Figure 4. Immunoglobulin levels are decreased in in vitro generated Lgals1^−/− plasmablasts.

B220⁺ splenic B cells from WT and Lgals1^−/− mice were enriched by magnetic selection and cultured during 4 days in the presence of 1μg/ml of LPS in complete RPMI medium with 10% FCS. The percentage (A) and the absolute cell number (B) of B220^lowCD138⁺ PB generated from 10⁶ B220⁺ spleen cells 4 days after stimulation were evaluated by flow cytometry. (C) GAL1 expression in non-stimulated and in LPS-stimulated B cells was determined by Q-PCR at days 1 and 2 after stimulation. (D, E) IgM and IgG3 secretion in culture supernatants from WT and Lgals1^−/− cells were quantified by ELISA. Cultures were performed in culture medium supplemented with 10% (D) or 5% (E) FCS and cells were allowed to secrete Ig for 16h. (F) The number of antibody-secreted cells (ASC) obtained from 1000 viable plated cells after WT and Lgas1^−/− cultures performed in 10% and 5% FCS, was determined by ELISPOT. Data are representative of more than 5 independent experiments. (G) Absolute number of B220^low CD138⁺ PB generated from 10⁶ B220⁺ spleen cells in the presence of 5% FCS and after 4 days of LPS stimulation. (H) Incorporation of 3H thymidine at day 2 and 3 (evaluated in cpm) by LPS stimulated B220⁺ cells in the presence of 10% and 5% FCS. Open and black squares correspond to WT and Lgals1^−/− mice, respectively.

* indicates significance versus WT mice with p<0.05.