急性髓性白血病患者异基因造血干细胞移植后γδT细胞免疫功能重建分析

Analysis of immune refunction of γδ T cells in patients with acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation

目的系统分析急性髓性白血病(AML)患者接受异基因造血干细胞移植前后γδT细胞免疫功能重建情况。方法利用反转录-多聚酶链反应(RT-PCR)方法检测12例AML患者移植前后外周血γδT细胞TRGV和TRDV各亚家族的谱系分布情况,进一步应用基因扫描分析各亚家族基因的互补决定区3(CDR3)的克隆性增殖情况。同时,采用实时荧光定量PCR方法对患者外周血γδT细胞的3个TRGV亚家族基因表达水平进行定量检测。收集8例健康成人外周血样作为对照。结果移植前后患者的TRGV亚家族谱系表达以及T细胞克隆性增殖情况无明显变化。移植前患者TRDV亚家族谱系存在明显限制性利用,部分亚家族不表达;其中TRDV3、TRDV4和TRDV8亚家族出现克隆性增殖,而移植后患者γδT细胞的TRDV亚家族谱系限制性利用情况虽有所改善,但TRDV3、TRDV4、TRDV5、TRDV6和TRDV8亚家族仍存在克隆性增殖情况。移植前后患者TRGVⅡ亚家族的表达水平均显著低于正常对照组;移植后患者的TRGV亚家族表达模式(TRGVⅠ>TRGVⅢ>TRGVⅡ)发生了明显改变,以TRGVⅠ亚家族的表达占优势。移植后无发生GVHD患者的TRGVⅠ亚家族的表达水平显著高于发生GVHD患者的TRGVⅠ亚家族的表达水平。结论移植后TRGVⅡ亚家族的免疫重建恢复较慢,且移植后TRGVⅠ亚家族表达水平较高提示较好的γδT细胞免疫重建。移植后克隆性增殖的γδT细胞可能是机体体内相关抗原的刺激下产生的反应性增殖性T细胞。

The current study was designed to investigate the function reconstitution of γδ T cells in the acute myeloid leukemia（AML） patients following allogenic hematopoietic stem cell transplantation（allo-HSCT） and to analysis the distribution and the clonality of TRGV and TRDV repertoire in peripheral γδ T cells of the AML patients.The distribution and the clonality of TRGV and TRDV repertoire in peripheral γδ T cells of 12 AML patients before and after allo-HSCT were observed by genescan technique.The expression levels of 3 TRGV subfamilies in peripheral γδ T cells of these patients were determined using real-time fluorescence quantitative PCR.Eight healthy individuals served as the normal controls.Data showed that there was no obvious difference in the distribution and clonality of TRGV subfamilies in patients before and after allo-HSCT,while restrictive utilization of TRDV subfamilies were observed in patients before allo-HSCT,and the oligoclonality of TRDV3,TRDV4 and TRDV8 were also detectable.Although restrictive utilization of TRDV subfamilies in peripheralγδ T cells from patients after allo-HSCT was improved,the oligoclonality of TRDV3,TRDV4,TRDV5,TRDV6 and TRDV8 were still detectable.The expression levels of TRGV Ⅱ subfamily in patients was significantly lower than that in controls.The expression patterns of TRGV subfamilies in patients after allo-HSCT demonstrated as TRGV Ⅰ 〉TRGV Ⅲ 〉TRGV Ⅱ,of which TRGV Ⅰsubfamily was dominant.The expression level of TRGV Ⅰ subfamily in patients without GVHD was significantly higher than that in patients with GVHD after allo-HSCT.Taken together,the immunologic reconstitution of TRGV Ⅱsubfamily in AML patients after allo-HSCT is slow,while the higher expression level of TRGV Ⅰ subfamily indicates a preferable reconstitution of γδ T cells.The oligoclonal proliferation of 78 T cells might be related to the host immune response to the associated antigen after allo-HSCT.