Female XBP1^+/+ and XBP1^CD19 mice were immunized i.p. with 100 µg MAP-DWEYS in CFA followed by booster immunizations with 100 µg MAP-DWEYS in IFA on days 14 and 28. (A and B) On day 35, serum was tested for anti-DWEYS (peptide; A) and anti-dsDNA (B) antibodies by ELISA of four serum dilutions. In both panels, each circle represents the mean ± SEM value for XBP1^+/+ (closed circles) or XBP1^CD19 (open circles) mice. (C) XBP1^+/+ and XBP1^CD19 mice from A and B were treated i.p. with 3 mg/kg LPS on days 52 and +54. Shown are representative images of IgG-specific IHC of kidney (top) and hippocampus (bottom) of mice 4–7 d after LPS treatment. XBP1^+/+ mice demonstrated anti-IgG binding to neurons in the CA1 region of the hippocampus. There was no anti-IgG binding in any of the XBP1^CD19 animals. Bars: (kidney) 200 µm; (hippocampus) 100 µm. All panels represent results from a single experiment with five mice per group. A second independent experiment reproduced these findings (not depicted).

XBP1^+/+ and XBP1^CD19 mice were immunized i.p. with a single 100-µg dose of NP-KLH in alum. (A) Representative flow cytometry of cell surface CD138 (y axis) and B220 (x axis) expression in splenocytes isolated from XBP1^+/+ and XBP1^CD19 mice at days 0, 5, and 14 after immunization. Mean percentage ± SEM of B220^int CD138⁺ cells is indicated. (B) B220^int CD138⁺ cells were isolated by FACS from a subset of splenocytes at day 5 after immunization. Cytospin preparations of cells were stained with modified Wright-Giemsa stain. (C) Splenocytes were isolated from mice on day 5 after immunization and incubated with Brefeldin A BODIPY, which selectively stains ER and Golgi organelles. Shown are representative flow cytometry histograms for B220^int CD138⁺ cells (solid line), B220⁺ CD138⁻ cells (long dashed line), and unstained cells (short dashed line). Brefeldin A-BODIPY mean fluorescence intensity (MFI) ± SEM is indicated for B220^int CD138⁺ cells (no difference in B220⁺ CD138⁻ cells). All experiments were performed with individual mice in at least three independent experiments.

XBP1^+/+ and XBP1^CD19 mice were immunized i.p. with a single 100-µg dose of NP-KLH in alum. At day 5 after immunization, splenocytes were isolated and sorted by FACS for B220⁺ CD138⁻ (B220) and B220^int CD138⁺ (CD138) populations. Panels show relative expression of indicated mRNA versus actin as measured by RT-PCR of cDNA prepared from indicated cell populations. In all panels, each bar represents the mean ± SEM value of individual XBP1^+/+ (open bar) and XBP1^CD19 (solid bar) mice in three independent experiments.

Memory B cells are present in normal numbers in XBP1^CD19 mice. (A, left) Representative expression of IgD and NP on [PI, CD4, CD8, F4/80, GR1]^neg splenocytes of day-14 mice immunized with Ribi (adjuvant) only (left) or NP-KLH in Ribi (right). Shown are XBP1^fl/fl CD19^+/+ (XBP1^+/+) mice (top) and XBP1^fl/fl CD19^cre/+ (XBP1^CD19) littermates (bottom). The inset represents the percentage of cells within profile (mean ± SEM; n = 3). (A, right) Total number of IgD^lo NP-specific B cells from day 0 (naive BALB/c), adjuvant only (day 14 Ribi only), day 5, and day 14 NP-KLH in Ribi immunized XBP1^+/+ mice (white columns) and XBP1^CD19 (black columns) mice (mean ± SEM; n = 3). (B, left) Representative expression of B220 and CD138 at the surface of IgD^lo NP-specific B cells at days 5 (left) and 14 (right) from XBP1^+/+ mice (top) and XBP1^CD19 littermates (bottom). The inset represents the percentage of cells within profile (mean ± SEM; n = 3). (B, right) Total number of NP-specific B cells compartment B220⁺, CD79b⁺ B220^lo, or CD138⁺ at day 5 (top) or day 14 (bottom) immunized XBP1^+/+ (white columns) and XBP1^CD19 (black columns) mice (mean ± SEM; n = 3; unpaired Student's t test; P > 0.05 for all data points). (C) [PI, CD4, CD8, F4/80, GR1]^neg IgD^lo NP⁺ CD138⁺ splenocytes of day-5 and day-14 immunized mice were sorted directly ex vivo into NP-specific Ig (IgM or IgG) revealing ELISPOT assays. Positives were scored manually under a dissection microscope. Each assay was done in triplicate from three separate animals (mean ± SEM; n = 3; unpaired Student's t test; *, P ≤ 0.05).