CD3^+、CD4^+和CD8^+T细胞Vβ亚家族中PD-1免疫表型检测方法的建立

Establishment of method for PD-1 immunophenotype detection in TCR Vβ repertoire of CD3^+,CD4^+ and CD8^+ T-cell subsets

目的:建立分析CD3^+、CD4^+和CD8^+T细胞Vβ亚家族中免疫抑制性受体程序性细胞死亡蛋白1(PD-1)分子免疫表型的方法,从而了解人体外周血T细胞谱系中PD-1表型细胞的分布频率。方法:收集健康个体(HI)外周血10例,利用抗CD45、CD3、CD4、CD8、PD-1等多色荧光流式单抗,以及T细胞受体(TCR) Vβ谱系试剂盒[IOTestBeta Mark TCR VβRepertoire Kit,共8管,每一管均为FITC和PE偶联的复合抗体(A～H),每一管复合抗体包含3个TCR Vβ亚家族的抗体],检测T细胞亚群TCR Vβ亚家族中PD-1的分布情况。结果:在10例HI的检测中,CD3^+、CD3^+CD4^+和CD3^+CD8^+T细胞亚群中检测的24个TCR Vβ亚家族总和符合试剂盒所提供的Quick Reference Card数据,初步结果显示,HI中CD3^+T细胞主要优势TCR谱系是Vβ2、Vβ3、Vβ8、Vβ9、Vβ5. 1、Vβ13. 1和Vβ13. 2;而在CD3^+CD4^+T细胞中的优势利用主要集中在Vβ2、Vβ3、Vβ8、Vβ9、Vβ5. 1和Vβ13. 1;在CD3^+CD8^+T细胞中则主要为Vβ1、Vβ2、Vβ3、Vβ9、Vβ13. 1和Vβ13. 2等亚家族。进一步分析显示PD-1^+细胞在HI的CD3^+、CD3^+CD4^+和CD3^+CD8^+T细胞24个TCR Vβ亚家族中均可以检测到,PD-1^+细胞在T细胞各亚群中分布频率有个体差异,在CD4^+T细胞中,以Vβ5. 2^+T细胞的分布频率较高,而在CD8^+T细胞中,以Vβ11^+和Vβ13. 6^+T细胞的分布频率较高。结论:本项工作利用健康人样本成功建立了多色荧光流式细胞术检测T细胞亚群TCR Vβ谱系中免疫抑制性受体PD-1分子免疫表型的方法,为进一步应用于分析白血病等病人TCR Vβ谱系的免疫抑制特点提供了新方法。

AIM:To establish the method for detecting the immunophenotype of immunosuppressive receptor programmed cell death protein1(PD-1)in T-cell receptor(TCR)Vβrepertoire of CD3^+,CD4^+and CD8^+T-cell subsets,therefore to evaluate the distribution of PD-1in T-cell repertoire from human peripheral blood(PB).METHODS:The PB samples from10cases of healthy individuals(HI)were collected.Using multi-colored fluorescence flow cytometry,the distribution frequency of PD-1in TCR Vβrepertoire was detected with a wide panel of anti-CD45,anti-CD3,anti-CD4,anti-CD8,anti-PD-1and24anti-TCR Vβrepertoire(IOTest■Beta Mark TCR VβRepertoire Kit,containing8tubes which labeled A-H,each tube is a composite antibody of FITC and PE coupling,each cocktail contains antibodies direc-ted to3different Vβsubfamilies)monoclonal antibodies.RESULTS:The total number of the24TCR Vβrepertoire detected in CD3^+,CD3^+CD4^+and CD3^+CD8^+T cells from10cases of HI was consistent with the Quick Reference Card data provided by the kit.The preliminary results showed that the frequency of Vβusage in CD3^+,CD4^+and CD8^+T cells was different.High usage of Vβ2,Vβ3,Vβ8,Vβ9,Vβ5.1,Vβ13.1and Vβ13.2was found in CD3+T cells,while high usage of Vβ2,Vβ3,Vβ8,Vβ5.1,Vβ9and Vβ13.1in CD3^+CD4^+T cells,and high usage of Vβ1,Vβ2,Vβ3,Vβ9,Vβ13.1and Vβ13.2in CD3^+CD8^+T cells were also observed.Further analysis showed that the expression of PD-1was detected in all24TCR Vβsubfamilies of CD3^+,CD3^+CD4^+and CD3+CD8+T cells.The higher frequency of PD-1^+T cells was CD4^+Vβ5.2^+T cells,whereas the higher frequency of PD1^+T cells in CD8^+Vβ11^+and CD8^+Vβ13.6^+T cells was detected.CONCLUSION:The method for detection of the immunosuppressive receptor PD-1in TCR Vβrepertoire of T-cell subsets is successfully established,which provides a new method for further analysis of immunosuppressive characteristics of TCR Vβrepertoire in the patients with leukemia.