AML-M5b外周血T细胞TCR Vβ亚家族表达及克隆性增殖特点

Expression of TCR V·β repertoire and clonal proliferation of activated T-cells in vitro from initially diagnosed leukemic patients(AML-M5b)

目的研究AML-M5b患者外周血T细胞TCR Vβ亚家族表达及克隆性增殖特点,并分析分选的患者T细胞经体外诱导、活化及短期培养后其TCR Vβ亚家族的利用及克隆性增殖情况。方法运用RT-PCR-基因扫描技术分析初治AML-M5b患者外周血T细胞TCR Vβ亚家族利用和克隆性增殖,免疫磁珠分选患者T淋巴细胞,体外利用自体白血病DC细胞联合CD3单抗、CD28对T细胞进行诱导、活化,并对诱导后T细胞进行TCR Vβ亚家族表达和克隆性增殖分析。结果9例患者T细胞在体外获得增殖,TCR Vβ分析显示患者外周血T细胞均表达部分Vβ亚家族,体外诱导前后的T细胞TCR Vβ亚家族表达和克隆性增殖特点不完全相同,有部分出现新的Vβ亚家族表达及克隆性增殖,有部分Vβ亚家族在诱导前后始终表现为克隆性增殖,体外杀伤性分析发现诱导后的T细胞对自体白血病细胞具有一定的识别作用。结论AML-M5患者TCR Vβ亚家族T细胞的倾斜性分布和克隆性增殖,部分Vβ亚家族T细胞在诱导前后始终保持克隆性增殖状态,可能是机体针对白血病相关抗原的特异性CTL,体外诱导活化及短期培养后患者T细胞TCR Vβ亚家族表达数量增加,并出现新的克隆性增殖Vβ亚家族T细胞,提示体外诱导活化能够产生白血病患者自体的白血病特异性CTL。

Objective To investigate the distribution and elonality of receptor （TCR） Vβ repertoire of the T- cells in initially diagnosed leukemic patients（AML- M5b） and analyze their elonal expansion when it activated in vitro by autologons leukemic dendritic cells. Methods By using the RT - PCR, the application of TCR Vβ repertoire was analyzed in T- cells from 9 leukemic patients. The CDR3 size of the detectable TCR Vβ subfamily was further determined by Genesean technique to evaluate the elonality of T cells. T cells were sorted from peripheral blood of the patients by immunomagnetie beads and activated in vitro stimilated by autologons leukemic dendritic cells combining anti - CD3 and anti - CD28 antibodies. And then the features of TCR Vβ subfamilies and its elonal characteristics of induced T cell were further analyzed. Results The restrict distribution TCR Vβ subfamily was found in all 9 patients. Clonal T- cell proliferation has been found in most eases investigated. T- cells from peripheral blood of initially diagnosed patients could be amplified 50 - 100 times in vitro. Both different expression of Vβ subfamilies and their elonal proliferation were partly observed before and after induction. Clonal expantion of the same TCR Vβ subfamilies was also found before and after induced in some patients. There were also new elonal proliferation of TCR Vβ subfamilies in induced and activated T - cells. T - cells activated in vitro could differentiate the autologous leukemic cells. Conclusion There is an obviously elonal proliferation of TCR Vβ subfamilies in leukemic patients（ AML- M5b） which may be the specific reaction of the body to leukemiaassociated antigens. The elonal proliferation can be efficiently induced by autologons leukemic dendritic cells with the combination of anti - CD3 and anti - CD28 antibodies in vitro. In patients with leukemia, autologons leukemic specific CTLs may also be produced by leukemic cells.