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

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