title: Next-generation-sequencing-spectratyping reveals public T-cell receptor repertoires in pediatric very severe aplastic anemia and identifies a β chain CDR3 sequence associated with hepatitis-induced pathogenesis.
abstract: Current diagnostic approaches that characterize T-cell deficiency by analyzing diversity of T-cell receptor sequences effectuate limited informational gain about the actual restrictiveness. For deeper insight into T-cell receptor repertoires we developed next-generation-sequencing-spectratyping, which employs high coverage Roche/454 sequencing of T-cell receptor (β)-chain amplicons. For automated analysis of high-throughput-sequencing data, we developed a freely available software, the TCR profiler. Gene usage, length, encoded amino acid sequence and sequence diversity of the complementarity determining region 3 were determined and comprehensively integrated into a novel complexity score. Repertoires of CD8(+) T cells from children with idiopathic or hepatitis-induced very severe aplastic anemia (n=7), children two months after bone marrow transplantation (n=7) and healthy controls (children n=5, adults n=5) were analyzed. Complexity scores clearly distinguished between healthy and diseased, and even between different immune deficiency states. The repertoire of aplastic anemia patients was dominated by public (i.e. present in more than one person) T-cell receptor clonotypes, whereas only 0.2% or 1.9% were public in normal children and adults, respectively. The CDR3 sequence ASSGVGFSGANVLT was highly prevalent in 3 cases of hepatitis-induced anemia (15-32% of all sequences), but was only low expressed in idiopathic aplastic anemia (2-5%, n=4) or healthy controls (<1%). Fifteen high frequent sequences were present exclusively in aplastic anemia patients. Next-generation-sequencing-spectratyping allows in-depth analysis of T-cell receptor repertoires and their restriction in clinical samples. A dominating clonotype was identified in hepatitis-induced anemia that may be associated with disease pathogenesis and several aplastic-anemia-associated, putatively autoreactive clonotypes were sequenced.
authors.affiliation: Department of Pediatric Oncology, Hematology and Clinical Immunology, University Children’s Hospital, Medical Faculty, Heinrich Heine University, Duesseldorf, Germany.
authors.forename: Pina F I
authors.initials: PF
authors.lastname: Krell
authors.forename: Susanne
authors.initials: S
authors.lastname: Reuther
authors.forename: Ute
authors.initials: U
authors.lastname: Fischer
authors.forename: Thomas
authors.initials: T
authors.lastname: Keller
authors.forename: Stephan
authors.initials: S
authors.lastname: Weber
authors.forename: Michael
authors.initials: M
authors.lastname: Gombert
authors.forename: Friedhelm R
authors.initials: FR
authors.lastname: Schuster
authors.forename: Corinna
authors.initials: C
authors.lastname: Asang
authors.forename: Polina
authors.initials: P
authors.lastname: Stepensky
authors.forename: Brigitte
authors.initials: B
authors.lastname: Strahm
authors.forename: Roland
authors.initials: R
authors.lastname: Meisel
authors.forename: Jens
authors.initials: J
authors.lastname: Stoye
authors.forename: Arndt
authors.initials: A
authors.lastname: Borkhardt
mesh_terms: D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D000741:Anemia, Aplastic / Q000175:diagnosis / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics*; D006505:Hepatitis / Q000175:diagnosis / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007223:Infant; D008969:Molecular Sequence Data; D011446:Prospective Studies; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D012720:Severity of Illness Index
chemical_list: D022801:Complementarity Determining Regions; D011948:Receptors, Antigen, T-Cell