Dataset Information

Unbiased analysis of TCR?/? chains at the single-cell level in human CD8+ T-cell subsets.

ABSTRACT: T-cell receptor (TCR) ?/? 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 TCR?/? 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 TCR?/? 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 TCR? and for TCR? 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 TCR?-?, (2) control of TCR?/? transcription with multiple transcriptional initiation sites, (3) altered utilization of TCR?/? chains in CD8(+) subsets, and (4) strong association between the clonal size of TCR?/? 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 TCR?/? repertoire, and provides new insights into the study of human TCR?/? chains.

SUBMITTER: Sun X

PROVIDER: S-EPMC3391256 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Unbiased analysis of TCRα/β chains at the single-cell level in human CD8+ T-cell subsets.

Sun Xiaoming X Saito Masumichi M Sato Yoshinori Y Chikata Takayuki T Naruto Takuya T Ozawa Tatsuhiko T Kobayashi Eiji E Kishi Hiroyuki H Muraguchi Atsushi A Takiguchi Masafumi M

PloS one 20120706 7

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 TC ...[more]

PMID: 22792299

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Project description:BackgroundmicroRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from cell surface receptors influence the expression of miRNAs in CD8+ T cells, and may have consequent effects on cell phenotype and function. We set out to investigate whether common gamma chain cytokines modulated human CD8+ T cell expression of miR-146a, which previous studies have associated with different stages of CD8+ differentiation. We also investigated how changes in miR-146a related to other miRNAs that alter with CD8+ differentiation status.MethodsWe treated human CD8+ T cells with the cytokines IL-2, IL-7 or IL-15 either at rest or after stimulation with anti-CD3 and anti-CD28. For some experiments we also purified human CD8+ T cell subsets ex vivo. Flow cytometry was used in parallel to assess cell surface memory marker expression. Total RNA from these cells was subjected to microarray analysis and real-time PCR for miRNA expression. Nucleofection studies were performed to assess potential mRNA targets of miR-146a.ResultsWe find that miR-146a is up-regulated in naïve CD8+ T cells exposed to IL-2 or IL-15, even in the absence of an activating T cell receptor stimulus, but not when IL-7 is also present. miR-146a expression correlates with a memory phenotype in both ex vivo and in vitro cultured cells although in our hands overexpression of miR-146a was not sufficient alone to drive a full memory phenotype. In ex vivo analysis, miR-146a was one of a small number of miRNAs that was differentially expressed between naïve and memory CD8+ T cells.ConclusionsmiR-146a is emerging as a critical regulator of immune system. Our data shows that miR-146a expression is strongly influenced by the cytokine milieu even in the absence of a T cell receptor stimulus. Our results have implications for studies designed to assess the function of miR-146a, help to define a fingerprint of miRNA expression in CD8+ T cell subsets and may be useful when designing optimal protocols for T cell expansion as efficacy of T cell immunotherapy is correlated with an 'early' memory phenotype.

Dataset Information

Unbiased analysis of TCR?/? chains at the single-cell level in human CD8+ T-cell subsets.

Publications

Unbiased analysis of TCRα/β chains at the single-cell level in human CD8+ T-cell subsets.

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