PERK-eIF2α UPR signaling is not required for plasma cell differentiation. (A) IP–Western blot analysis of expression and activation of IRE1α and PERK in primary B cells upon LPS stimulation. B220⁺ primary B cells from the spleens of 2-month-old WT mice were cultured in vitro in the presence of LPS (20 μg/ml) for the indicated time intervals. To generate controls for IRE1α and PERK proteins, WT, ire1α^–/–, and perk^–/– MEFs and primary B cells were treated with thapsigargin (Thap; 0.2 μM) for 3 hours. Equivalent amounts of cellular lysates were applied for each sample. p-, phosphorylated. (B–C) Kinetics of induction of spliced XBP1 and secreted IgM transcripts (B) and EDEM and CHOP transcripts (C) in primary B cells upon LPS treatment. Primary B cells were isolated from spleens of WT adult mice and were then stimulated with LPS (20 μg/ml) in vitro for the indicated time intervals. Levels of transcripts were determined by quantitative real-time RT-PCR. (D) FACS analysis of B220 and IgD in splenocytes from rag2^–/– mice reconstituted with WT (eIF2α SS) or eIF2α phosphorylation–defective (eIF2α AA) fetal liver cells. Fetal hematopoietic liver cells (4 × 10⁶) from E13.5 embryos were used for transplantion. (E) Western blot analysis of phosphorylated and total eIF2α proteins in the reconstituted B cells. B220⁺ cells were sorted from the spleens of rag2^–/– mice reconstituted with eIF2α AA or SS fetal liver and then treated with 0.2 μM thapsigargin for 3 hours. Thapsigargin-treated eIF2α AA and SS MEFs were used as controls. (F) Levels of serum IgM and IgG1 in the rag2^–/– mice reconstituted with eIF2α SS or eIF2α AA fetal liver cells. Blood samples were collected from the reconstituted rag2^–/– mice at 1 month after transplantation. Levels of serum IgM and IgG1 were determined by ELISA. (G) The proposed model depicts that IRE1α is required for both the early and the late stages of B lymphocyte development. At the very early stage, IRE1α plays a role in the induction of the rag1, rag2, and TdT genes; thus IRE1α is required for VDJ recombination and BCR formation. At the late stage, IRE1α kinase and RNase activities regulate the terminal differentiation of B cells to plasma cells through splicing of XBP1 mRNA.