Diphtheria toxin mediated depletion of B cells in B-DTR mice.

<p>The mice were treated once daily for 4 days with a dose of 25 ng/g body weight of diphtheria toxin. The decrease of B cells in comparison to T cells is shown in spleen, lymph nodes, bone marrow and peritoneal cavity; dot blots in the left panel and the corresponding bar charts in the right panel.</p

eYFP expression in CD19-Cre/eYFP mice.

<p>A. CD19-Cre/eYFP mice were analyzed for eYFP expression in the B cell compartment of spleen, lymph nodes, Peyer's patches, peritoneal cavity and bone marrow. The shown histograms are gated on live lymphocytes and CD19. The filled histogram curve represents CD19-Cre/eYFP mice and the unfilled control mice. Percentages of eYFP⁺ CD19⁺ cells are shown in the histograms, upper number/percentage B-DTR mice, lower panel shows cells of control mice. B. To identify the CD19⁺ eYFP⁻ B-cell fraction in bone marrow of CD19-Cre/eYFP mice, bone marrow cells were stained for AA4.1 and CD23.</p

Total serum immunoglobulin levels after depletion of B cells.

<p>B-DTR mice and control mice were injected daily four times with 25 ng/g body weight DT. Mice were bled just before DT injection and in weekly intervals thereafter. Shown are the total immunoglobulin levels at the indicated time points. The values are shown as average of groups of at least five mice.</p

Efficiency of B cell depletion.

<p>A. Number of marginal zone, follicular, immature and mature B cells in spleen. B. Depletion of B cells in bone marrow, gated for B220⁺. C. Depletion of transitional B cells in spleen, gated for CD19⁺ cells. D. Depletion of mature and immature B cells in spleen, gated for CD19⁺ cells. E. Depletion of marginal zone and follicular B cells gated for CD19⁺ cells. F. Depletion of B1a and B2 B cells in peritoneal cavity, gated for CD19⁺. G. Depletion of resting B cells in peritoneal cavity gated for CD19⁺ the percentages of cells in the different shown B cell subpopulations are indicated.</p

Plasma cells identified with Syndecan-1 in cytospins.

<p>Bone marrow cells from NP-CG immunized and boosted cells were analyzed for plasma cells. Plasma cells are shown in red with a surface staining of syndecan-1, the remaining cell population was counter stained with Hoechst (blue). The bar chart represents plasma cells found in the regarding groups (n = 5). The pictures show representative sections of the cytospins.</p

Experimental setup.

<p>Experimental setup.</p

Interaction effects calculated by multiple linear regression.

<p>This schematic visualization of second order linear regression models interaction effects. The diagram of the linear regression model includes two main covariates (strain <i>H</i> and stimulation with <i>Γ</i>) and their interaction covariate <i>H∶Γ</i>. The main covariates can assume two values (<i>H</i>: C57BL/6 or BALB/c; <i>Γ</i>: IFN-γ stimulation or no stimulation). The arrows indicate the estimated effects β. The pink and turquoise arrows reflect the aggravating or alleviating interaction effects as deviations from the additive model. A second order linear model can dissect the effects arising from two perturbations and their interaction by looking at the magnitude and significance of its regression covariates. Most importantly, the interaction covariate can indicate either an alleviating (weaker than expected from the single intervention effects) or aggravating (stronger than expected) interaction. The linear model includes two main covariates <i>H</i> and <i>Γ</i> and their interaction covariate <i>Η∶Γ</i>.</p

Schematic visualization for the interpretation of the eruption plot.

<p>The results of two models can be compared in the eruption plot. The arrows of an eruption plot can have different sizes and directions. This scheme helps to interpret the arrow. Effect size is displayed along the x-axis and the significance on the y-axis. The red area shows the region of interest (ROI).</p

Linear regression models.

<p>Linear regression models.</p

Cluster and pathway analysis.

<p>A: the effect estimates of Model 3 were subjected to a hierarchical cluster analysis. Genes are displayed in the rows, which showed a significant global effect (F-test p-value <0.05 after FDR correction and at least one of the covariates having +/−1.5 fold change). The three columns are the covariates <i>Η</i>, <i>Γ</i>, and <i>Η∶Γ</i>. The column <i>strain</i> shows differences between C57BL/6 and BALB/c, up-regulation shown in red and down-regulation shown in green. The column <i>Γ</i> shows in red up-regulation in BALB/c and in green down-regulation upon IFN-γ stimulation. The third column helps to distinguish alleviating and aggravating effects. Aggravating effects are represented in pink and alleviating effects in turquoise. P-values are plotted separately in a heatmap. The order of the genes is given by the effect estimate clustering. P-values are given in −log₁₀ scale and start from 0 displayed in colors ranging from blue to white. B: The results of a pathway enrichment analysis of cluster 6 as a bar plot. The direction of regulation of the genes of cluster 6 is indicated by the color bar. Gene Ontology ‘Biological Process’ terms and KEGG pathway categories (p<0.01) are sorted from bottom (most significant) to top. To reduce redundancy, similar terms are represented by the most significant and specific term. For complete list of functional annotations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091840#pone.0091840.s010" target="_blank">Table S2</a>. The right side shows the results of a TFBS analysis of this gene cluster. The two most significantly represented TFBS are given by the name of the transcription factor, the motif, and the p-value.</p