Induction of CD40 Expression and Enhancement of Monoclonal Antibody Production on Murine B Cell Hybridomas by Cross‐Linking of IgG Receptors

Abstract

The 55-6 murine B cell hybridoma line not constitutively expressing CD40 was treated with increasing amounts of intact anti-mouse surface immunoglobulin G antibody (anti-mIgG) either not preincubated or preincubated for 48 h with lipopolysaccharide (LPS). In vitro, cross-linking of surface immunoglobulin G (sIgG) with the whole molecule of anti-IgG antibodies induced the expression of CD69, CD40, and CD19 surface antigens on 55-6 cells. The effect of sIgG ligation was dose-dependent, and preincubation with LPS enhanced their responsiveness to anti-mIgG stimulation. The expression of these surface molecules reached the maximum value during the first part of the cell cycle, corresponding to the position of the G1 peak of the DNA distribution. Stimulation of cells with anti-mIgG did not induce changes either in the number of viable cells or in the fraction of cells undergoing proliferation (mitosis). However, preincubation of 55-6 cells with LPS for 48 h before stimulation with anti-mIgG increased both the maximum specific growth rate (micromax) and the percentage of cells in the G2/M phase, in comparison with non-preincubated cells. Moreover, on cells preincubated with LPS prior to anti-mIgG treatment, specific IgG2a production rate was enhanced significantly compared to that obtained in control cultures. The correlation between the antibody production rate and the amount of IgG that is detectable on the cell surface was analyzed by flow cytometry. A good correlation between secreted and surface IgG was observed, and the results of cell cycle analyses demonstrated that the 55-6 hybridoma cell line has a substantially higher sIgG content in G1 phase.

Effects of Synchronization on CD40 Expression and Antibody Production in Hybridoma Cells Stimulated with Anti-mIgG

“…In previous experiments with the 55-6 hybridoma cell line (16) we demonstrated that both CD40 expression and surface IgG content, as detected with immunofluorescent techniques, were maximum during the G1 cell cycle phase on cells stimulated with anti-mIgG. Moreover, a correlation between the specific antibody secretion rate and surface IgG content was observed.…”

“…The cell line used, a mouse-mouse B cell hybridoma, designated 55-6 (ATCC: CRL-2156), produces IgG2a monoclonal antibodies to human immunodeficiency virus (HIV) glycoprotein 120 (gp120). The cells were grown in static T-culture flasks in the medium previously described by Martín-López et al, (16).…”

Effects of hydroxyurea on monoclonal antibody production induced by anti-mIgG and LPS stimulation on murine B cell hybridomas

“…These data showed that LPS and antimIgG, which are widely considered as activatingagent of B cells (Kearney and Lawton 1975;Klaus et al 1984;Lenschow et al 1991;Oliver et al 1999), may trigger the same metabolic routes in hybridoma as in B cells to increase their proliferation and mAbs production. Moreover, this stimulatory effect of LPS and anti-mIgG was higher during G1 phase of the cell cycle (Martín-López et al 2007b).…”

“…The cells were grown in static T-culture flasks in the medium previously described (Martín-López et al 2007b). …”

“…To assess the changes in cell-cycle distribution cellular DNA content were analyzed by PI staining and flow cytometry. The following protocols were previously described (Martín-López et al 2007b). Data analysis employed software from Coulter Corporation (System II TM ) and histograms were analysed with the algorithm developed by Watson et al (1987).…”