多参数TOX免疫表型法的建立及在母细胞性浆细胞样树突状细胞肿瘤中的应用

Establishment of multi-parameter immunophenotypic assay for TOX expression and its application for the patient with BPDCN

目的:建立多参数流式检测胸腺细胞选择相关高迁移率族蛋白(TOX)在PD-1、Tim-3和CD244耗竭性T细胞中的免疫表型法,以及其在1例罕见CD4-CD56^(+)表型的血液恶性肿瘤母细胞性浆细胞样树突状细胞肿瘤(BPDCN)患者中的应用分析。方法:收集1例BPDCN患者骨髓(BM)和外周血(PB),2例非肿瘤手术患者和1例健康者BM以及11例健康者(HI)PB分别作为BM和PB对照组,利用多色流式单抗CD45、CD3、CD4、CD8、CD25、FoxP3、TOX、PD-1、Tim-3、CD244及相应同型对照进行流式细胞检测。结果:流式检测结果提示,BPDCN患者BM和PB中TOX^(+)CD3^(+)、TOX^(+)CD4^(+)和TOX^(+)CD8^(+)T细胞比例均比对照组升高;BPDCN患者BM和PB中TOX^(+)PD-1^(+)CD8^(+)、TOX^(+)Tim-3^(+)CD8^(+)和TOX^(+)CD244^(+)CD8^(+)耗竭性T细胞中的分布比例也显著高于对照组。此外,在BPDCN患者BM和PB的CD4^(+)CD25^(+)FoxP3^(+)Treg细胞中TOX^(+)细胞分布比例高于对照组;同时TOX^(+)细胞在PD-1^(+)CD4^(+)CD25^(+)FoxP3^(+)Treg、Tim-3^(+)CD4^(+)CD25^(+)FoxP3^(+)Treg和CD244^(+)CD4^(+)CD25^(+)FoxP3^(+)Treg耗竭性T细胞中的分布比例也明显高于对照组。结论:成功建立多参数流式细胞术检测PD-1、Tim-3和CD244耗竭性T细胞亚群中TOX免疫表型法,TOX可能是BPDCN患者T细胞免疫调节的重要转录因子。

Objective:To establish a multi-parameter flow cytometry method for detecting the immunophenotypes of thymocyte selection-associated high mobility group box protein(TOX)expression and co-expression with programmed death 1(PD-1),T cell immunoglobulin domain and mucin domain-containing molecule 3(Tim-3)and CD244 in T cell subsets,and analyze the immune suppression characteristic in a patient with blastic plasmacytoid dendritic cell neoplasm(BPDCN).Methods:The peripheral blood(PB)and bone marrow(BM)of one patient with BPDCN were collected,while BM of non-tumor surgery(2 cases and 1 healthy donor),PB of 11 healthy individuals severed as control.The monoclonal antibodies CD45,CD3,CD4,CD8,CD25,FoxP3,TOX,PD-1,Tim-3,CD244 and corresponding isotype controls were employed in polychromatic flow cytometry detection.Results:The proportion of TOX^(+)CD3^(+),TOX^(+)CD4^(+)and TOX^(+)CD8^(+)T cells in BM and PB of the patient with BPDCN was higher than that of healthy control.Moreover,the percentage of TOX^(+)PD-1^(+)CD8^(+),TOX^(+)Tim-3^(+)CD8^(+)and TOX^(+)CD244^(+)CD8^(+)cells in BM or PB from the BPDCN patient was also higher than that from control.In addition,an increased percentage of TOX^(+)cells among CD4^(+)CD25^(+)FoxP3^(+)Treg cells in BM or PB of the BPDCN patients was observed in comparison with healthy individuals.And the number of TOX^(+)cells among the PD-1^(+)CD4^(+)CD25^(+)FoxP3^(+)Treg,Tim-3^(+)CD4^(+)CD25^(+)FoxP3^(+)Treg and CD244^(+)CD4^(+)CD25^(+)FoxP3^(+)Treg was also higher than that of healthy individuals.Conclusion:The detection of TOX expression and co-expression with PD-1,Tim-3 and CD244 in T cell subsets are successfully established using multi-parameter flow cytometry,and TOX may be an important transcription factor in T cell immune regulation of BPDCN patient.