Article Figures & Data
Figures
- Fig 1
Treatment with MnTBAP reduces superoxide levels in activated B cells. Splenocytes from naïve C57BL/6 mice (A) or those infected with 2 × 105 PFU of LCMV Armstrong 8 days prior and treated with either vehicle or 5 mg/kg of MnTBAP (B) were incubated with HE and stained with anti-B220 and anti-IgD antibodies. The HE levels are plotted as histograms, with the MFI indicated in the upper right corners of the plots. (C) Quantification of HE levels in activated/memory phenotype B cells from multiple mice was done by dividing the HE MFI of activated memory phenotype B cells (B220+ IgDlo) in infected mice by the HE MFI of unactivated B cells (B220+ IgDhi) in naïve mice. Six mice were analyzed in a minimum of two independent experiments. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
- Fig 2
MnTBAP treatment results in decreased expansion of virus-specific IgM and IgG ASC during acute viral infection. C57BL/6 mice were treated with vehicle or 5 mg/kg of MnTBAP. Four hours later, mice were infected with 2 × 105 PFU of LCMV Armstrong. A maintenance dose of vehicle or MnTBAP was administered every 24 h for 8 days. At the indicated time points, mice were sacrificed and the numbers of virus-specific IgM (A and C) and IgG (B and D) ASC in the spleen and bone marrow were quantitated by ELISPOT assay. (E) Quantification of the number of plasmablasts in vehicle- and MnTBAP-treated mice following staining with anti-B220, anti-CD19, anti-IgD, and anti-CD138. (F) Naïve or LCMV-infected mice received a daily dose of vehicle or MnTBAP every 24 h for 8 days. On day 8, mice were sacrificed and spleens were removed. The total IgG ASC were quantitated by ELISPOT assay. (G) The number of LCMV-specific IgG memory B cells was quantified by limiting dilution assay at day 38 postinfection in vehicle- and MnTBAP-treated mice. (H) Serum virus-specific IgG antibody titer was determined on day 38 postinfection by ELISA. The dashed line represents the limit of detection. N.D., not detectable. (I) Affinity was determined by ELISA and was quantified as the concentration of NaSCN required to reduce the absorbance of serum virus-specific IgG by 50%. The averages and standard deviations are shown. Five to nine mice were analyzed in a minimum of two independent experiments. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
- Fig 3
MnTBAP treatment decreases the number of germinal center B cells. C57BL/6 mice were treated with either vehicle or 5 mg of MnTBAP/kg. After 4 h, mice were infected with 2 × 105 PFU of LCMV Armstrong. A maintenance dose was administered every 24 h for 8 days. At the indicated time points, mice were sacrificed, the spleen was removed, and cells were stained with anti-B220, anti-GL7, and anti-Fas. The numbers of B220+ GL7+ Fas+ germinal center B cells were quantitated, and the averages and standard deviations are shown. Five to six mice were examined in a minimum of two independent experiments. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
- Fig 4
Decreased cytokine production by virus-specific CD4+ T cells from mice treated with MnTBAP. C57BL/6 mice were treated with either vehicle or 5 mg of MnTBAP/kg. After 4 h, mice were infected with 2 × 105 PFU of LCMV Armstrong. A maintenance dose was administered every 24 h for 8 days. At the indicated time points, mice were sacrificed and the spleen was removed. Splenocytes were stimulated with GP61-80 peptide for 5 h at 37°C. Following stimulation, cells were stained with anti-CD4, anti-IFN-γ, and either anti-TNF-α (A) or anti-IL-2 (B). The dot plots are gated on CD4+ T cells, and the numbers in each plot indicate the percentage of CD4+ T cells present in the quadrants. (C) The number of IFN-γ+ CD4+ T cells was quantitated in the spleen. For each virus-specific CD4+ T cell population, the percentage of IFN-γ+ cells that produced TNF-α (D) and IL-2 (E) was determined. The averages and standard deviations are plotted. Five to six mice were analyzed in a minimum of two independent experiments. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
- Fig 5
Reduced number of functional TFH cells in mice treated with MnTBAP during an acute viral infection. C57BL/6 mice were treated with either vehicle or 5 mg of MnTBAP/kg. After 4 h, mice were infected with 2 × 105 PFU of LCMV Armstrong. A maintenance dose was administered every 24 h for 8 days. At the indicated time points, mice were sacrificed and the spleen was removed. Splenocytes were stained with anti-CD4, anti-CD44, antiCD62L, and anti-CXCR5 and anti-SLAM (A) or anti-GL-7 (B). The numbers of TFH cells (A) and germinal center (GC) TFH cells (B) were quantitated, and the averages and standard deviations are shown. (C) To determine the effect of MnTBAP administration on IL-21 production by CD4+ T cells, mice were sacrificed at the indicated time points and stimulated with PMA and ionomycin for 4 h at 37°C. Following stimulation, cells were stained with anti-CD4 and IL-21R. The number of IL-21+ CD4+ T cells was quantitated in the spleen, and the averages and standard deviations are shown. Five to six mice were analyzed in a minimum of two independent experiments. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
- Fig 6
Reduced ASC and CD4+ T cells in MnTBAP-treated mice are due to decreased proliferation and increased Bim-mediated cell death. C57BL/6 mice were treated with either vehicle or 5 mg of MnTBAP/kg. After 4 h, mice were infected with 2 × 105 PFU of LCMV Armstrong. A maintenance dose was administered every 24 h for 8 days. (A) Proliferation was assessed by giving the mice BrdU (0.8 mg/ml) in their drinking water beginning at day 6 postinfection. Mice were sacrificed on day 7 postinfection, and spleens were harvested. Splenocytes were stained with anti-BrdU, anti-CD138 anti-B220, anti-CD19, and anti-IgD (A) or with anti-BrdU, anti-CD4, and anti-CD44 (B). The plots are gated on B220hi-int CD19hi-int IgD− CD138+ plasmablasts (A) or CD4+ CD44hi (B) cells. The numbers in the plots indicate the percentages of plasmablast or activated CD4+ T cells that are BrdU+. The averages and standard deviations are plotted in panel C. C57BL/6 and Bcl2l11−/− mice were treated with either vehicle or 5 mg of MnTBAP/kg and infected as described previously. Mice were sacrificed at day 8 postinfection, and the spleens were removed. The numbers of antigen-specific IgM (D) and IgG (E) ASC were quantitated by ELISPOT assay. (F) Splenocytes were stimulated with GP61-80 peptide for 5 h at 37°C. Following stimulation, cells were stained with anti-CD4 and anti-IFN-γ. The number of IFN-γ+ CD4+ T cells was quantitated in the spleen. (G) Viral titers in the spleens of wild-type (WT) and Bcl2l11−/− mice were determined by plaque assay on day 8 postinfection. The averages and standard deviations are shown. Four to six mice were analyzed in a minimum of two independent experiments. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
