Increased proliferation of T_em cells upon removal of TR2.

<p>(A) Absolute number of CD4⁺ T cells in spleens of tam-iCD4TR2 and control mice 1, 2, 4, and 6 wk p.a. Mice were treated with tamoxifen for 5 consecutive days (mean ± SEM, 9 mice per group, analysed in three independent experiments). (B) Percentage of T_em, cells in the spleen of tam-iCD4TR2, and control mice at indicated time points (percentage out of CD4⁺ T cell, mean ± SEM, 9 mice per group, analysed in three independent experiments). (C) The percentages of BrdU⁺ T_em cells isolated from spleens of tam-iCD4TR2 and control mice, gated on CD4⁺ T cells (mean ± SEM, 9 mice per group, analysed in three independent experiments). (D) The percentage of T_n and T_em cells in the spleen of thymectomised tam-iCD4TR2 and control mice at indicated time points (mean ± SEM, 9 mice per group, analysed in two independent experiments). (E) Rag1^−/− mice were reconstituted with T-cell–depleted bone marrow from WT CD45.1⁺ and CD45.2⁺ iCD4TR2 or TR2 mice in 1∶1 ratio and treated with tamoxifen for 5 consecutive days 5 wk postreconstitution. Scheme of the experimental setup. (F) The percentage of CD4⁺ T_em of total CD4⁺ T cells from LNs are shown (left panel) and the percentage of BrdU⁺ T_em cells isolated from LNs (right panel) (mean ± SEM, 10 mice per group, analysed in three independent experiments). (G) Flow cytometric analysis of the expression of IFN-γ and IL-4 by splenic CD4⁺ T cells from tam-iCD4TR2 and control mice 2 wk p.a. (representative data of two independent experiments). Quantitative RT-PCR of T-bet mRNA in sorted splenic CD4⁺ T cell. These data are representative results of two independent experiments (right panel).</p

Validation of CD4-CreER^t2/tamoxifen-mediated TR2 ablation.

<p>(A) Quantitative RT-PCR of TR2 mRNA in FACS-sorted splenic CD4⁺ T cell subsets. These data are representative results of three independent experiments. (B) Quantitative RT-PCR of TR2 mRNA in FACS-sorted CD4⁺ T cells from mesenteric lymph nodes and lamina propria. These data are representative results of three independent experiments. (C) Flow cytometric analysis of TR2 expression by CD4⁺ and CD8⁺ T cells from peripheral blood. These data are representative results of four independent experiments. (D) Western blot analysis of pSmad2 and Vinculin in lysates of magnetically purified splenic CD4⁺ T cells from tam-iCD4TR2 and control mice 2 wk p.a.. The cells were cultured for 40 min in the presence of antiCD3/CD28 either with or without 20 ng/ml TGFβ-1. These are representative data of two independent experiments. (E) Flow cytometric analysis of TR2 expression by thymocytes 2 wk p.a. These data are representative results of two independent experiments. (F) Flow cytometric analysis of expression of CD4 and CD8 by thymocytes (left panel) as well as Foxp3 and CD25 by CD4⁺ SP thymocytes (right panel) from tam-iCD4TR2 and control mice at 2 wk p.a. These data are representative results of three independent experiments.</p

Development of lethal autoimmunity after thymic deletion of TR2.

<p>Tamoxifen-treatment of Rag1^−/− mice started 3 d before reconstitution with T-cell–depleted bone marrow from iCD4TR2 or control mice (A–G). (A) Flow cytometric analysis of TR2 expression by CD4⁺ and CD8⁺ T cells at day 34. Representative data of two independent experiments. (B) Body weight was monitored during the whole experiment (mean ± SEM, 5 mice per group, representative data of two independent experiments). (C) Kaplan-Meyer survival graph for all animals of experiments. (D) Representative micrographs of H&E- and anti-CD3-stained tissue sections of indicated organs at day 34. The size bar indicates 100 µm. (E) Flow cytometric analysis of the expression of CD44 and CD62l by CD4⁺ and CD8⁺ T cells. The percentage of T_n and T_em cells in the spleen of experimental and control chimeric mice (mean ± SEM, 6 mice per group, analysed in two independent experiments). (F) Percentage and number of splenic T_reg cells at day 34 (mean ± SEM, 5 mice per group; representative data of two independent experiments). (G) Flow cytometric analysis of FoxP3 and CTLA-4 by indicated splenic CD4⁺ T cells subsets at day 34 (representative data of three independent experiments). Mean florescence intensity of CTLA-4 expression by splenic T_reg cells (right panel, mean ± SEM, 4 mice per group; representative data of three independent experiments). (H) Rag1^−/− mice were reconstituted with T-cell–depleted bone marrow from iCD4TR2 or control mice. Tamoxifen treatment of recipients started 5 wk postreconstitution and body weight was monitored during the whole experiment (mean ± SEM, 5 mice per group; representative data of two independent experiments). (I) The percentage of T_em, cells in the spleen of experimental and control chimeric mice (mean ± SEM, 6 mice per group, analysed in two independent experiments). (J) Tamoxifen-treatment of Rag1^−/− mice started 3 d before reconstitution with T-cell–depleted bone marrow from iCD4TR2 or control mice. At day 15 posttransfer treatment with anti-CD8 antibody or isotype control started. Shown is a Kaplan-Meyer survival graph for all animals in the experiments (representative data of two independent experiments). (K) Representative micrographs of H&E-stained lung sections in the terminal stage of the disease. The size bar indicates 100 µm.</p

T_reg cells lacking TR2 are functional.

<p>(A) <i>In vitro</i> suppression assay: sorted conventional CD45.1⁺CD4⁺ T cells were stimulated with anti-CD3 (2 µg/ml) and cocultured with sorted WT T_reg cells or tam-iCD4TR2 T_reg cells (isolated 14 d p.a.) at various ratios. Thymidine was added for the last 24 h of culture. Analysis was performed after 96 h. Percent suppression as mean ± SD (analysed in two independent experiments). (B) <i>In vitro</i> suppression assay: sorted conventional CD45.1⁺CD4⁺ T cells were labelled with CFSE, stimulated with anti-CD3 (2 µg/ml), and cocultured with sorted wt T_reg cells or tam-iCD4TR2 T_reg cells (isolated 14 d p.a.) at various ratios. FACS analysis was performed after 96 h. These data are representative results of two independent experiments. (C and D) <i>In vivo</i> suppression assay: Development of colitis in Rag1^−/− mice after transfer of conventional CD4⁺ T cells alone or in combination with tam-iCD4TR2 (mice treated for 5 d, cells isolated 1 wk p.a.) T_reg cells or iCD4TR2 T_reg cells. Change in body weight after 8 wk posttransfer (mean, 3 mice per group, representative data of two independent experiments). (D) Representative micrographs of H&E-stained small intestine sections from <i>in vivo</i> suppression experiments isolated from Rag1^−/− mice 8 wk after transfer of the indicated cells. Scoring of colitis severity according to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001674#pbio.1001674-Asseman1" target="_blank">[60]</a>. (E) Criss-cross <i>in vitro</i> suppression assay: sorted conventional tam-iCD4TR2 and WT T cells were cocultured with sorted tam-iCD4TR2 and wt T_reg cells at various ratios. Analysis was performed after 96 h (representative data of two independent experiments). (F) Development of colitis in Rag1^−/− mice after adoptive transfer of conventional tam-iCD4TR2 and wt T cells alone or in combination with tam-iCD4TR2 T_reg cells. Change in body weight after 8 wk posttransfer (mean ± SEM, 3 mice per group, representative data of two independent experiments).</p

Deregulated proliferation control upon removal of TR2.

<p>(A) Flow cytometric analysis of CD69 expression by CD4⁺ splenic T cells isolated 2 wk p.a. These data are representative results of three independent experiments. (B) Analysis of sensitivity to activation through measurement of proliferation. Sorted CD4⁺ T cells were cultured for 72 h and stimulated with different anti-CD3 concentrations. Thymidine was added for the last 24 h of culture (mean ± SEM, 4 mice per group, analysed in two independent experiments). (C) Flow cytometric analysis of cytoplasmic calcium by ratiometric measurement of Indo-1–labelled cells from tam-iCD4TR2 and control mice. TCR crosslinking was performed after 15 s. On the left mean ratio of the baseline (representative data of two independent experiments). (D) Flow cytometric analysis of IL-2, CD25, and CD122 expression by splenic CD4⁺ T cells isolated from tam-iCD4TR2 and control mice. These data are representative results of two independent experiments. (E and F) Proliferation analysis of sorted CD4⁺ T cells cultured for 72 h with anti-CD3 (0.6 µg/ml) and anti-CD25 (PC61) (E) or indicated cytokines (F). Thymidine was added for the last 24 h of culture (mean ± SEM, 4 mice per group, analysed in two independent experiments).</p

Increased proliferation of regulatory T cells upon removal of TR2.

<p>(A) The percentage of T_reg cells (left panel) and number of T_reg cells (right panel) in the spleen of tam-iCD4TR2 and control mice at indicated time points (mean ± SEM, 9 mice per group, analysed in two independent experiments). (B) The percentage of T_reg cells in the indicated organs of tam-iCD4TR2 and control mice (mean ± SEM, 5 mice per group, analysed in two independent experiments). (C) The percentage of T_reg cells in the spleens of thymectomised tam-iCD4TR2 and control mice 20 wk p.a. (mean ± SEM, 9 mice per group, analysed in two independent experiments). (D) The percentage of T_reg (left panel) and absolute number of Treg cells (right panel) within the LN CD4⁺ T cells of the indicated CD45.1⁺ or CD45.2⁺ bone marrow–derived cells (experiment described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001674#pbio-1001674-g005" target="_blank">Figure 5E</a>, mean ± SEM, 10 mice per group, analysed in three independent experiments). (E) The percentage of BrdU⁺ T_reg cells isolated from LN (experiment described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001674#pbio-1001674-g005" target="_blank">Figure 5E</a>, mean ± SEM, 10 mice per group, analysed in three independent experiments). (F) Flow cytometric analysis of Nrp-1 and Foxp3 expression by CD4⁺ T cells (left panel) and the percentage of Nrp-1⁺ and Nrp-1⁻ T_reg cells within the LN CD4⁺ T cells of tam-iCD4TR2 and control mice 2 wk p.a. (right panel) (mean ± SEM, 9 mice per group, analysed in two independent experiments). (G) Flow cytometric analysis of Ki-67 expression by Nrp-1⁺ T_reg cells (left panel) and the percentage of Ki-67⁺Nrp-1⁺ T_reg cells within the LN CD4⁺ T cells of tam-iCD4TR2 and control mice 2 wk p.a. (right panel) (mean ± SEM, 9 mice per group, analysed in two independent experiments). (H) Flow cytometric analysis of the expression of CTLA4 by T_reg and Foxp3 by CD4⁺CD25⁺ T cells isolated from spleen of mixed bone marrow chimeras. These data are representative results of three independent experiments (left panel). Flow cytometric analysis of the expression of ICOS by splenic T_reg cells from tam-iCD4TR2 and control mice at indicated time point p.a. Representative data of three independent experiments.</p

Cks1 regulates proliferation in oncogene-induced AML by suppressing p27^Kip1.

<p><b>A</b>, 5-FU-mobilized bone morrow from mice of the indicated genotype was infected with <i>Myc-GFP</i> virus and 2×10⁶ GFP-positive cells were transplanted with an infection rate of about 40% into lethally irradiated recipients. Immunoblot analysis of the indicated proteins was performed 3 weeks after transplantation. <b>B</b>, Flow cytometric anti-BrdU staining of GFP-positive Gr1/CD11b-positive bone marrow cells. 1 mg BrdU/g body weight was intraperitoneally injected 12 hours before bone marrow harvest. The bars represent the mean ± standard deviation of n = 3 or 5 individual mice per genotype. <b>C</b>, Survival curve of mice transplanted with <i>Myc-GFP</i>-infected bone marrow of the indicated <i>Cks1</i> and <i>p27^Kip1</i> genotype. All mice died from AML. The median survival is not significantly different between the genotypes.</p

Targeted Cks1 overexpression is not sufficient to induce B cell lymphoproliferation or lymphoma.

<p>Data from two experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037433#pone-0037433-g002" target="_blank">Figure 2</a>) are combined. One experiment with 2×10⁶ transplanted cells and an infection rate of 15% (n = 4 or 5 recipient mice) and one with 1×10⁶ cells and an infection rate of 45% (n = 2 or 3 recipient mice). <b>A</b>, Flow cytometric analysis of the percentage of GFP-positive B cells. Leucocytes were gated on B220+ cells. The analysis shows a divergence in the groups, some animals loose GFP positivity in the B cell compartment over time irrespective of the transduction efficiency. <b>B</b>, Left panel: Flow cytometric results of the bone marrow (BM) and spleen (S) for GFP positivity (of all cells, after lysis of erythrocytes). Right panel: percentage of GFP-positive cells in the B220 compartment of bone marrow (BM) and spleen (S). The analysis was performed on the day of indisposition and sacrifice. <b>C</b>, Representative images of flow cytometric analysis of GFP-positive B cells in the bone marrow. The genetic group is indicated. <b>D</b>, Survival of the <i>CD19-Cre;Stop-Cks1-GFP</i> group (median: 444 days) was identical to the survival of the <i>CD19-Cre;Stop-GFP</i> group (median: 391 days). All mice died from irradiation induced reasons but not B cell lymphoma.</p

Ubiquitous Cks1 overexpression in the bone marrow is not sufficient to induce myeloproliferative disease or leukemia.

<p>4×10⁶ GFP-positive cells were transplanted with an infection rate of 30%. <b>A</b>, Cks1 mRNA levels in the indicated bone marrow subsets of transplanted mice were analyzed by realtime PCR at day 160 after transplantation. Shown is the relative expression of Cks1 as compared to the B cell expression of the GFP mice as a control sample set at 1. <i>GFP</i> group: n = 2; <i>Cks1-GFP</i> group: n = 3. The bars represent the mean relative expression ± standard deviation. realtime PCR was performed in duplicates. <b>B</b>, Immunoblot analysis of the bone marrow of transplanted mice on the day of indisposition and sacrifice. <b>C</b>, The left panel shows the GFP positivity of the bone marrow (BM) and spleen (S). The three right panels show the percentages of B-, T- and myeloid cells. <b>D</b>, Representative flow cytometric dot blot images of the myeloid cell compartment of <i>GFP</i>, <i>Cks1-GFP</i> and <i>Myc-GFP</i> animals on the day of their sacrifice. <b>E</b>, Survival analysis of lethally irradiated recipient mice that were transplanted with <i>GFP</i>, <i>Cks1-GFP</i> or <i>Myc-GFP</i> infected bone marrow. None of the <i>GFP</i> or <i>Cks1-GFP</i> mice died of leukemia, while all <i>Myc-GFP</i> mice succumbed to AML. Median survival: <i>GFP</i> group, 469 days; <i>Cks1-GFP</i> group, 541.5 days; <i>Myc-GFP</i> group, 52 days. Only the survival of <i>Myc-GFP</i> group is significantly different from the other groups.</p