Original Article
TCR Repertoire Analysis by Next Generation Sequencing Allows Complex Differential Diagnosis of T Cell–Related Pathology

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Abstract

Clonotype analysis is essential for complete characterization of antigen-specific T cells. Moreover, knowledge on clonal identity allows tracking of antigen-specific T cells in whole blood and tissue infiltrates and can provide information on antigenic specificity. Here, we developed a next generation sequencing (NGS)-based platform for the highly quantitative clonotype characterization of T cells and determined requirements for the unbiased characterization of the input material (DNA, RNA, ex vivo derived or cell culture expanded T cells). Thereafter we performed T cell receptor (TCR) repertoire analysis of various specimens in clinical settings including cytomegalovirus (CMV), polyomavirus BK (BKV) reactivation and acute cellular allograft rejection. Our results revealed dynamic nature of virus-specific T cell clonotypes; CMV reactivation was linked to appearance of new highly abundant antigen-specific clonalities. Moreover, analysis of clonotype overlap between BKV-, alloantigen-specific T cell–, kidney allograft- and urine-derived lymphocytes provided hints for the differential diagnosis of allograft dysfunction and enabled appropriate therapy adjustment. We believe that the established approach will provide insights into the regulation of virus-specific/anti-tumor immunity and has high diagnostic potential in the clinical routine.

Key words:

Acute cellular rejection
BKV
differential diagnosis
kidney transplantation
next generation sequencing
polyoma

Abbreviations

ACR
acute cellular rejection
BKV
polyomavirus BK
BKVAN
BKV-associated nephropathy
CDR3
complementarity determining region 3
CMCV
cumulative mean CV
CMV
cytomegalovirus
CV
coefficient of variation
gDNA
genomic DNA
MH
Morisita–Horn similarity index
NGS
next generation sequencing
OPP
overlapping peptide pools
PBMC
peripheral blood mononuclear cells
PCR
polymerase chain reaction
TCR
T cell receptor

Cited by (0)

These authors contributed equally.