State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
J Transl Med. 2011 Dec 8;9:210. doi: 10.1186/1479-5876-9-210.
T cell receptor (TCR) reflects the status and function of T cells. We previously developed a gene melting spectral pattern (GMSP) assay, which rapidly detects clonal expansion of the T cell receptor β variable gene (TCRBV) in patients with HBV by using quantitative real-time reverse transcription PCR (qRT-PCR) with DNA melting curve analysis. However, the molecular profiles of TCRBV in peripheral blood mononuclear cells (PBMCs) and CD8+, CD8- cell subsets from chronic severe hepatitis B (CSHB) patients have not been well described.
Human PBMCs were separated and sorted into CD8+ and CD8- cell subsets using density gradient centrifugation and magnetic activated cell sorting (MACS). The molecular features of the TCRBV CDR3 motif were determined using GMSP analysis; the TCRBV families were cloned and sequenced when the GMSP profile showed a single-peak, indicative of a monoclonal population.
The number of skewed TCRBV in the CD8+ cell subset was significantly higher than that of the CD8- cell subset as assessed by GMSP analysis. The TCRBV11 and BV7 were expressed more frequently than other members of TCRBV family in PBMCs and CD8+, CD8- subsets. Also the relatively conserved amino acid motifs were detected in the TCRBV22, BV18 and BV11 CDR3 in PBMCs among patients with CSHB.
The molecular features of the TCRBV CDR3 were markedly different among PBMCs and CD8+, CD8- cell subsets derived from CSHB patients. Analysis of the TCRBV expression in the CD8+ subset was more accurate in assessing the status and function of circulating T cells. The expression of TCRBV11, BV7 and the relatively conserved CDR3 amino acid motifs could also help to predict and treat patients with CSHB.
T 细胞受体（TCR）反映 T 细胞的状态和功能。我们之前开发了一种基因熔解谱图（GMSP）分析方法，该方法通过使用带有 DNA 熔解曲线分析的定量实时逆转录 PCR（qRT-PCR），快速检测乙型肝炎病毒（HBV）患者 T 细胞受体β可变基因（TCRBV）的克隆扩增。然而，慢性重症乙型肝炎（CSHB）患者外周血单个核细胞（PBMC）和 CD8+、CD8-细胞亚群中的 TCRBV 分子谱尚未得到很好的描述。
使用密度梯度离心和磁激活细胞分选（MACS）分离和分选人 PBMC，并分为 CD8+和 CD8-细胞亚群。使用 GMSP 分析确定 TCRBV CDR3 基序的分子特征；当 GMSP 谱显示单峰时，指示单克隆群体，克隆和测序 TCRBV 家族。
通过 GMSP 分析评估，CD8+细胞亚群中的偏倚 TCRBV 数量明显高于 CD8-细胞亚群。TCRBV11 和 BV7 在 PBMC 和 CD8+、CD8-亚群中比 TCRBV 家族的其他成员表达更频繁。在 CSHB 患者的 PBMC 中，TCRBV22、BV18 和 BV11 CDR3 中也检测到相对保守的氨基酸基序。
CSHB 患者的 PBMC 和 CD8+、CD8-细胞亚群中的 TCRBV CDR3 分子特征明显不同。分析 CD8+亚群中 TCRBV 的表达更能准确评估循环 T 细胞的状态和功能。TCRBV11、BV7 和相对保守的 CDR3 氨基酸基序的表达也有助于预测和治疗 CSHB 患者。
