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.SIS-3 cost 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 MNS biological activity 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.
