Lung transcriptome comparison of mutant versus wild type animals following OVA challenge.

<p>(A) Summarized heat map of genes regulated after OVA challenge in all three mutant mouse lines compared to the respective challenged control littermates. Genes with similar expression patterns are grouped together (A to G). Color code indicates the mean fold changes of the respective genes in one group for each OVA challenged mutant mouse compared to the mean of the corresponding challenged wild type littermates. Orange represents up- and blue down-regulation in the mutant mice. The column “# genes” displays the number of genes that are included in each gene group A to G (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134503#pone.0134503.s003" target="_blank">S2D Fig</a> shows the corresponding data for individual genes). (B) Venn diagram representing the overlap of gene regulation. (C) Summarized heat map of overlapping gene expression in at least 2 of the 3 mutant mouse lines. Grey boxes indicate that there is no significant regulation of these genes in the respective mutant mouse lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134503#pone.0134503.s003" target="_blank">S2E Fig</a> shows the data for individual genes of this panel).</p

Criteria for selection of mutant mouse line.

<p>^apublished data</p><p>^boriginal data from GMC screen</p><p>Criteria for selection of mutant mouse line.</p

Immunoglobulin isotype levels in mutant mice before and after OVA challenge.

<p><b>(</b>A) <i>Cox4i2</i>^{<i>tm1Hutt</i>}, (B) <i>Ifit2</i>^{<i>tm1</i>.<i>1Ebsb</i>} and (C) <i>Prdm11</i>^{<i>tm1</i>.<i>1ahl</i>}. Box plots show levels of immunoglobulin isotypes in the respective mutant mice and corresponding wild type littermates after OVA challenge. Grey boxes display the interquartile range of each immunoglobulin isotype under standard conditions (+/+: wild type,-/-: respective mutant mice; p-value * < 0.05; n = 10–12 per group).</p

Cytometric and cytokine analyses in BAL after OVA challenge.

<p>(A to C) <i>Cox4i2</i>^{<i>tm1Hutt</i>}, (D to F) <i>Ifit2</i>^{<i>tm1</i>.<i>1Ebsb</i>} and (G to K) <i>Prdm11</i>^{<i>tm1</i>.<i>1ahl</i>}. (A, D, and G) Total cell count in BAL following OVA challenge. (B, C, E, F, H, and I) Relative cell count of eosinophils (Eos), T cells, B cells, neutrophils (Neutro) and macrophages (Macro) as indicated in panels following OVA challenge. (J) Relative cell count of CD4⁺ T cells in BAL following OVA challenge. (K) Quantification of cytokines in BAL following OVA challenge (p-value * < 0.05; ** < 0.01; *** < 0.001; n = 10–12 per group).</p

<i>In vitro</i> splenocyte proliferation.

<p><i>In vitro</i> proliferation response was investigated in <i>Ifit2</i>^{<i>tm1</i>.<i>1Ebsb</i>} and <i>Prdm11</i>^{<i>tm1</i>.<i>1ahl</i>} splenocyte cultures. (A) For B-cell activation, <i>Ifit2</i>^{<i>tm1</i>.<i>1Ebsb</i>} splenocytes were cultured with 1 μg/ml anti-CD40 and 1 ng/ml IL-4 or 5 μg/ml anti-CD40 and 10 ng/ml IL-4 (n = 6 per group). (B) For T-cell stimulation, <i>Prdm11</i>^{<i>tm1</i>.<i>1ahl</i>} splenocytes were cultured with 20 U/ml IL-2 and either 0.05 μg/ml, 0.5 μg/ml or 5.0 μg/ml of anti-CD3 for T cell activation (n = 5 per group).</p

Comparative analysis of transcriptome data under standard conditions.

<p>(A) Summarized heat map from HCL analysis of genes regulated in the three mutant mouse lines. Genes with similar expression patterns are grouped together (A to E). Color code indicates the mean fold change of the respective genes in one group for each mutant mouse compared to the mean of the corresponding wild type littermate group. Orange represents up- and blue down-regulation in the respective mutant mice. The column “# genes” gives the number of genes that are included in each group A to E. (B) Venn diagram of differentially expressed genes common or specific in <i>Cox4i2</i>^{<i>tm1Hutt</i>}, <i>Ifit2</i>^{<i>tm1</i>.<i>1Ebsb</i>} and <i>Prdm11</i>^{<i>tm1</i>.<i>1ahl</i>} mutant mice.</p

Total IgE levels in plasma before and after OVA challenge.

<p>Box plots show total IgE levels after OVA challenge in mutant mouse lines and corresponding wild type littermates. Grey boxes show the interquartile ranges of total IgE levels under standard conditions of the respective groups (p-value ** < 0.01, n = 11–31 per group).</p

Cell autonomous function for CIP2A in T-cell activation.

<p>(A) CIP2A protein expression from activated WT or CIP2A^HOZ CD8 T-cells. Mean + S.E.M of CIP2A protein expression using β-actin as a normalization control is shown. (B) CD4⁺ cells from Zap70^+/- or ATP analogue HXJ2-sensitive Zap70^(AS) mice were stimulated <i>in vitro</i> with plate-bound anti-CD3 and anti-CD28 antibodies in the presence or absence of HXJ42 (1 μM). Cells were harvested at indicated time-points and shown is real-time PCR analysis of CIP2A transcript levels relative to actin as normalized to the Zap70^+/- unstimulated sample. Shown is a representative of two independent experiments with identical results. (C) Cell surface staining of CD69 from CD4⁺CD62L⁺ T-cells isolated from WT or CIP2A^HOZ mice stimulated with anti-CD3 and anti-CD28 for 24h. The mean + S.E.M. of three independent experiments is shown. Student's t test. (D) Number of viable splenocytes determined by CellTiter-Glo Assay 7 days post-stimulation with IL-2 (20U/ml) and anti-CD3 (1.25, 2.5 or 5 μg/ml). Blue bars indicate medians, circle individual data points (n = 6 for WT & CIP2A^HOZ cells). * p<0.05, ** p<0.01, Student’s t-test. (E) Human CD4⁺ T-cells isolated from umbilical cord blood pooled from 5–6 individuals were nucleofected with scramble nontargeting siRNA or CIP2A siRNA. Cells were rested for 48hrs and activated with anti-CD3 and anti-CD28 for 24h. The mean + S.E.M. of three independent experiments is shown. Student's t test.</p

Characterization of CIP2A^HOZ lymphocytes.

<p>Characterization of CIP2A^HOZ lymphocytes.</p

Impaired T-cell activation in CIP2A^HOZ mice <i>in vivo</i>.

<p>(A-H) Flow cytometry analysis of splenocytes from 3 WT and 4 CIP2A^HOZ mice five days after recall infection with high-dose <i>L</i>.<i>m</i>.-OVA. (A) Representative flow cytometry analysis for CD4⁺ and CD8⁺ T cells. (B) Percentage of CD4⁺ and CD8⁺ splenocytes from analysis described in (A). * p < 0.05, Two-tailed T-test. (C) Representative dot plots of antigen-specific CD8⁺ T cells identified by H-2K^b/SIINFEKL multimer staining. Dot plots are gated on living CD45⁺ CD3⁺ CD8⁺ cells. (D) Bar chart numbers indicate H2-Kb/SIINFEKL multimer+ cells as percentages of CD8+ T cells. T-test. (E-H) Analysis of OVA-specific CD8⁺ T lymphocytes from control and mutant mice 5 days after recall infection with high-dose <i>L</i>.<i>m</i>.-OVA and unchallenged control mice. (E) CD62L and CD127 surface expression on OVA-specific (H-2K^b/SIINFEKL multimer⁺) allows the characterization of secondary T-cell subsets: central memory phenotype (T_CM, CD127⁺ CD62L⁺) and effector memory phenotype (T_EM, CD127⁺ CD62L^-) can be observed in immunized, unchallenged mice; whereas immunized challenged mice present two main populations of effector T cells differentiated by their CD127 expression. (F) Bar chart numbers indicate percentages of effector phenotype CD8⁺ T-cells in regards of total splenocytes or OVA-specific CD8⁺ T lymphocytes (right). * p < 0.05, Two-tailed T-test. (G) Cytokine production by antigen-specific T-cells from CIP2A^HOZ and WT mice on day 5 after recall infection with high-dose <i>L</i>.<i>m</i>.-OVA. (H) Proportion of T_EM and T_CM from total splenocytes. * p < 0.05, Two-tailed T-test.</p