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

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(MTT)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尤统计学意义.结论 WT1肽特异性CTL能够以HLA-1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.

Elimination of leukemic CD34 + progenitor cells by using cytotoxic T lymphocytes specifically targeting WT1-derived peptide: an experimental study in vitro

Objective To investigate the effects of targeting elimination of the leukemic CD34 + progenitor cells by using cytotoxic T lymphoeytes (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 cytotoxie T cells (CTLs) specifically for WT1 peptide, and restricted by HLA-A *0201 (Group A).The specific lysis effects of WTI peplide 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 (MTI') chromatometry assay.The colony-forming unit-granulocyte 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 methylcellulose medium colony-forming unit assay.The CTLs elicited by DCs without WTi 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 cases with HLA-A * 0201+/WTI+ leukemic bone marrow CD34 + progenitor cells and NB4 leukemia cells, the specific lysis levels were 55.3% ± 2.8%, 67.1% ±3.2%,49.4% ± 3.8% and 55.0% ± 3.7% respectively, all significantly higher than those upon the 3 cases with HLA-A * 0201-/WT1+ leukemic bone marrow CD34 + progenitor cells, normal healthy peripheral blood CD34 + cells of the healthy persons, andleukemia cell of the lines K562 and U937 (the specific lysis levels were all < 20%).The specific lysis level of Group A CTLs was significantly higher than those of Group B and Group C CTLs (both P < 0.01).The relative colony formation rates of WTI-CTLs in Group A upon the 2 cases with HLA-A * 0201+/WT1+ leukemic CD34 + progenitor cells were 17.8%±4.0% and 20.8% ± 3.41% respectively, both significantly lower than those of the none WT1-CTLs in Group B(88.9% ± 3.4% and 91.8% ± 5.7% respectively, both P ＜0.01).There was no significant difference in the relative colony formation rate upon HLA-A * 0201+/WT1- leukemic bone marrow CD34 + progenitor cells and the HLA-A * 0201-/WT1- or HLA-A * 0201+/WT1- normal peripheral blood CD34 + progenitor cells between the CTLs of Groups A and B.Conclusions The CTLs specific for WT1 and restricted by HLA-A * 0201 can exert specific lysis upon leukemic CD34 + progenitor cells highly expressing WT1 gene, and can inhibit the CFU-GM colony formation of such leukemic CD34 + progenitor cells.The product of expression of WT1 gene may be used as a target in the leukemic CD34 + cells.