CHB患者外周血及肝组织中T淋巴细胞受体β链互补决定区3谱系分析
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摘要
目的：1.分析慢性乙型肝炎(Chronic Hepatitis B, CHB)患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)及肝脏组织T淋巴细胞受体(T cell receptor, TCR)β链各家族互补决定区3(complementarity determining region3,CDR3)谱系的变化,比较CHB患者PBMC及肝脏组织TCRBV家族CDR3克隆增生异常率；2.初步探讨肝脏组织病理学改变及血清HBVDNA载量与TCRCDR3谱系漂移的关系；3.对出现的单或寡克隆性增生T细胞TCRCDR3区进行基因和氨基酸组成分析,为CHB患者的个体化治疗寻找共同的特异性(Hepatitis B virus, HBV)抗原表位奠定基础。方法：1.采集4例正常献血员抗凝静脉血5mL、11例CHB患者外周抗凝静脉血5mL及相应肝组织为研究对象。密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cell, PBMC),提取PBMC及肝组织总RNA,逆转录合成cDNA,根据TCRβ链24个可变区基因片段(BV)家族设计相应的上游特异性引物,在恒定区(BC)设计一条共用的荧光标记的下游引物,采用荧光定量PCR (fluorescence quantitative polymerase chain reaction, FQ-PCR)溶解曲线法扩增出包含完整的CDR3区的24个TCRBV家族,继而分析各样本PCR产物形成的24个TCRBV家族溶解曲线谱型图；2.选取谱型图上呈单／寡峰形的TCRBV家族PCR产物相同条件进行PCR,经1.5%琼脂糖凝胶电泳鉴定后进行基因测序,利用分子生物信息学软件DNAtools6.0等分析C D R 3区的基因和氨基酸组成；3.比较11例CHB患者外周血及肝组织TRBV各家族CDR3克隆增生异常率,同时对11例CHB患者PBMC及肝组织TRBV家族CDR3克隆增生异常率与HBV-DNA载量、肝组织学炎症分级、纤维化分期分别做Spearman's相关分析。结果：1.4例正常人外周血TCRβ链24个家族CDR3谱型图大多数呈多峰形,即各家族呈现不同的CDR3多态性,其24个家族逆转录-聚合酶链式反应(reverse transcription polymerase chain reaction, RT-PCR)产物行1.5%琼脂糖凝胶电泳时均发现一条模糊条带。11例CHB患者PBMC及肝组织TCRβ链各家族CDR3溶解曲线谱型图出现数量不等,形态不一的单峰、寡峰、偏峰,部分家族表达频率极低或缺失,在1.5%琼脂糖凝胶电泳图上多数家族预测范围大小处呈现一条模糊条带,部分家族出现清晰条带或无条带；2.肝组织TRBV家族的克隆增生异常率显著高于外周血的异常率(X2=18.50,P<0.01)；3.11例CHB患者PBMC TRBV家族CDR3克隆增生异常率与HBV-DNA载量、肝组织学炎症分级、纤维化分期做Spearman's相关分析,显示上述各项指标与CHB患者PBMCTRBV家族CDR3克隆增生异常率无相关性；4.11例CHB患者肝组织TRBV家族CDR3克隆增生异常率与HBV-DNA载量、肝组织学炎症分级、纤维化分期采用Spearman's相关分析发现CHB患者肝组织TRBV家族CDR3克隆增生异常率与炎症分级呈负相关,相关系数r=-0.65,P<0.05；其他各项无相关性；5.对谱型图上呈单/寡克隆性增生的部分家族进行TCRCDR3区测序发现,患者2外周血TRBV10与其肝组织TRBV8、TRBV11有完全相同CDR3序列；外周血中的患者2TRBV10、患者4TRBV11,肝组织中的患者2TRBV8、患者4TRBV11有相同基序" T D T Q Y";外周血中的患者3TRBV19、TRBV20,肝组织中患者6TRBV11有相同基序"Q P Q H "。结论：1.CHB患者炎症活动期外周血和肝组织多个TCRBV家族谱型存在克隆性增生,且绝大部分单/寡克隆性增生的T细胞不具有相同的CDR3氨基酸序列,提示慢性乙型肝炎炎症活动期多个抗原表位参与了T细胞免疫应答。2.CHB患者炎症活动期肝组织免疫应答较外周血细胞免疫应答有明显差异。3.其中一例患者在外周血及肝组织中不同TRBV家族有完全相同TCRCDR3区,推测为该个体针对同一HBV抗原表位的T细胞克隆应答,为CHB患者的T细胞应答机制和个体化治疗、HBV的T细胞表位研究提供了初步的基础。
Objectives By analyzing the spectral patterns of Complementarity Determining Regions 3 (CDR3) length distribution for T-cell receptor beta chain variable (TCRBV) gene families of infiltrating T cells in the livers and peripheral blood mononuclear cell (PBMC), we evaluated the difference of T cells clone expansion in the livers and PBMC of patients with chronic hepatitis B (CHB).The relationship among the abnormal rate of clonal expansion of TCRBV gene families with the liver pathological changes,HBVDNA loading was investigated. The nucleotide sequences of TCRβchain CDR3 of expended PBMC and hepatic tissues were further determined in order to find the common Hepatitis B virus (HBV) specific epitopes. Methods:PBMC was isolated by Ficoll-Hypaque density gradient centrifugation and hepatic tissues were collected from the 11 patients with CHB. Total RNA was extracted from PBMC of 11 patients and 4 normal control (NC), and from hepatic tissues of 11 CHB patients, then total RNA was reversely transcripted into cDNA. The TCR CDR3 transcripts was amplified by fluorescence quantitative polymerase chain reaction (FQ-PCR) using specific primer for 24 TCRBV families and one BC region primer (fluorescence labeled). The gene melting spectral pattern (GMSP) of 24 TRBV gene families were obtained from melting curve. When the GMSP of 24 TRBV gene families in the PBMC and hepatic tissue of patients showed a single peak (the monoclonal/oligoclonal TCRβT cells), the PCR products which showed a single peak (clonal expansion) were amplified again at the same PCR condition, but the BC region primer was not labeled by fluorescence. Nucleotide sequences of TCR CDR3 of the PCR products were analyzed using DNA tools 6.0 and so on. The data was compared by chi square test and spearman's analysis. Results:The GMSP of 24 TRBV families in the PBMC from 4 NC showed a diverse multimodal peak. Compared to NC, the GMSP of 24 TRBV families in the 11 CHB patients, no matter in the PBMC or hepatic tissue, showed either a single peak or prominent melting peaks, some patients even showed disappeared for certain TCRBV families, while the FQ-PCR products of most of 24 TRBV families showed a blur band in the prediction of products size on 1.5% agarose gel by GoldView staining and parts of TRBV family PCR products exhibited a disappeared band or a clear band in patients with CHB. The abnormal rate of clonal expansion of TCRBV gene families in the hepatic tissue was significantly higher than that in the PBMC (X2=18.50, P<0.01). There were no significant correlations of the abnormal rate of clonal expansion of TCRBV gene families in the PBMC with the HBVDNA loading, degrees of liver inflammation and the stages of liver fibrosis in CHB patients. The abnormal rate of clonal expansion of TCRBV gene families in hepatic tissue showed a significant negative correlation with the degrees of liver inflammation in patients with CHB (r=-0.65,P<0.05). The abnormal rate of clonal expansion of TCRBV gene families in hepatic tissue showed no significant correlations with the HBVDNA loading, the liver fibrosis stages in CHB patients. The sequence results of single peak patterns from GMSP showed that TRBV10 in PBMC of patient-2 and TRBV8, TRBV11 in hepatic tissue of patient-2 had a shared TCR CDR3βsequence. TRBV10 of patient-2, TRBV11 of patient-4 in PBMC and TRBV8,TRBV11 of patient-2, TRBV11 of patient-4 in hepatic tissue shared the same motif" T D T Q Y".TRBV19、TRBV20 of patient-3 in PBMC and TRBV11 of patient-6 in hepatic tissue shared the same motif " Q P Q H ". Conclusions:T cells in the peripheral blood and hepatic tissue in patients with CHB showed a clonal expansion compared to NC. The significant difference of T cellular immunologic response between in PBMC and in hepatic tissue of CHB patients. The shared TCR CDR3 sequence suggested a T cell clonal expansion which was driven by homo-epitope antigen in the same CHB patient.Our results might be shed light on the individualized immunotherapy of CHB and the design of a HBV-epitope vaccine. Keywords Chronic Hepatitis B; T-cell receptor; Complementarity Determining Region 3;FQ-PCR
Objectives By analyzing the spectral patterns of Complementarity Determining Regions 3 (CDR3) length distribution for T-cell receptor beta chain variable (TCRBV) gene families of infiltrating T cells in the livers and peripheral blood mononuclear cell (PBMC), we evaluated the difference of T cells clone expansion in the livers and PBMC of patients with chronic hepatitis B (CHB).The relationship among the abnormal rate of clonal expansion of TCRBV gene families with the liver pathological changes,HBVDNA loading was investigated. The nucleotide sequences of TCRβchain CDR3 of expended PBMC and hepatic tissues were further determined in order to find the common Hepatitis B virus (HBV) specific epitopes. Methods:PBMC was isolated by Ficoll-Hypaque density gradient centrifugation and hepatic tissues were collected from the 11 patients with CHB. Total RNA was extracted from PBMC of 11 patients and 4 normal control (NC), and from hepatic tissues of 11 CHB patients, then total RNA was reversely transcripted into cDNA. The TCR CDR3 transcripts was amplified by fluorescence quantitative polymerase chain reaction (FQ-PCR) using specific primer for 24 TCRBV families and one BC region primer (fluorescence labeled). The gene melting spectral pattern (GMSP) of 24 TRBV gene families were obtained from melting curve. When the GMSP of 24 TRBV gene families in the PBMC and hepatic tissue of patients showed a single peak (the monoclonal/oligoclonal TCRβT cells), the PCR products which showed a single peak (clonal expansion) were amplified again at the same PCR condition, but the BC region primer was not labeled by fluorescence. Nucleotide sequences of TCR CDR3 of the PCR products were analyzed using DNA tools 6.0 and so on. The data was compared by chi square test and spearman's analysis. Results:The GMSP of 24 TRBV families in the PBMC from 4 NC showed a diverse multimodal peak. Compared to NC, the GMSP of 24 TRBV families in the 11 CHB patients, no matter in the PBMC or hepatic tissue, showed either a single peak or prominent melting peaks, some patients even showed disappeared for certain TCRBV families, while the FQ-PCR products of most of 24 TRBV families showed a blur band in the prediction of products size on 1.5% agarose gel by GoldView staining and parts of TRBV family PCR products exhibited a disappeared band or a clear band in patients with CHB. The abnormal rate of clonal expansion of TCRBV gene families in the hepatic tissue was significantly higher than that in the PBMC (X2=18.50, P<0.01). There were no significant correlations of the abnormal rate of clonal expansion of TCRBV gene families in the PBMC with the HBVDNA loading, degrees of liver inflammation and the stages of liver fibrosis in CHB patients. The abnormal rate of clonal expansion of TCRBV gene families in hepatic tissue showed a significant negative correlation with the degrees of liver inflammation in patients with CHB (r=-0.65,P<0.05). The abnormal rate of clonal expansion of TCRBV gene families in hepatic tissue showed no significant correlations with the HBVDNA loading, the liver fibrosis stages in CHB patients. The sequence results of single peak patterns from GMSP showed that TRBV10 in PBMC of patient-2 and TRBV8, TRBV11 in hepatic tissue of patient-2 had a shared TCR CDR3βsequence. TRBV10 of patient-2, TRBV11 of patient-4 in PBMC and TRBV8,TRBV11 of patient-2, TRBV11 of patient-4 in hepatic tissue shared the same motif" T D T Q Y".TRBV19、TRBV20 of patient-3 in PBMC and TRBV11 of patient-6 in hepatic tissue shared the same motif " Q P Q H ". Conclusions:T cells in the peripheral blood and hepatic tissue in patients with CHB showed a clonal expansion compared to NC. The significant difference of T cellular immunologic response between in PBMC and in hepatic tissue of CHB patients. The shared TCR CDR3 sequence suggested a T cell clonal expansion which was driven by homo-epitope antigen in the same CHB patient.Our results might be shed light on the individualized immunotherapy of CHB and the design of a HBV-epitope vaccine. Keywords Chronic Hepatitis B; T-cell receptor; Complementarity Determining Region 3;FQ-PCR
引文
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[8]Lim A,Baron V, ferradini L,et al. Combination of MHC-peptide multimer-based T cell sorting with the Immunoscope permits sensitive exvivo quantitation and follow-up of human CD8+T cell immune responses[J].Immunology Methods 2002;241(1-2):177-94.
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[11]Yao XS,Zhang GW,Ma L,et al. Analysis of the CDR3 length of TCR alphabeta T cells in the peripheral blood of patients with chronic hepatitis B[J].Hepatol Res.2006,35:10-18.
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