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

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

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

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

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

(A) Lymphocytes of spleen (SP) and inguinal LNs were analyzed for the expression of IgM and IgD by flow cytometry

Numbers indicate the mean percentages and SD of gated populations. SP: follicular (FO) B cells (IgMIgD), marginal zone (MZ), and transitional B cells (IgMIgD); LN: IgMIgD. Values were calculated from seven independent experiments. (B) Flow cytometric analysis of follicular B cells (FO; CD21CD23) and marginal zone B cells (MZ; CD21CD23) in the spleen. Numbers indicate mean percentages and standard deviations of B220 B cells displaying a MZ B or FO B cell phenotype. Values were calculated from seven independent experiments. (C) Absolute numbers of FO and MZ B cells in the spleens of LMP1/CD40 and control mice. (D) Splenocytes were stained with antibodies specific for the indicated activation and adhesion markers. Histograms show surface expression of the indicated markers on gated B220 B cells from LMP1/CD40 (continuous line) and control (dotted line) mice. Lymphocytes isolated from LNs showed a similar extent of activation (not depicted).<p><b>Copyright information:</b></p><p>Taken from "Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1317-1329.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413030.</p><p></p

(A) Southern blot analysis to examine IgH gene configuration with an IgH-specific probe

Genomic DNA was prepared and digested with EcoRI from splenic cells of LMP1/CD40 mice that developed neoplasias (34, 142, 176, 197, 230, 247, 365, and 221), one young LMP1/CD40 mouse (900; 3 mo of age), as well as a control mouse (905). The sequence analysis of the amplified IgH genes is shown in Fig. S6. (B) Representative histological analyses of diseased LMP1/CD40 mice and age matched control mice. (1) HE-stained normal spleen. (2) HE-stained spleen representative for diseased LMP1/CD40 mice, showing prominent nodular tumor infiltrates. (3) Lymphoma infiltrate within the spleen of a representative diseased LMP1/CD40 mouse. B cells were visualized by immunohistochemistry using an antibody specific for B220. (4) Higher magnification of section described in 3. (5) Dispersed reactive T cells within the lymphoma infiltrate were detected by an anti-CD3–specific antibody. A representative highly magnified section is shown. Bars: (1 and 2) 400 μm; (3) 100 μm; (4, 5, and inset) 25 μm. (C) Flow cytometric analysis of splenic cells for the expression of IgM and IgD. The gates were set according to the age-matched controls. For the diseased LMP1/CD40 mice, a shift toward one or two main populations could be observed, as shown for three representative samples. Fig. S6 is available at .<p><b>Copyright information:</b></p><p>Taken from "Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1317-1329.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413030.</p><p></p

(A) Whole-cell extracts of splenic B cells of LMP1/CD40 (L/C) and control (co) mice from three independent preparations (1–3) were loaded on one gel and analyzed for Jnk (Jnk2, 54 kD; Jnk1, 46 kD), Erk (Erk1

44kD; Erk2, 42 kD), p38/MAPK (38 kD) and the corresponding phosphorylated forms with specific antibodies. Equal protein loading was controlled by Ponceau S staining. The graph shows the mean values of the fold induction of the signals from the unphosphorylated and phosphorylated forms of Jnk, Erk, and p38/MAPK compared with the signals in control cells. Mean values and SDs were calculated from at least five independent experiments. SDs are shown by error bars. *, P < 0.05; **, P < 0.001, calculated by the two-tailed Student's test. (B) After CD40 triggering, LMP1/CD40-expressing cells are damped in activation compared with control cells. Splenic B cells of LMP1/CD40 and control mice were stimulated with α-CD40 antibody for the indicated time points, and Western blots were performed as described in A. Equal protein loading was controlled by tubulin staining. One representative experiment out of three is shown. (C) The improved survival of LMP1/CD40-expressing B cells is dependent on continuous Erk phosphorylation. Splenic B cells of LMP1/CD40 and control mice (CD19-Cre) were cultured for up to 5 d with the Mek1/2 inhibitor U0126 (dotted line) and the Jnk-inhibitor SP600125 (dashed line; triangle and square, respectively). As control, B cells from LMP1/CD40 and control mice were cultured in the presence of DMSO (continuous lines, rectangle, and circle, respectively). Percentages of living cells (Topro negative) were determined by flow cytometry at day 1, 3, and 5. The bars show mean percentages of living cells of three independent experiments. Error bars show the SD.<p><b>Copyright information:</b></p><p>Taken from "Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1317-1329.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413030.</p><p></p

(A) Targeting strategy for the insertion of a conditional allele into the murine -locus

Cre-mediated recombination leads to deletion of the stop cassette and expression of under transcriptional control of the endogenous -promoter. The scheme of the targeting construct shows the EcoRI recognition sites and the location of the probe. The expected fragments after EcoRI digestion and hybridization with the labeled probe are indicated by the thin lines. SAS, splice acceptor site; STOP, Stop cassette, XbaI, site of insertion. (B) Southern blot analysis of EcoRI-digested genomic DNA showing the deletion efficiency of the stop cassette in B cells of the spleen (SP; lane 3) and the BM (lane 6) of LMP1/CD40 mice. DNA from B cells of control mice (lanes 2 and 5) and mice heterozygous for (lane 1 and 4) was included. B cells were purified by using magnetic beads against CD19. After deletion of the stop cassette, the rosa26 probe detects a 5.2-kb fragment (LMP1/CD40). The 15- and 7.1-kb fragments represent the wild-type allele ( locus) and the targeted allele (), respectively. The deletion efficiency was ∼50% in the BM and almost complete in the spleen. (C) LMP1/CD40 protein expression. Western blots were prepared from spleen lysates of LMP1/CD40;CD19-Cre (lanes 1 and 2) and control mice (lane 3). As control, protein lysates of 293 cells transiently transfected with a LMP1/CD40 expression vector were included in the analysis (lane 4). The 56-kD LMP1/CD40 chimeric protein was detected by an anti–human CD40 antibody. ns, nonspecific band.<p><b>Copyright information:</b></p><p>Taken from "Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1317-1329.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413030.</p><p></p