Figure 2.
Division-linked Id3 down-regulation is required for plasma cell differentiation. (a) Id3 expression in B cells isolated from Id3GFP/+ reporter mice gated according to their division history or plasma blast phenotype after 4 d in culture with LPS and IL-4 (left), geometric means of fluorescence intensity (GMFI) per division (right). (b) Id3 transcript amounts identified by RNAseq across cell divisions for WT B cells (division 0–7) and in vitro differentiated plasmablasts (PB). (c and d) B cells were transduced with either a control vector or a vector that forces overexpression of Id3. Representative flow cytometric analysis (c) after culture in LPS and IL-4 for 4 d. Frequency of plasmablasts (Syndecan-1+) and switched (IgG1+) cells within the retrovirally transduced population identified by Cherry fluorescence, gated on divided cells. Quantification of plasma blasts as percentage of B cells ***, P = 0.0002 (d). Flow cytometric analyses in a and c are representative of three to seven independent experiments with a total of three to seven biological replicates. Data in d are cumulative of seven independent experiments. Graphs show mean ± SEM; statistical significance (d) was determined by Student’s t test.