplantation. Patients that experienced an acute rejection episode early after transplantation had more alloreactive T cells before transplantation compared to control patients. These results illustrate that we developed a promising tool for the measurement of alloreactive T cells and therefore for the assessment of the immunological risk after kidney transplantation. The further development of this testing method shall improve the translatability in clinical routine diagnostics. Ultimately, the assay performance needs to be tested in larger trials.In den vergangenen Jahrzehnten hat sich die Prognose von Patienten und Patientinnen mit Nierentransplantat signifikant verbessert. Dies ist vor allem auf ein verlängertes Transplantatüberleben durch den Einsatz von sehr effektiven Immunsuppressiva und eine starke Verminderung von Abstoßungsreaktionen zurückzuführen. Allerdings erhöht die immunsuppressive Medikation das Risiko von infektiösen, kardiovaskulären und malignen Erkrankungen, welche die häufigsten Ursachen für das Versterben trotz funktionierendem Transplantat darstellen. Durch eine personalisierte immunsuppressive Therapie könnte Komplikationsrate durch die Immunsuppression möglichst gering gehalten werden, während gleichzeitig das Transplantat langfristig geschützt wird. Für die Einführung personalisierter Medizin wird allerdings ein Marker benötigt, welcher zuverlässig das Risiko einer zellulären Abstoßungsreaktion abbildet und so die immunsuppressive Therapie leiten kann. Die Hauptakteure der zellulären Rejektion sind alloreaktive T Zellen. Vor allem Gedächtnis-T Zellen, welche gegen Proteine auf den Transplantatzellen gerichtet sind, können die kurz- und langfristige Funktionalität des Transplantats vermindern. Das Ziel dieser Doktorarbeit war somit, zu erforschen, wie sich die Reaktivität der T Zellen gegen das Transplantat bestimmen lässt, um anhand dieser Ergebnisse einen Tests für das Risiko von Abstoßungsreaktionen zu entwickeln. Um alloreaktive T Zellen zu aktivieren und ihre Frequenz zu bestimmen, werden Zellen des Organspenders für die Stimulation benötigt. Wir konnten als Erste den Einsatz von Transplantatzellen aus dem Urin des Empfängers für diesen Zweck beschreiben. Dazu kultivierten wir Tubulusepithelzellen aus dem Urin und konnten zeigen, dass diese HLA-Proteine, die Zielmoleküle für alloreaktive T Zellen, exprimieren. Sowohl alloreaktive T-Helferzellen als auch zytotoxische T Zellen von Transplantatempfängern reagierten auf die Tubulusepithelzellen des Nierenspenders, und taten dies sogar stärker als auf Milzzellen des Spenders, einer alternativen Quelle von Stimulatorzellen. In einer Proof of Concept Studie hatten Patienten, welche schon vor der Transplantation eine höhere Anzahl an gegen das Transplantat gerichteten T Zellen hatten, eine schlechtere Nierenfunktion im Verlauf nach der Transplantation. Patienten, welche eine frühe Abstoßungsreaktion nach der Transplantation entwickelten, hatten mehr alloreaktive T Zellen vor Transplantation als Kontrollpatienten. Diese Ergebnisse zeigen, dass wir ein vielversprechendes Verfahren zur Messung von alloreaktiven T Zellen und damit der Bestimmung des immunologischen Risikos nach einer Nierentransplantation entwickeln konnten. In der weiteren Entwicklung des Testverfahrens soll die Anwendbarkeit in der Routinediagnostik von nierentransplantierten Patienten verbessert werden. Größere Studien müssen dann durchgeführt werden, um die diagnostische Leistungsfähigkeit zu testen

Generation of HBsAg-reactive T- and B-cells following HBV vaccination in serological non-responders under hemodialysis treatment

HBV vaccination is recommend for hemodialysis patients, but only 50–60% of the patients show seroconversion. HBV vaccine-induced generation of HBV reactive T and B cells could be detected regardless of their capacity to mount a serological response, indicating that patients without seroconversion are potentially protected by their HBV-reactive T cell pool. Hemodialysis patients (HDP) are at high risk of hepatitis B virus (HBV) infection. Hence, stringent precautions including HBV vaccination are recommended for seronegative HDP [1]. Although a four times higher vaccination dose is used in HDP compared to healthy individuals, only 50–60% seroconvert [2]. Reasons for the low seroconversion rate remain unknown

Immune monitoring facilitates the clinical decision in multifocal COVID-19 of a pancreas-kidney transplant patient

The optimal management in transplant recipients with coronavirus disease 2019 (COVID-19) remains uncertain. The main concern is the ability of immunosuppressed patients to generate sufficient immunity for antiviral protection. Here, we report on immune monitoring facilitating a successful outcome of severe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated pneumonia, meningoencephalitis, gastroenteritis, and acute kidney and pancreas graft failure in a pancreas-kidney transplant recipient. Despite the very low numbers of circulating B, NK, and T cells identified in follow-up, a strong SARS-CoV-2 reactive T cell response was observed. Importantly, we detected T cells reactive to Spike, Membrane, and Nucleocapsid proteins of SARS-CoV-2 with majority of T cells showing polyfunctional proinflammatory Th1 phenotype at all analyzed time points. Antibodies against Spike protein were also detected with increasing titers in follow-up. Neutralization tests confirmed their antiviral protection. A correlation between cellular and humoral immunity was observed underscoring the specificity of demonstrated data. We conclude that analyzing the kinetics of nonspecific and SARS-CoV-2-reactive cellular and humoral immunity can facilitate the clinical decision on immunosuppression adjustment and allow successful outcome as demonstrated in the current clinical case. Although the antiviral protection of the detected SARS-CoV-2-reactive T cells requires further evaluation, our data prove an ability mounting a strong SARS-CoV-2-reactive T cell response with functional capacity in immunosuppressed patients