Ex vivo PC differentiation system using B1-8^hisplenic B cells. (A) The dynamics of IRF4 expression and class-switched IgG1 frequency in response to differing BCR signaling intensities. Splenic B cells were purified from B1-8^hi mice and stimulated with IL-2, 4, and 5 and CD40L with indicated concentrations of NP(40)-ficoll. IRF4 expression was analyzed at 72 hours with costaining of CD138 and IgG1. Data are representative of 2 independent experiments. (B) Heat map showing alteration of gene expression along PC differentiation. B1-8^hi splenic B cells were isolated and stimulated with IL-2, 4, and 5, CD40L, and 10⁻² ng/mL of NP(40)-ficoll, and messenger RNA (mRNA) was extracted at indicated time followed by RNA-seq. Cluster 1 (up-late), genes upregulated along differentiation; cluster 2 (up-early), genes transiently upregulated around 60 hours; cluster 3 (down), genes downregulated along differentiation. Each cluster contains 402, 60, and 603 genes with indicated genes, respectively. (C) Heat maps showing transcripts of Aicda from panel B and RT-PCR of germ line and postswitched immunoglobulin gene. For RT-PCR, results are presented relative to the abundance of transcripts encoding β2-microglobulin (Β2m), and the average expression is from 1 experiment using 3 mice. (D) Heat maps showing transcripts of indicated IRF4 direct target genes with that of Irf4 and Irf8 from panel B. For panels B-D, genes are indicated with each color: red, cluster 1; green, cluster 2; blue, cluster 3, respectively.