人类全血中调节性B细胞检测技术的建立及应用
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摘要
建立了一种全血、多参数的流式检测技术用以分析人类外周血调节性B细胞(regulator B cells,Breg)相关分子标志物,即CD19~+TGF-β~+、CD19~+IL-10~+、CD19~+CD25~+,比较不同刺激条件CpG+PIB及LPS+PIB对检测结果的影响,并探讨Breg细胞与实体肿瘤的相关性.实验结果显示,人全血经5～6 h刺激因子刺激后能够有效地检测到Breg细胞,且两种检测条件均能够获得较好的检测效果,其中CpG+PIB刺激条件有更高的检测效率.应用该检测方法分别检测健康人20例及肿瘤患者60例,结果提示肿瘤患者CD19~+IL-10~+细胞比例(1.05±0.78)%与健康人(0.45±0.23)%相比显著升高(P=0.001);而肿瘤患者CD19~+TGF-β~+细胞比例(0.96±0.87)%与健康人(0.91±0.40)%相比没有显著差异(P=0.764),肿瘤患者CD19~+CD25~+细胞比例(18.16±10.57)%与健康人(18.83±6.22)%相比没有显著差异(P=0.729),但Ⅳ期肿瘤CD19~+TGF-β~+细胞比例(1.23±1.16)%以及CD19~+CD25~+细胞比例(23.41±11.70)%均高于健康组.该方法的建立为探讨Breg细胞与疾病免疫状态的相关性提供了一种可行的研究方法.
A whole blood and multi-parameter flow cytometry technique was developed to analyze the molecular markers of regulatory B cells(Breg) in human peripheral blood, including CD19~+TGF-β~+, CD19~+IL-10~+ and CD19~+CD25~+. The effects of different stimulating conditions, CpG+PIB and LPS+PIB, on Breg cells were compared. Furthermore, the correlation between Breg cells and solid tumors was explored. The experimental results show that human Breg cells can be effectively detected from peripheral blood cells after stimulation for 5-6 hours. Both stimulating conditions can obtain good results, however CpG+PIB have higher stimulating efficiency. Then, this method was applied in clinical samples for detecting Breg cells in 20 healthy people and 60 tumor patients. It showed that the proportion of CD19~+IL-10~+ cells in tumor patients(1.05±0.78)% was significantly higher than that in healthy people(0.45±0.23)%(P=0.001). Although, the proportion of CD19~+TGF-β~+ cells in tumor patients(0.96±0.87)% was not significantly different from that in healthy subjects(0.91±0.40)%(P=0.764), and the proportion of CD19~+CD25~+ cells in tumor patients(18.16±10.57)% was not significantly different from healthy people(18.83±6.22)%(P=0.729). However, we observed that stage IV tumor patients had higher proportion of CD19~+TGF-β~+ cells(1.23±1.16)% and CD19~+CD25~+ cells(23.41±11.70)% than healthy group. The establishment of this method may provide a feasible research method for exploring the correlation between Breg cells and immune status of diseases.
A whole blood and multi-parameter flow cytometry technique was developed to analyze the molecular markers of regulatory B cells(Breg) in human peripheral blood, including CD19~+TGF-β~+, CD19~+IL-10~+ and CD19~+CD25~+. The effects of different stimulating conditions, CpG+PIB and LPS+PIB, on Breg cells were compared. Furthermore, the correlation between Breg cells and solid tumors was explored. The experimental results show that human Breg cells can be effectively detected from peripheral blood cells after stimulation for 5-6 hours. Both stimulating conditions can obtain good results, however CpG+PIB have higher stimulating efficiency. Then, this method was applied in clinical samples for detecting Breg cells in 20 healthy people and 60 tumor patients. It showed that the proportion of CD19~+IL-10~+ cells in tumor patients(1.05±0.78)% was significantly higher than that in healthy people(0.45±0.23)%(P=0.001). Although, the proportion of CD19~+TGF-β~+ cells in tumor patients(0.96±0.87)% was not significantly different from that in healthy subjects(0.91±0.40)%(P=0.764), and the proportion of CD19~+CD25~+ cells in tumor patients(18.16±10.57)% was not significantly different from healthy people(18.83±6.22)%(P=0.729). However, we observed that stage IV tumor patients had higher proportion of CD19~+TGF-β~+ cells(1.23±1.16)% and CD19~+CD25~+ cells(23.41±11.70)% than healthy group. The establishment of this method may provide a feasible research method for exploring the correlation between Breg cells and immune status of diseases.
引文
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[9] Blair P A,Nore F,et al.CD19+ CD24hi CD38hi B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic lupus erythematosus patients[J].immunity,2010,32(1):129-140.
[10] Flores-Borja F,Bosma A,Ng D,et al.CD19+ CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation[J].Science translational medicine,2013,5(173):173ra23.
[11] Amu S,Tarkowski A,D?Rner T,et al.The Human Immunomodulatory CD25+ B Cell Population belongs to the Memory B Cell Pool[J].Scandinavian journal of immunology,2007,66(1):77-86.
[12] Kessel A,Haj T,Peri R,et al.Human CD19+ CD25hi gh B regulatory cells suppress proliferation of CD4+ T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells[J].Autoimmunity reviews,2012,11(9):670-677.
[2] Matsushita T.Regulatory B Cells in Mouse Models of Systemic Lupus Erythematosus (SLE)[J].Methods in molecular biology (Clifton,N.J.),2014,1190:195-205.
[3] Balkwill F,Montfort A,Capasso M.B regulatory cells in cancer[J].Trends in Immunology,2013,34(4):169-173.
[4] Zhou J,Min Z,Zhang D,et al.Enhanced frequency and potential mechanism of B regulatory cells in patients with lung cancer[J].Journal of Translational Medicine,2014,12(1):304-314.
[5] De A C,Tejeraalhambra M,Alonso B,et al.New regulatory CD19(+)CD25(+) B-cell subset in clinically isolated syndrome and multiple sclerosis relapse.Changes after glucocorticoids[J].Journal of Neuroimmunology,2014,270(1-2):37-44.
[6] Inoue S,Leitner W W,Golding B,et al.Inhibitory effects of B cells on antitumor immunity[J].Cancer research,2006,66(15):7741-7747.
[7] Evans J G,Chavez-Rueda K A,Eddaoudi A,et al.Novel suppressive function of transitional 2 B cells in experimental arthritis[J].The Journal of Immunology,2007,178(12):7868-7878.
[8] Yanaba K,Bouaziz J D,Haas K M,et al.A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses[J].Immunity,2008,28(5):639-650.
[9] Blair P A,Nore F,et al.CD19+ CD24hi CD38hi B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic lupus erythematosus patients[J].immunity,2010,32(1):129-140.
[10] Flores-Borja F,Bosma A,Ng D,et al.CD19+ CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation[J].Science translational medicine,2013,5(173):173ra23.
[11] Amu S,Tarkowski A,D?Rner T,et al.The Human Immunomodulatory CD25+ B Cell Population belongs to the Memory B Cell Pool[J].Scandinavian journal of immunology,2007,66(1):77-86.
[12] Kessel A,Haj T,Peri R,et al.Human CD19+ CD25hi gh B regulatory cells suppress proliferation of CD4+ T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells[J].Autoimmunity reviews,2012,11(9):670-677.