Abstract
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.
Highlights
CD8+ T cells play an important role in adaptive immunity against virus-infected cells and tumor cells [1,2,3]
These results indicate that the integration of 59-RACE
The transcriptional initiation sites (TISs) in individual TCRa/b variable segment (TRAV and TRBV) were analyzed, and the results showed that subsets of TRAV and TRBV transcripts started from more than 2 positions (Figure 3B and 3C), suggesting that there are multiple binding sites for transcription factors that tightly control the expression of TCRa/b chains restricted in T lymphocytes [27,28]
Summary
CD8+ T cells play an important role in adaptive immunity against virus-infected cells and tumor cells [1,2,3]. Effector CD8+ T cells have direct effector functions such as cytotoxic activity and cytokine production in response to the target cells, whereas memory CD8+ T cells do not show these functions, but have the ability to proliferate and secrete large amounts of cytokines when the cells are stimulated by antigens [6]. T-cell receptor (TCR)a/b chains are heterodimeric membrane proteins expressed on the surface of CD8+ T-cells, and they contribute to direct recognition of antigen peptide presented on the major histocompatibility complex (MHC) in the target cells [7,8]. The specificity of antigen recognition for diverse peptideMHC (pMHC) complexes depends on the 3 complementarity determining regions (CDRs) of both TCRa and TCRb chains. The diversity of CDR3 is further generated by the deletion and insertion of nucleotides within the junction of V-J and V-D-J in TCRa and TCRb chains, respectively [9,10,11]
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