肾母细胞瘤基因衍生肽诱导细胞毒性T淋巴细胞对白血病患者骨髓CD34＋细胞的体外杀伤效应  

Elimination of leukemic CD34 ＋ progenitor cells by using cytotoxic T lymphocytes specifically targeting WTl-derived peptide： an experimental study in vitro

作　　者：顾伟英[1] 陈子兴[2] 邱国强[1] 胡绍燕[2] 贾丽[1] 吴炜[1] 何军[2] 岑建农[2] 贺白[1] 

机构地区：[1]苏州大学附属第三医院常州市第一人民医院血液科,213003 [2]苏州大学附属第一医院江苏省血液病研究所

出　　处：《中华医学杂志》2008年第48期3401-3406,共6页National Medical Journal of China

基　　金：江苏省医学重点学科建设与人才战略专项资金资助项目（K0609）

摘　　要：目的探讨肾母细胞瘤基因（WT1）衍生肽负载树突状细胞（DC）诱导细胞毒性T淋巴细胞（CTL）对白血病CD34＋细胞的体外清除效应。方法合成一段针对HLA—A＊0201锚位的WT19聚肽，体外负载来源于HLA—A＊0201^＋健康人的DC后，诱导产生WT1肽特异性CTL（A组），以噻唑盐（MTF）比色法观察其对WT1表达阳性白血病患者（HLA—A；0201^＋者3例，HLA-A＊0201^-者3例）骨髓CD34＋细胞、健康人（HLA—A＋0201^＋者2例，HLA-A＊0201^-者1例）外周血CD34＋细胞和白血病NB4、K562及U937细胞株的体外杀伤效应，粒细胞-巨噬细胞系集落形成试验观察其对白血病患者骨髓CD34＋细胞和健康人外周血CD34＋细胞粒细胞-巨噬细胞系集落形成单位（CFU—GM）形成的影响。设立单独DC诱导CTL（B组）和IL-2诱导T细胞（C组）作为对照。结果在效靶比为20：1时，A组CTL对3例HLA-A$0201^＋白血病患者骨髓CD34＋细胞和NB4细胞的杀伤活性（分别为55．3％±2．8％，67．1％±3．2％、49．4％±3．8％和55．0％±3．7％）明显高于对3例HLA-A＊0201^-白血病患者骨髓CD34＋细胞、健康人外周血CD34＋细胞及K562、U937细胞的杀伤活性（均〈20％），并明显高于B组和C组CTL（均P〈0．01）。2例HLA—A＊0201^＋白血病患者骨髓CD34＋细胞经A组CTL处理后CFU-GM集落相对形成率分别为17．8％±4．0％和20．8％±3．4％，明显低于经B组CTL处理后（分别为88．9％±3．4％和91．8％±5．7％，均P〈0．01）；HLA—A＊0201^-白血病患者骨髓CD34＋细胞、健康人外周血CD34＋细胞经A组和B组CTL处理后CFU-GM集落相对形成率差异无统计学意义。结论WT1肽特异性CTL能够以HLA—I类抗原限制方式杀伤高表达WT1基因的白血病CD34＋细胞，且能特异性抑制其CFU—GM集落形成，WT1基因的表达产物可以作为清除白血病CD34＋细胞靶点。Objective To investigate the effects of targeting elimination of the leukemic CD34 ＋ progenitor cells by using cytotoxic T lymphocytes （CTLs） specifically against Wilms tumor gene （ WT1 ） - derived peptide. Methods A 9-mer WT1 peptide （CMTWNQMNL） containing HLA-A ＊ 0201-binding anchor motifs was synthesized. The suspended cells were collected from the culture of the peripheral blood mononuclear cells and divided into 2 groups ： Group D （ pure T cells） and Group C （ IL2 ＋ T cells）. The dendritic cells （DCs） generated from the peripheral blood mononuclear cells of an HLA-A ＊ 0201-positive healthy donor were repeatedly loaded with WT1 peptide so as to elicit cytotoxic T cells （CTLs） specifically for WT1 peptide, and restricted by HLA-A ＊ 0201 （ Group A）. The specific lysis effects of WT1 peptide specific CTLs upon leukemic bone marrow CD34 ＋ progenitor cells positively expressing WT1 （3 being HLA-A ＊ 0201^＋ and 3 being HLA-A ＊ 0201^- ）, peripheral blood CD34 ＋ cells from healthy persons （2 being HLA-A ＊ 0201^＋ and 1 being HLA-A ＊ 0201^ - ）, and leukemia cells of the lines of NB4 （ HLA-A ＊ 0201^＋/WT1 ~ ）, U937 （ HLA-A ＊ 0201^ ＋/WT1^ - ）, and K562（ HLA-A ＊ 0201^-/WT1^ ＋ ） were measured by methyl thiazolyl tetrazolium （MTT） chromatometry assay. The colony-forming unit-granuloeyte macrophage （CFU-GM） forming capability of the leukemic bone marrow CD34 ＋ progenitor cells and peripheral blood CD34 ＋ cells from healthy persons treated with WT1 peptide specific CTLs were also determined by methyleellulose medium colony-forming unit assay. The CTLs elicited by DCs without WT1 peptide loading （Group B）and T cells cultured with IL-2 （Group C） were taken as controls. Results The CTLs specific for WT1 peptide and restricted by HLA-A ＊ 0201 （ Group A） exerted a specific lysis upon the 3 eases with HLA-A ＊ 0201^＋/WT1^＋ leukemic bone marrow CD34 ＋ progenitor cells and NB4 leukemia cells, the specific lysis l

关 键 词：白血病 基因 肾母细胞瘤 抗原 CD34+ 免疫疗法 T淋巴细胞 细胞毒性 

分 类 号：R686[医药卫生—骨科学]