牙鲆T细胞抗原受体(TCR)β链同源cDNA的克隆

从牙鲆外周血淋巴细胞cDNA文库中克隆TCRβ1319bp同源cDNA序列并对其基因结构进行生物信息学分析。RT-PCR其编码区为942bp,编码314个氨基酸,编码区包括V、D、J和C区4个基因片段。TCRVβ片段主要包括从21～115位氨基酸残基的95个氨基酸序列,Jβ区与哺乳动物的Jβ区高度保守,在位于Vβ和Jβ之间的Dβ区中没有发现8碱基保守序列(GGACAGGG),CDR3β氨基酸序列为GTRILYE。牙鲆TCRCβ片段共有179个氨基酸残基,缺失位于Cβ细胞外区和临近区域的2个5～9氨基酸弹性区域。牙鲆TCRCβ片段的铰链区有6个氨基酸残基,缺少半胱氨酸位点,含Lys271保守位点。经RT-PCR检测,在脾、外周血和前肾中都发现TCRβ的表达,肌肉和肝中均未克隆到目的片段。

用TCRβ可变区基因阵列反映疾病的淋巴细胞克隆变化

将正常脐带血TCRβ(T细胞受体 β)重排基因制备成基因阵列 ,观察能否反映肿瘤或自身免疫病患者的淋巴细胞克隆的变化。方法 采用RT -PCR扩增混合 1 0例脐带血的cDNA不同家族的TCRβ可变区基因全部序列 ,经测序胶或SS CP电泳后 ,电转移到尼龙膜上 ,制成TCRβ可变区基因阵列。用同位素α - 32 PdCTP标记正常成人、脐带血、待检患考的TCRβ基因 ,与基因阵列杂交。结果 :从测序胶排列的基基阵列发现 ,正常成人及脐带血呈正常高斯分布 ,即呈多克隆性 ;一些肿瘤患者T淋巴细胞克隆有少数几个克隆的异常增生 ,即呈寡克隆性 ;1例急性T淋巴细胞白血病患者仅在Vβ1 6见单一的条带扩增 ,呈单克隆性。结论 :该基因阵列可以反映正常人淋巴细胞的克隆分布和疾病时出现的淋巴细胞变化。

半滑舌鳎外周血T淋巴细胞的分离、鉴定及TCRβ基因免疫应答分析

为开展半滑舌鳎(Cynoglossus semilaevis)免疫学研究提供细胞平台,利用密度梯度离心法分离半滑舌鳎外周血淋巴细胞,采用短期细胞培养法分离悬浮淋巴细胞,悬浮淋巴细胞在含有0.3μg/m L的PHA的DMEM完全培养基,于24℃条件下可连续培养3—4d左右,采用自制的尼龙毛柱可将悬浮淋巴细胞中的非黏附淋巴细胞和黏附淋巴细胞成功分离;利用流式细胞仪结合特异抗体检测对非黏附淋巴细胞和黏附淋巴细胞进行鉴定,结果表明,非黏附细胞与鼠抗人FTIC-CD3单克隆抗体特异结合,为T样淋巴细胞;黏附细胞和鼠抗人FTICCD19单抗特异结合,为B样淋巴细胞。T细胞表面抗原受体TCRβ基因可特异性的在非黏膜细胞中表达,而在黏附细胞中不表达,证明分离获得的非黏膜细胞为T淋巴细胞,采用q RT-PCR(Quantitative Real-Time PCR)方法检测TCRβ基因表达,结果表明,TCRβ基因在半滑舌鳎肝、脾、头肾、后肾、小肠、胃、血液、鳃、皮肤、肌肉、心脏、脑、卵巢组织中均有表达,其中在肠、胃、脾、头肾中表达量较高;鳗弧菌感染后TCRβ基因在肝、脾、鳃中呈现明显的上调表达,且表达峰值出现在感染后72—96h,表明TCRβ基因在获得性免疫应答中起重要作用。

直肠癌肿瘤浸润性淋巴细胞中T细胞受体基因谱型特征

目的 分析直肠癌肿瘤浸润性淋巴细胞 (tumor -infiltatinglymphocytes ,TIL)克隆的TCRβ基因谱型及CDR3区氨基酸序列 ,了解直肠癌TIL抗肿瘤细胞克隆的基因特征。方法 采用RT -PCR方法扩增直肠癌肿瘤组织、术前外周血和术后外周血T淋巴细胞TCTβ基因的 2 4个不同V基因片段 ,经过特殊的测序凝胶分离和银染色后 ,确定它们的TCRβ基因表达谱型。通过对PCR产物直接测序的方法 ,分析克隆性增殖的特征和CDR3区氨基酸序列。结果 直肠癌肿瘤组织TIL的TCRβ基因谱型中具有克隆性表达 ,表明直肠癌TIL是呈寡克隆性增生的淋巴细胞 ;来自 2例不同患者的肿瘤组织TIL中单克隆性表达的TCRβV11基因具有安全相同的DNA和氨基酸序列 ;其余四个寡克隆的CDR3区存在共同保守的氨基酸序列G/xGGN/xY(D)EQ ,这些序列可能直接接触直肠癌肿瘤组织相关抗原。结论 直肠癌肿瘤组织TIL细胞中存在特异性识别肿瘤相关抗原肽的T淋巴细胞克隆。

猪T细胞受体β链的基因结构及其多样性分析

设计猪TCRβ链基因的一对特异性引物,用RT-PCR法从合作小型猪外周血、淋巴结和脾淋巴细胞中高通量克隆和鉴定了82个猪TCRβ基因(简称STB)。测序分析表明,克隆的猪TCRβ链的基因都含有可变的信号肽区和V区、高变的D区和J区以及恒定的C区的基因片段,绝大部分基因间的核苷酸序列组成都不完全相同,且具有十分复杂的多态性和多样性,基因间的相似性在36.6%~99.9%,这与TCRβ链的基本基因结构特征相一致。猪TCRβ基因的分子结构、遗传演化关系和归类分析发现,在同一类基因分子中其信号肽区、FR1区和CDR1区、FR2区和CDR2区以及FR3区之间的差异主要由V基因片段的多态性引起,而CDR3区的差异主要由其D、J基因片段的多样性造成。同时发现猪TCRβ与人类的遗传演化关系较近,大部分基因序列都能找到与人类对应的TRBV、TRBD、TRBJ基因片段,但又存在一定的差异性。这表明,猪TCRβ基因既有应对外界复杂微生物环境的免疫多样性分子遗传基础,也有物种间的相似性与特异性。

Changes in the T-cell receptor Vβ gene repertoire after allogeneic hematopoietic stem cell transplantation

Background We distinguished graft-versus-host disease (GVHD) from graft-versus-leukemia (GVL) effects and to investigate the distribution of T-cell receptor (TCR) Vβ gene repertoire in individuals with leukemia before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods Peripheral blood mononuclear cells (PBMC) were obtained from 10 normal individuals, 8 donors and 11 patients with leukemia before and after transplantation. Polymerase chain reaction (PCR) amplification of complementarity-determining region 3 (CDR3) of 24 TCR Vβ genes was used to examine serial samples of PBMC. The PCR products were further analyzed by genescan to evaluate clonality of T cells.Results The 24 TCR Vβ gene repertoire displayed highly diverse and polyclonal spectratypes in all normal individuals and 4 of 8 donors. Anoth er 4 donors expressed part of the 24 TCR Vβ subfamily and 1 donor had oligoclonality. The expressions of the 24 TCR Vβ subfamilies were skewed and restric ted in 11 leukemia patients before and after transplantation. Some absences of 24 TCR Vβ subfamily expression were quite similar between the recipients pro-transplantation and related donors. The number of subfamilies expressed increased over time post-transplantation, but the restricted expressions of the subfamily could last 6-30 months after transplantation. All patients with GVHD and some without GVHD exhibited T cell clonal expansion. The expansive T cell clone was distributed in Vβ 2-3, 16-17, 18-19, 21 and Vβ 23 in patients with GVHD and in Vβ 7, 9, 16 and 19 in patients without GVHD. One patient with syngeneic-HSCT (syn-HSCT) had Vβ 15 and 16 T cell expansion after transplantation. One patient displayed Vβ 18 T cell expansion after donor lymphocyte infusion (DLI).Conclusions Normal individuals express the entire 24 TCR Vβ ge ne repertoire and have polyclonal distribution. However, the TCR Vβ gene repertoire is only partially expressed in some donors. The TCR Vβ gene repertoire is restrictedly expressed in a skew fashion in patients with leukemia before and after transplantation. The number of TCR Vβ gene subfamilies increases over time post- transplantation. GVHD and GVL effects may induce the proliferation of T cell clones.Clinical GVL response may be distinguished from GVHD alloreactivity through the host MHC antigen.

T cell receptor Vβ repertoire usage and clonal expansion of T cells in chronic myelogenous leukemia

Background In general,it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients. As we know,few studies have dealt with the distribution of oligoclonal T cells in leukemia,so we investigated the distribution and clonality of TCR Vβ repertoire of T cells in patients with chronic myelogenous leukemia (CML) in chronic phase. Methods The complementarity determining region 3 (CDR3) of TCR Vβ24 subfamily genes were amplified in peripheral blood mononuclear cells from 27 cases with CML using reverse transcription-polymerase chain reaction (RT-PCR). In order to observe the distribution of TCR Vβ repertoire,the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vβ T cells. The PCR products of the oligoclonal T cells from three cases were analyzed by direct sequencing to define the sequence of CDR3.Results The expression pattern of TCR Vβ repertoire in different individuals are different. Vβ2-21 subfamilies could be detected in CML cases. The frequent usage Vβ repertoire in CML was Vβ1,Vβ2 orVβ13. Most of the PCR products from 27 patients displayed polyclonality,while a part of the PCR products from 21 out of 27 samples displayed clonal expansion pattern. The clonal expanded T cells in CML could be found in Vβ16 subfamilies. The frequent usage of Vβ genes in clonal expansion was Vβ3,Vβ13 or Vβ21. Multiple Vβ clonal expansion was a general phenomenon in the same patient. The CDR3 sequence of Vβ21 oligoclonal T cells from 3 cases showed some difference in splice regions and in the usage of J segments.Conclusions These results indicated that clonal expanded T cells could be found in patients with CML and were tendentious in Vβ3,Vβ13 and Vβ21 subfamilies that may be related to the specific immune response for leukemia cell associated antigen.