Next generation sequencing based assessment of the alloreactive T cell receptor repertoire in kidney transplant patients during rejection: a prospective cohort study.

BACKGROUND: Kidney transplantation is the optimal treatment in end stage renal disease but the allograft survival is still hampered by immune reactions against the allograft. This process is driven by the recognition of allogenic antigens presented to T-cells and their unique T-cell receptor (TCR) via the major histocompatibility complex (MHC), which triggers a complex immune response potentially leading to graft injury. Although the immune system and kidney transplantation have been studied extensively, the subtlety of alloreactive immune responses has impeded sensitive detection at an early stage. Next generation sequencing of the TCR enables us to monitor alloreactive T-cell populations and might thus allow the detection of early rejection events. METHODS/DESIGN: This is a prospective cohort study designed to sequentially evaluate the alloreactive T cell repertoire after kidney transplantation. The TCR repertoire of patients who developed biopsy confirmed acute T cell mediated rejection (TCMR) will be compared to patients without rejection. To track the alloreactive subsets we will perform a mixed lymphocyte reaction between kidney donor and recipient before transplantation and define the alloreactive TCR repertoire by next generation sequencing of the complementary determining region 3 (CDR3) of the T cell receptor beta chain. After initial clonotype assembly from sequencing reads, TCR repertoire diversity and clonal expansion of T cells of kidney transplant recipients in periphery and kidney biopsy will be analyzed for changes after transplantation, during, prior or after a rejection. The goal of this study is to describe changes of overall T cell repertoire diversity, clonality in kidney transplant recipients, define and track alloreactive T cells in the posttransplant course and decipher patterns of expanded alloreactive T cells in acute cellular rejection to find an alternative monitoring to invasive and delayed diagnostic procedures. DISCUSSION: Changes of the T cell repertoire and tracking of alloreactive T cell clones after combined bone marrow and kidney transplant has proven to be of potential use to monitor the donor directed alloresponse. The dynamics of the donor specific T cells in regular kidney transplant recipients in rejection still rests elusive and can give further insights in human alloresponse. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03422224 , registered February 5th 2018.

Molecular pathogenesis of post-transplant acute kidney injury: assessment of whole-genome mRNA and miRNA profiles.

Acute kidney injury (AKI) affects roughly 25% of all recipients of deceased donor organs. The prevention of post-transplant AKI is still an unmet clinical need. We prospectively collected zero-hour, indication as well as protocol kidney biopsies from 166 allografts between 2011 and 2013. In this cohort eight cases with AKI and ten matched allografts without pathology serving as control group were identified with a follow-up biopsy within the first twelve days after engraftment. For this set the zero-hour and follow-up biopsies were subjected to genome wide microRNA and mRNA profiling and analysis, followed by validation in independent expression profiles of 42 AKI and 21 protocol biopsies for strictly controlling the false discovery rate. Follow-up biopsies of AKI allografts compared to time-matched protocol biopsies, further baseline adjustment for zero-hour biopsy expression level and validation in independent datasets, revealed a molecular AKI signature holding 20 mRNAs and two miRNAs (miR-182-5p and miR-21-3p). Next to several established biomarkers such as lipocalin-2 also novel candidates of interest were identified in the signature. In further experimental evaluation the elevated transcript expression level of the secretory leukocyte peptidase inhibitor (SLPI) in AKI allografts was confirmed in plasma and urine on the protein level (p<0.001 and p = 0.003, respectively). miR-182-5p was identified as a molecular regulator of post-transplant AKI, strongly correlated with global gene expression changes during AKI. In summary, we identified an AKI-specific molecular signature providing the ground for novel biomarkers and target candidates such as SLPI and miR-182-5p in addressing AKI.