Quantitative Assessment of Human T-Cell Repertoire Recovery After Allogeneic Hematopoietic Stem Cell Transplantation

摘要

A method and an apparatus are provided for determining T-cell repertoire recovery after allo-HSCT or identifying patients at high risk of infection. Combination of 5′-RACE PCR with deep sequencing was used to quantify TCR diversity in 33 individuals using a single oligonucleotide pair. Analysis of duplicate blood samples revealed highly reproducible detection of expanded TCR clonotypes. After 6 months, recipients of cord blood grafts without anti-thymocyte globulin therapy approximated the TCR diversity of healthy subjects, whereas recipients of T-cell-depleted peripheral blood stem cell grafts had a 28-fold and 14-fold lower CD4+ and CD8+ T-cell diversity, respectively. After 12 months, these differences had leveled out for the CD4+, but not the CD8+ T-cell compartment.

主权项

1. A method for determining T-cell receptor β clonotype diversity and frequency in a subject, comprising
a) obtaining a blood sample from the subject b) isolating CD4+ and CD8+ T-lymphocytes, or subsets of CD4+ and CD8+ T-lymphocytes c) extracting total RNA from the cells isolated in step b), d) generating cDNA from the total RNA, e) amplifying the cDNA, f) sequencing the amplified cDNA, g) identifying T-cell receptor β clonotypes in the cDNA sequences and quantifying the diversity of the clonotypes and the clonotype frequency of each clonotype in the sample.