Bortezomib treatment enhances T cell apoptosis by inhibiting NF-κB activation.

<p>(<b>A</b>) The percentages of Annexin-V/7-AAD⁺ CD4⁺ and CD8⁺ T cells in mesenteric lymph nodes 7 days after the administration of DSS. Bar graphs indicate mean (± SEM) percentages of mesenteric lymph node Annexin-V/7-AAD⁺ CD4⁺ and CD8⁺ T cells following bortezomib or control treatment in one representative experiment with 4 mice per group. (<b>B</b>) IκBα mRNA expression in mesenteric lymph nodes CD4⁺ and CD8⁺ T cells. Transcript levels were quantified by real-time PCR analysis and were normalized with an internal control. Values represent means (± SEM) from ≥4 mice of each group. A, B) Significant differences between sample means are indicated; *<i>P</i><0.05; **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Bortezomib treatment affects cytokine production in DSS-induced colits.

<p>Cytokine mRNA expression in the colon (<b>A</b>) and mesenteric lymph nodes (<b>B</b>) in mice treated with bortezomib or control during DSS-induced colitis. Colon tissue and mesenteric lymph nodes were collected from control- or bortezomib-treated mice 7 days following DSS administration. Transcript levels were quantified by real-time PCR analysis and were normalized with an internal control. Values represent means (± SEM) from ≥4 mice of each group. Significant differences between sample means are indicated; *<i>P</i><0.05, **<i>P</i><0.01. Results represent one of two independent experiments producing similar results.</p

Bortezomib suppressed the severity of DSS-induced colitis.

<p>Mice ingested either DSS solution or normal drinking water. Mice were treated with 200 µl of bortezomib (0.75 mg/kg) or PBS (control) intravenously twice weekly, starting 2 days prior to DSS administration. The severity of intestinal injury was evaluated by quantitatively measuring body weight (<b>A</b>) and DAI scores (<b>B</b>). DAI scores were based on weight loss, stool consistency, and bleeding. Values represent means (±SEM) from ≥4 mice per group. Significant differences between sample means are indicated: *<i>P</i><0.05; **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Profile of infiltrating cells in the colon and mesenteric lymph nodes during DSS-induced colitis in mice treated with bortezomib or control.

<p>(<b>A</b>) The numbers of neutrophils, CD4⁺ T cells, CD8⁺ T cells, B220⁺ B cells, and F4/80⁺ macrophages per one field of view (×200) in the colon were counted. (<b>B</b>) Bortezomib affects CD4⁺ and CD8⁺ T cell numbers in mesenteric lymph nodes during DSS-induced colitis. Bar graphs indicate CD4⁺ T cells, CD8⁺ T cells, and B220⁺ cells in mesenteric lymph nodes 7 days after induction of colitis. A, B) Values represent means (± SEM) from ≥4 mice per group. Significant differences between sample means are indicated: *<i>P</i><0.05. Similar results were obtained in at least two independent experiments.</p

Bortezomib treatment reduces IFN-γ production by CD4⁺ and CD8⁺ T cells in mesenteric lymph nodes during DSS-induced colitis.

<p>IFN-γ production by mesenteric lymph node CD4⁺ (<b>A</b>) or CD8⁺ (<b>B</b>) T cells 7 days following DSS administration as determined by intracellular cytokine staining with flow cytometry analysis. Bar graphs indicate mean (± SEM) percentages and numbers of draining lymph node IFN-γ-producing CD4⁺ or CD8⁺ T cells following bortezomib or control treatment in one representative experiment with three mice per group. Significant differences between PBS-treated mice versus other group are indicated; *<i>P</i><0.05, **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

CD154^TGCD22^−/− B10 cells are regulatory.

<p>(<b>A</b>) B10 cells in CD154^TGCD22^−/− mice are found predominately within the spleen CD1d^hiCD5⁺ B cell population. Splenocytes from WT and CD154^TGCD22^−/− mice were cultured for 5 h with L+PIM to induce IL-10 expression, with the cells analyzed for cell surface CD19, CD1d and CD5, and intracellular IL-10 expression. CD19⁺IL-10⁺ and CD19⁺IL-10⁻ B cells (left panels, gated regions) were further gated to show relative CD1d and CD5 expression (merged dot plots, right panels). Bar graphs show the mean (±SEM) B10 cell frequencies within the indicated populations for ≥4 mice of each genotype based on the gated regions indicated in the merged dot plots. Mean B10 cell frequencies significantly different between cell populations from the same genotype are indicated by asterisks (**p≤0.01), and for the same population between genotypes by crosses (††p<0.01). (<b>B</b>) Analysis of CD154^TGCD22^−/− spleen B cell purity within the CD1d^hiCD5⁺ (B10-rich) and CD1d^loCD5⁻ populations following cell sorting of splenocytes stained for CD19, CD1d and CD5. These cells were subsequently used for the adoptive transfer experiments described in (C). (<b>C</b>) B10 cells from CD154^TGCD22^−/− mice reduce EAE disease severity. Purified CD1d^hiCD5⁺ or CD1d^loCD5⁻ spleen B cells from naïve CD154^TGCD22^−/− mice (B) were either adoptively transferred into WT recipient mice immediately (non-activated) or were cultured with agonistic CD40 mAb for 48 h with LPS added during the final 5 h of culture before transfer. Other recipient mice received PBS alone (Control). One day after cell transfers, EAE was induced by MOG immunization. Values represent mean (±SEM) clinical EAE scores from 5 mice per group, with significant differences from PBS control mice indicated: *p<0.05.</p

Impaired IgG and GC responses in CD154^TGCD22^−/− mice.

<p>(<b>A</b>) Serum IgM and IgG levels of 4 and 12 mo-old WT, CD22^−/−, CD154^TG, and CD154^TGCD22^−/− mice. Symbols represent serum concentrations for individual mice as determined by ELISA, with means indicated by horizontal bars. (<b>B</b>) Serum autoAbs reactive with dsDNA, ssDNA, or histone proteins in 12 mo-old mice. ELISA OD values for IgM (upper panels) and IgG (lower panels) autoAbs are shown for individual mice, with means indicated by horizontal bars. Sera from 2 mo-old WT C57BL/6 and 6 mo-old MLR<i>lpr</i> mice were used as negative and positive controls, respectively. (<b>C</b>) Impaired IgG responses to a TD Ag. WT (<i>n</i> = 3), CD22^−/− (<i>n</i> = 4), CD154^TG (<i>n</i> = 6), and CD154^TGCD22^−/− (<i>n</i> = 8) mice were immunized with DNP-KLH in adjuvant on day 0, and boosted on day 21. The graph shows mean (±SEM) DNP-specific IgG levels as determined by ELISA. Images on the right represent immunofluorescence staining of frozen spleen sections from all genotypes harvested 7 days after the boost phase of DNP-KLH immunization. Merged images show the presence of B220⁺ B cells (red) and GC GL7⁺B220⁺ B cells (yellow). Enlarged regions from these sections indicate typical GC structures present within the follicles of WT and CD22^−/− mice, and detectable GL7⁺B220⁺ B cells within the follicles of CD154^TG mice, but not in CD154^TGCD22^−/− mice (representative regions are shown for comparison). (A–C) Means significantly different from WT are indicated by asterisks (*p≤0.05, **p≤0.01), and between other indicated groups by crosses (†p<0.05, ††p<0.01). (<b>D</b>) Reduced GC B cells in CD154^TGCD22^−/− mice as quantified by flow cytometry analysis. Mice were immunized with NP-CGG in alum, with spleens analyzed for GL7⁺B220⁺ B cells on day 10. Contour plots show mean (±SEM) GL7⁺ cell frequencies among total B220⁺ cells from <i>n</i> = 3 mice of each genotype. Bar graphs show mean (±SEM) GL7⁺ B cell numbers from naive (open bars) and immunized (filled bars) mice. In the contour plots, mean B10 cell frequencies significantly lower than those of WT mice are indicated by asterisks (**p≤0.01). In the bar graphs, means significantly different between naïve and immunized mice of the same genotype are indicated by asterisks (*p≤0.05); crosses for CD154^TGCD22^−/− mice indicate that mean GL7⁺ cell numbers were significantly reduced relative to all other genotypes (††p<0.01).</p

Lymphocyte subsets in WT, CD22^−/−, CD154^TG, and CD154^TGCD22^−/− mice^a.

a<p>Numbers represent mean (±SEM) values from ≥4 littermates of each genotype. Asterisks indicate values significantly different from WT mice (*p<0.05; **p<0.01). PLN, peripheral LN; MLN, mesenteric LN; PC, peritoneal cavity.</p>b<p>B10 and B10pro cells were identified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022464#pone-0022464-g003" target="_blank">Figure 3</a>.</p>c<p>IL-10-competent T cells were identified following 5 h stimulation with PMA/ionomycin.</p

Spleen B10 and B10pro cells expand dramatically in CD154^TGCD22^−/− mice.

<p>(<b>A</b>) Blood, spleen, inguinal LN, and peritoneal cavity B10 cell numbers in WT, CD22^−/−, CD154^TG, and CD154^TGCD22^−/− mice. Leukocytes from the indicated tissues were cultured with LPS, PMA, and ionomycin for 5 h to induce IL-10 production, with monensin included in the cultures to block IL-10 secretion (L+PIM stimulation). The cells were then stained for cell surface CD19 and cytoplasmic IL-10 with flow cytometry analysis. Monensin treated cells served as negative controls for IL-10 expression. Representative contour plots gated on CD19⁺ cells are shown with the percentage of IL-10⁺ cells indicated. Bar graphs represent mean (±SEM) B10 cell frequencies (%) and numbers (#) from ≥3 mice of each genotype. (<b>B</b>) Similar B10 cell surface phenotypes among mouse genotypes. Splenocytes were cultured with L+PIM for 5 h and then stained for cell surface molecules and cytoplasmic IL-10. Histograms indicate cell surface molecule expression by IL-10⁺ (thick lines) and IL-10⁻ (thin lines) B cells. Dashed histograms represent isotype-matched control mAb staining. Results are representative of ≥3 mice of each genotype analyzed. (<b>C</b>) Spleen B10+B10pro cell frequencies and numbers. Splenocytes were cultured with an agonistic CD40 mAb, with L+PIM added during the final 5 h of 48 h cultures. Cytoplasmic IL-10⁺ B cells were identified as in (A). Bar graphs show means (±SEM) from ≥3 mice of each genotype. (A,C) Significant differences from WT mice are indicated: *p<0.05, **p<0.01. Means significantly different between CD154^TGCD22^−/− and CD154^TG mice are indicated: †p<0.05, ††p<0.01.</p

B cell development in CD154^TGCD22^−/− and CD154^TG mice.

<p>(<b>A</b>) B cell CD154 expression in WT, CD22^−/−, CD154^TG and CD154^TGCD22^−/− mice. Blood and spleen B220⁺ B cells were assessed for CD154 expression by immunofluorescence staining with flow cytometry analysis. Dashed lines delineate borders between CD154⁺ and CD154⁻ cells as determined using WT lymphocytes. (<b>B</b>) Ectopic CD154 expression is B cell-restricted in CD154^TGCD22^−/− mice. CD154 expression by blood and spleen B220⁺ and B220⁻ mononuclear cells was assessed by immunofluorescence staining with flow cytometry analysis. Results represent those obtained in 6 pairs of mice. (<b>C</b>) Increased CD5 expression by B cells from CD22^−/− and CD154^TGCD22^−/− mice. Spleen B cells were assessed for CD5 expression by immunofluorescence staining. Gates indicate percentages of CD5⁺ B cells among total B220⁺ cells. (<b>D</b>) Spleen CD1d^hiB220⁺ B cell localization. Tissue sections from WT, CD22^−/−, CD154^TG, and CD154^TGCD22^−/− mice were stained with B220 (FITC, green) and CD1d (PE, red) mAbs. Merged images highlight CD1d^hiB220⁺ cells (yellow). (A–C) Results are representative of ≥3 mice of each genotype. (<b>E</b>) Spleen CD1d^hiB220⁺ B cell numbers in WT, CD22^−/−, CD154^TG and CD154^TGCD22^−/− mice as determined by immunofluorescence staining. Results represent the mean (±SEM) from ≥3 mice of each genotype. (<b>F</b>) CD154^TGCD22^−/− B cells are hyper-responsive to CD40 signals. Spleen B cells from WT, CD22^−/−, CD154^TG, and CD154^TGCD22^−/− mice were cultured with mitogenic CD40 mAb or anti-IgM Ab for 72 h, with [³H]-thymidine incorporation assessed during the final 18 h of culture. Values represent means (±SEM) of triplicate cultures. (E–F) Means significantly different from WT values are indicated by asterisks (**p<0.01), and between other genotypes by crosses (††p<0.01). (<b>G</b>) CD154^TGCD22^−/− B cell proliferation in response to LPS stimulation. Cell division of CFSE-labeled B cells from WT, CD22^−/−, CD154^TG and CD154^TGCD22^−/− mice following LPS stimulation was quantified after 72 h in culture by flow cytometry. (F–G) Results are representative of 2 independent experiments with similar results. (<b>H</b>) Enhanced survival by CFSE-labeled B cells from CD154^TG and CD154^TGCD22^−/− mice after 10 days in culture without mitogenic stimulation as assessed by flow cytometry. Results represent 3 independent experiments producing similar results.</p