Unbiased analysis of TCRα/β chains at the single-cell level in human CD8+ T-cell subsets.
T-cell receptor (TCR) α/β chains are expressed on the surface of CD8(+) T-cells and have been implicated in antigen recognition, activation, and proliferation. However, the methods for characterization of human TCRα/β chains have not been well established largely because of the complexity of their structures owing to the extensive genetic rearrangements that they undergo. Here we report the development of an integrated 5'-RACE and multiplex PCR method to amplify the full-length transcripts of TCRα/β at the single-cell level in human CD8(+) subsets, including naive, central memory, early effector memory, late effector memory, and effector phenotypic cells. Using this method, with an approximately 47% and 62% of PCR success rate for TCRα and for TCRβ chains, respectively, we were able to analyze more than 1,000 reads of transcripts of each TCR chain. Our comprehensive analysis revealed the following: (1) chimeric rearrangements of TCRδ-α, (2) control of TCRα/β transcription with multiple transcriptional initiation sites, (3) altered utilization of TCRα/β chains in CD8(+) subsets, and (4) strong association between the clonal size of TCRα/β chains and the effector phenotype of CD8(+) T-cells. Based on these findings, we conclude that our method is a useful tool to identify the dynamics of the TCRα/β repertoire, and provides new insights into the study of human TCRα/β chains.
Center for AIDS Research, Kumamoto University, Honjo, Kumamoto, Japan.
Xiaoming
X
Sun
Masumichi
M
Saito
Yoshinori
Y
Sato
Takayuki
T
Chikata
Takuya
T
Naruto
Tatsuhiko
T
Ozawa
Eiji
E
Kobayashi
Hiroyuki
H
Kishi
Atsushi
A
Muraguchi
Masafumi
M
Takiguchi
D000328:Adult; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism*; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D059010:Single-Cell Analysis / Q000379:methods; D024363:Transcription Initiation Site; D060152:V(D)J Recombination
D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta