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

Constantin Aschauer, Kira Jelencsics, Karin Hu, Andreas Heinzel, Julia Vetter, Thomas Fraunhofer, Susanne Schaller, Stephan Winkler, Lisabeth Pimenov, Guido A Gualdoni, Michael Eder, Alexander Kainz, Heinz Regele, Roman Reindl-Schwaighofer, Rainer Oberbauer, Constantin Aschauer, Kira Jelencsics, Karin Hu, Andreas Heinzel, Julia Vetter, Thomas Fraunhofer, Susanne Schaller, Stephan Winkler, Lisabeth Pimenov, Guido A Gualdoni, Michael Eder, Alexander Kainz, Heinz Regele, Roman Reindl-Schwaighofer, Rainer Oberbauer

Abstract

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.

Keywords: Alloreactivity; Kidney transplant; Next generation sequencing; Rejection; T cell receptor.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Study Flowchart. All eligible patients receiving a kidney transplant from donors evaluated at our center will be included and TCR repertoire of total PBMCs and alloreactive T cells will be analyzed. TCR repertoire in the periphery and in biopsies will be assessed at surveillance biopsies and TCMR. DSA: donor reactive antibodies, TCR: T cell receptor, PBMC: peripheral blood mononuclear cells, KTX: kidney transplant, TCMR: T cell mediated rejection
Fig. 2
Fig. 2
Detectable effect size versus sample size. Depicted is the relationship between number of samples in the study and minimal detectable difference in population means for standard deviations of 0.05 (red), 0.1 (blue) and 0.15 (pink). SD: Standard deviation
Fig. 3
Fig. 3
Bioinformatic Flowchart. A pipeline for the analysis of T cell repertoires. A: First, barcode analysis is done as well as separating the sequences belonging to the different individuals (B). C: Reads that cannot be assigned to a specific individual are stored in a separate FASTQ file for later investigation regarding the origin of these sequences. D: Adapters and barcodes from sequences are trimmed. During this step the UMIs are determined and stored in a separate file (D1). E: Afterwards the sequences are clustered with respect to their reference genes and UMIs. F/G: Clonotypes are assembled and clonality, diversity and repertoire overlap analysis is performed

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