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Establishment of activation and differentiation of memory B cells into antigen-specific secreting cells from peripheral blood mononuclear cells in vitro

埃利斯波特 外周血单个核细胞 抗体 CD40 抗原 免疫学 分子生物学 乙型肝炎表面抗原 记忆B细胞 体外 化学 生物 B细胞 细胞毒性T细胞 乙型肝炎病毒 病毒 生物化学
作者
Dongmei Bi,Xiaojian Han,Jianing Shi,Ye Liu
出处
期刊:Int J Immunol 卷期号:38 (4): 305-310
标识
DOI:10.3760/cma.j.issn.1673-4394.2015.04.001
摘要

Objective To isolate antigen specific antibody-secreting cells ( plasma cells ) for development of antibody drug, we established efficient methods for the activation and differentiation of memory B cells into plasma cells in human peripheral blood lymphocytes (PBMCs) in vitro. Methods PBMCs were isolated from peripheral blood of two healthy donors received hepatitis B vaccine (named Z and L, vaccination time for 3 years and 25 years, respectively.) .We actived and induced PBMCs by adding cytokines(IL-2 and IL-4) and TLR activators (CpG or R848) for 6 days.Secreted antibodies in the culture supernatans were measured by enzyme-linked immunosorbent assay (ELISA). Antibody secreting cells (ASCs) were detected by enzyme-linked immuno spot assay (ELISPOT). Results IgG concentrations in group C treated with CpG DNA 2006 combined with anti-CD40 antibody and IL-2 and in group R treated with R848 combined with IL-2 were 80.87 ng/mL and 85.97 ng/mL, respectively.These IgG levels were significantly higher copmpared with that in control group(t=23.318, t=60.639, both P<0.05). The results showed that group C and R were efficiently actived, and the group R was significantly more than the group C. Both activation methods could induce B cell differentiation into plasma cells in vitro.Further, to analyze whether antigen specific plasma cells could be induced using the established motheds, we activated memory B cells in PBMCs vaccinated with hepatitis B vaccine.We detected HBsAg specific IgG secreting cells at different cell densities(104 to 105 cells/culture), the results showed that the group R was significantly more than the control group(t=5.031, t=11.561, both P<0.05). Conclusion We established the optimal method for B cell activation in vitro.This method could efficiently induce activation and differentiation of memory B cells from PBMCs into antigen-specific plasma cells. Key words: Memory B lymphocytes; Antigen specific antibody secreting cells; In vitro activation; Peripheral blood mononuclear cells
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