白血病过继免疫治疗时外周血T淋巴细胞诱导培养后的克隆性变化  

Changes of T-cell Clonality after Induction-cultivation of Peripheral T Lymphocytes in Adoptive Immunotherapy for Leukemias

作　　者：刘岩[1] 顾江英[1] 欧媛[2] 李绵洋[2] 王赫[1] 靳娴[1] 陶秀艳[1] 刘兆利[1] 马杏凡 王秀丽[1] 马思琨 康蕊[1] 蔡鹏[1] 童春容[1] 朱平[2] 

机构地区：[1]北京市道培医院特检中心,北京100049 [2]北京大学第一医院血液科,北京100034

出　　处：《中国实验血液学杂志》2009年第3期621-626,共6页Journal of Experimental Hematology

基　　金：国家科技部国际科技合作重大项目(编号2006DFB31430);国家自然科学基金(编号30470939)资助

摘　　要：本研究分析白血病细胞免疫时自体树突细胞(DC)和多种细胞因子诱导外周血淋巴细胞后T细胞克隆性的变化。采集21例化疗缓解后行细胞免疫治疗的白血病患者的骨髓和外周血,加入细胞因子培养,获得树突细胞(DC)和激活T细胞。用RT-PCR扩增代表T细胞克隆性的T细胞受体β链可变区(T-cell receptor β variable region,TCRBV),用TCRBV基因扫描的方法观察培养前后T细胞克隆的特征性变化;对T细胞克隆的TCRBV进行序列分析。流式细胞术(FCM)检测CD3+、CD4+、CD8+、CD56+和CD4+CD25str+FOXP3+,确定其细胞毒T细胞、辅助T细胞、调节T细胞和NK-T细胞的比例变化。结果表明:临床缓解后的白血病患者的T细胞呈寡克隆分布,分属在24个TCRBV家族中一些TCRBV基因家族的淋巴细胞克隆消失,仅少数保持多克隆分布,其中TCRBV24基因家族均为克隆性的分布(100%)。分析患者的TCRBV24基因序列特征时发现,3例在CDR3区共用motif VAG,2例共用GGG,表明这些TCRBV24有可能识别同一抗原,其中1例(例5)患者除TCRBV24在CDR3区有motif GGG外,TCRBV8也有GGG,也可能会识别同一抗原。外周血淋巴细胞经过13天的体外抗原和细胞因子培养后,培养前出现的一些寡克隆以及克隆完全缺失的状态随即出现了明显变化,培养后都出现完整多克隆分布。将培养前后T细胞进行流式细胞术分析显示,培养后淋巴细胞数目为培养前的3.38±1.20倍,CD3+数培养前为(71.1±11.8)%细胞,培养第13天后为(95.4±3.2)%,明显增加;CD4+数培养前为(26.7±11.4)%,培养第13天后为(27.0±13.1)%,无明显变化(p>0.01);CD8+数培养前为(35.7±12.9)%,培养第13天后为(55.5±13.8)%,明显增加(p<0.01);CD3+CD56+数培养前为(3.1±1.6)%,培养第13天后为(9.8±6.1)%,增加2倍多(p<0.01);CD4+CD25str+FOXP3+数培养前为(0.12±0.1)%,培养第13天后为(0.22±0.18)%(p<0.01)。结论:T淋巴细胞诱导培养方法的主要作用是促进CTL和NK细胞增殖,缓解后白血病患This study was purposed to analyze the changes of T-ceU clonality after induction of peripheral T lymphocytes by autogenous DC and cytokines in the preparation of adoptive immunotherapy for leukemias. The bone marrow and peripheral blood from 21 leukemia patients at remission stage after treatment and subjected to adoptive immunotherapy were collected. Their DCs and T-cells were stimulated with cytokines and then were mixed to activate T-cells. T-cell receptor β variable region （TCRBV） families were amplified by RT-PCR, and genescan method and sequencing of the PCR products were used to observe the clonality changes of T-cells before and after the induction and cultivation of T-cells. The flow cytometry was used to identify CD3 ＋ , CD4＋ , CD8＋ , CD3 ＋ CD56 ＋ and CD4 ＋ CD25str＋ FOXP3 ＋ cells to disclose the ratio change of cytotoxic T lymphocytes （ CTL）, helper T-cells, regulatory T-cells and NK T-cells before and after induction and cultivation of T-cells. The results showed that in the 21 patients, most of the 24 TCRBV families presented as oligoclonal distribution on genescan, several families were not expressed, and only a few families remained polyclonal. TCRBV24 was found to be oligoclonal in all of the 21 patients. DNA sequence analysis of TCRBV24 revealed a common motif of VAG in CDR3 in 3 cases and a common motif of GGG in CDR3 in 2 cases. In patient 5, both TCRBV 24 and TCRBV8 contained the same motif of GGG in CDR3. The identical motif in these patients may suggest that these T-cells recognize the same antigen. The peripheral lymphocytes demonstrated recovery of clonal profile on genescan from oligoclonal profile and absence of several families before the induction and cultivation to typical polyclonal profile in all TCRBV families after the induction by DC and cytokines for 13 days. After the induction and cultivation, the number of lymphocytes increased to 3.38 ± 1.20 times. CD3 ＋ , CD4 ＋ , CD8＋ , CD3 ＋ CD56＋ and CD4＋ CD25str＋ FOX P3＋cells were 71.1 ±11.8%, 2

关 键 词：白血病 过继免疫治疗 T细胞克隆 T细胞受体亚家族(TCRBV) 基因扫描 

分 类 号：R733.7[医药卫生—肿瘤]