In treated, but not in DprE1-IN-2 web control mice. B and T cells stained respectively with B220 and CD3 and counterstained with dapi (E) ZK 36374 biological activity plasma BAFF levels elevated in treated mice. (F) CD20 mRNA expression decreased in treated mice. n = 7? mice per group * p,0.05 control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gBAFFR-mab Treatment in Atherosclerosis ManagementFigure 2. Body weight, plasma lipids, B1a and other lymphocytes 18325633 unchanged in ApoE2/2 mice treated with anti-BAFFR antibody. A) Body weights, (B) Plasma lipids and (C) Peritoneal CD22+CD5+ B1a cells. (D) Peritoneal CD22+, CD5+ B1a B cells identified by FACS analysis. Red circle 12926553 represents CD22+ CD5+ B1a cells (E) CD4+, CD8+, NK1.1+ (NK), NK1.1+ TCRb+ (NKT) and CD4+ CD25+ foxp3+ regulatory T cells (T-reg) in spleens. n = 7? mice per each group control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gBAFFR-mab Treatment in Atherosclerosis ManagementFigure 3. Atherosclerosis ameliorated in ApoE2/2 mice treated with anti-BAFFR antibody accompanied by immunohistochemical demonstration of reduced B cells, CD4+T cells, CD8+T cells and IL1b in aortic lesions. (A) Oil Red-O stained lipid accumulation and total lesion area at aortic roots (B) CD68+ macrophage accumulation. Immunohistochemical analysis show decreased CD19+ B cells, CD4+ T cells, CD8+ T cells (C) and IL1b (D) in atherosclerotic lesions. n = 7? mice per each group * p,0.05 control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gBAFFR-mab Treatment in Atherosclerosis ManagementFigure 4. Reduced aortic inflammation and plasma immunoglobulins in anti BAFFR antibody treated- ApoE2/2 mice. Real-time PCR analysis shows (A) Cytokines- IL1b, TGFb, TNFa and IFNc- decreased, but (B) MCP1, MIF and VCAM were unaffected. ELISAs show (C) plasma total immunoglobulins decreased and (D) MDA-oxLDL specific antibodies unaffected. n = 7? mice per each group * p,0.05 control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gBAFFR-mab Treatment in Atherosclerosis ManagementBAFFR-mab Treatment in Atherosclerosis ManagementFigure 5. Anti-BAFFR antibody selectively depletes mature B cells, not B1a cells in ApoE2/2 mice with established atherosclerosis. (A) ApoE2/2 mice fed a HFD for 6 weeks to generate atherosclerosis, followed by three doses of anti-BAFFR antibody while feeding a HFD for a further 6 weeks. At end of experiment, FACS analysis show (B-C) mature B2 B cells in spleens selectively depleted. Representative FACS image show (C) mature and immature B cells in spleen stained with CD93 and CD19 antibodies Blue circle represents CD93+ CD19+ immature B cells and red circle CD932 CD19+ mature B cells. (D) Spleen B cell zones disrupted in BAFFR-depleted mice compared to control group. (E) Plasma levels of BAFF determined by ELISA increased in anti-BAFFR-antibody treated ApoE2/2 mice. (F) mRNA expression of CD20 in spleens reduced in BAFFR-treated mice. n = 7? mice per each group * p,0.05 control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gCD4 and CD8 were manually counted under light microscopy and corrected to total lesion areas as quantified by Optima software [12].CD20 (AS) 59-GACAGAATGCCCAAGAACAC-Statistical AnalysisStatistical significance was calculated by 2-tailed Student t test or Mann-Whitney U test, depending on whether the data were normally distributed, as asse.In treated, but not in control mice. B and T cells stained respectively with B220 and CD3 and counterstained with dapi (E) Plasma BAFF levels elevated in treated mice. (F) CD20 mRNA expression decreased in treated mice. n = 7? mice per group * p,0.05 control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gBAFFR-mab Treatment in Atherosclerosis ManagementFigure 2. Body weight, plasma lipids, B1a and other lymphocytes 18325633 unchanged in ApoE2/2 mice treated with anti-BAFFR antibody. A) Body weights, (B) Plasma lipids and (C) Peritoneal CD22+CD5+ B1a cells. (D) Peritoneal CD22+, CD5+ B1a B cells identified by FACS analysis. Red circle 12926553 represents CD22+ CD5+ B1a cells (E) CD4+, CD8+, NK1.1+ (NK), NK1.1+ TCRb+ (NKT) and CD4+ CD25+ foxp3+ regulatory T cells (T-reg) in spleens. n = 7? mice per each group control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gBAFFR-mab Treatment in Atherosclerosis ManagementFigure 3. Atherosclerosis ameliorated in ApoE2/2 mice treated with anti-BAFFR antibody accompanied by immunohistochemical demonstration of reduced B cells, CD4+T cells, CD8+T cells and IL1b in aortic lesions. (A) Oil Red-O stained lipid accumulation and total lesion area at aortic roots (B) CD68+ macrophage accumulation. Immunohistochemical analysis show decreased CD19+ B cells, CD4+ T cells, CD8+ T cells (C) and IL1b (D) in atherosclerotic lesions. n = 7? mice per each group * p,0.05 control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gBAFFR-mab Treatment in Atherosclerosis ManagementFigure 4. Reduced aortic inflammation and plasma immunoglobulins in anti BAFFR antibody treated- ApoE2/2 mice. Real-time PCR analysis shows (A) Cytokines- IL1b, TGFb, TNFa and IFNc- decreased, but (B) MCP1, MIF and VCAM were unaffected. ELISAs show (C) plasma total immunoglobulins decreased and (D) MDA-oxLDL specific antibodies unaffected. n = 7? mice per each group * p,0.05 control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gBAFFR-mab Treatment in Atherosclerosis ManagementBAFFR-mab Treatment in Atherosclerosis ManagementFigure 5. Anti-BAFFR antibody selectively depletes mature B cells, not B1a cells in ApoE2/2 mice with established atherosclerosis. (A) ApoE2/2 mice fed a HFD for 6 weeks to generate atherosclerosis, followed by three doses of anti-BAFFR antibody while feeding a HFD for a further 6 weeks. At end of experiment, FACS analysis show (B-C) mature B2 B cells in spleens selectively depleted. Representative FACS image show (C) mature and immature B cells in spleen stained with CD93 and CD19 antibodies Blue circle represents CD93+ CD19+ immature B cells and red circle CD932 CD19+ mature B cells. (D) Spleen B cell zones disrupted in BAFFR-depleted mice compared to control group. (E) Plasma levels of BAFF determined by ELISA increased in anti-BAFFR-antibody treated ApoE2/2 mice. (F) mRNA expression of CD20 in spleens reduced in BAFFR-treated mice. n = 7? mice per each group * p,0.05 control IgG treated mice; BAFFR-antibody treated mice. doi:10.1371/journal.pone.0060430.gCD4 and CD8 were manually counted under light microscopy and corrected to total lesion areas as quantified by Optima software [12].CD20 (AS) 59-GACAGAATGCCCAAGAACAC-Statistical AnalysisStatistical significance was calculated by 2-tailed Student t test or Mann-Whitney U test, depending on whether the data were normally distributed, as asse.