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The Characteristics of Umbilical Cord Blood T-cell Repertoire Based on Detection of T-cell Receptor Genes Rearrangement

Author: ChenShaoHua
Tutor: LiYangQiu
School: Jinan University
Course: Hematological Diseases
Keywords: umbilical cord blood αβ~+ andγδ~+ T cell TCR Vα/Vβsubfamilies TCR Vγ/Vδsubfamilies TCRζgene FACS genescan real-time RT-PCR
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 26
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Abstract

Objective:To analyze the characteristics of umbilical cord blood(UCB) T cell repertoire based on the detection of T-cell receptor(TCR) gene rearrangement.This study is expected to provide more detailed and more comprehensive information on immunological characteristics of UCB T cells for its application.Method:The frequency of CD3~+-,CD4~+-,CD8~+-,αβ~+- andγδ~+- T cells were examined in umbilical cord blood(UCB) by FACS.The CDR3 size of TCR Vα/Vβand TCR V_γ/Vδsubfamily genes were analyzed in mononuclear cells(MCs) from 10 to 16 UCB samples respectively,using RT-PCR and genescan technique.Quantitative detection of the expression level of TCR V_γsubfamily genes were performed from 16 UCB cases by real-time PCR.T cells from 3 cases UCB were stimulated and amplified by rhIL-2,PHA,CML cell or PML-RARαpeptide in vitro using liquid T-cell culture technique,to detect the usage and clonality feature of TCR Vβsubfamilies.CD4~+-and CD8~+-T cells were sorted from MCs of 12 UCB samples using the MACS magnetic bead separation system.Real-time PCR with SYBR Green I technique was used for detecting TCRζgene expression level in MCs,sorted CD4~+- and CD8~+-T cells from UCB.The mutation or polymorpheirm of TCRζgene was determined on the change of melting curve and nucleotide sequencing.Peripheral blood(PB) samples from total of 60 healthy adult individuals served as controls.Result:(1)The mean percentages of CD3~+/MCs,CD3~+CD8~+/MCs,CD3~+ TCRαβ~+/MCs, CD3~+TCR_γδ~+/MCs and CD3~+TCR_γδ~+/CD3~+ cells from UCB were significantly lower than that from healthy adult individuals,while the CD3~+TCRαβ~+/CD3~+ cells was higer in UCB than that from healthy adult individual PB group.(2) The selected usage of TCR repertoire could be identified in TCR Vα/Vβand TCR V_γ/Vδof UCB.The majority of TCR Vα/Vβsubfamily T cells in UCB displayed polyclonality,whereas the clonal expanded T cells were detected in different TCR V_γ/Vδsubfamilies,occasional oligoclonal peaks are identified in some TCR Vαsubfamilies.The pattern of TCR V_γ,subfamily expression level in UCB was V_γⅠ＞V_γⅢ＞V_γⅡ,in contrast,V_γⅡ＞V_γⅠ＞V_γⅢwas found in PBMCs.(3) The expression and polyclonality pattern of all 24 TCR Vβsubfamily T cells could be detected after stimulation with rhIL-2 or PHA respectively,whereas the restricted expression of TCR Vβsubfamilies and Vβ21,Vβ13,Vβ14 and Vβ16 clonal expansion T cells were found in cultured T cells induced by CML cells or PML-RARαpeptide respectively.(4) TCRζgene was expressed in all cases from both UCB and healthy adults.The relative mRNA expression level of TCRζgene in mononuclear cells(6.7%±5.56%),CD4~+T cells(6.74%±2.0%) and CD8~+T cells (6.88%±1.76%) from UCB were significantly higher than that from healthy adults (P=0.000,P=0.034,P=0.000) respectively.Neither mutation nor polymorpherim of TCRζgene was identfied in all samples.Conclusion:It is the first report providing a detailed profile of the UCB T-cell repertoire in China.The skewed distribution of Vα/Vβor V_γ/Vδsubfamily T cells could be found in UCB,which might be responsible for lower incidence of GVHD in cord blood hematopoietic stem cells transplantation.The clonal expansion of TCR subfamilies in UCB T cells remains unknown,it might be due to clonal absence during T-cell repertoire development.However,UCB T cells may have the potential capability of proliferation in different TCR Vβsubfamily T cells,and the ability of restricted usage and clonal expansion,when T cells were induced by leukemia associated antigen such as CML cells or PML-RARαpeptide.In addition,the SYBR Green I real-time technique for quantitative detection of TCR V_γand TCRζchain expression levels was established successfully.This is,to our knowledge,the first description in the expression feature of TCR V_γand TCRζchain gene in UCB.

The Characteristics of Umbilical Cord Blood T-cell Repertoire Based on Detection of T-cell Receptor Genes Rearrangement

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