Flow cytometric analysis of ILC3s using <i>RORc</i>-eYFP fatemap mice.

<p>(a) Gating strategy for ILC3s in <i>RORc</i>-eYFP mice: after exclusion of doublets, live CD45⁺ B220⁻ CD3⁻ TCRβ⁻ YFP⁺ cells were selected. (b) The population gated in panel a contains a significant fraction of CD5⁺ cells both in spleen, lung and colon. (c) In <i>RORc</i>-eYFP-RAG^−/− mice all YFP⁺ CD45⁺ live cells stain negative for CD3, TCRβ and also CD5. Representative plots for n = 3 (mean +/− s.e.m.).</p

CD5⁺ positive cells within the ILC3 gate express high levels of <i>TRAC</i>.

<p>Normalized fold expression for <i>TRAC</i> and <i>TRDC</i> comparing CD5⁺ and CD5⁻ cells within the ILC3 gate (“CD5⁺ ILC3s”, pregated on CD45⁺ B220⁻ CD11b⁻ CD3⁻ TCRβ⁻ YFP⁺ cells), αβ and γδ T cells, monocytes (Mono) purified from <i>RORc</i>-eYFP mice and ILC3s derived from <i>RORc</i>-eYFP <i>RAG1</i>^−/− mice (ILC3s <i>RAG1</i>^−/−). Results are presented relative to those obtained from ILC3s of <i>RORc</i>-eYFP <i>RAG1</i>^−/− mice (mean +/− s.e.m.).</p

Comparable cuprizone-induced demyelination after oligodendroglial deletion of JunB and c-Jun.

<p>(a) Representative images of JunB^f/f/c-Jun^f/f controls and JunB^Δol/c-Jun^Δol double mutants that received cuprizone for 6 weeks. (b) The number of microglia and extent of demyelination was evaluated in matched sections (n = 3–5 mice per group, n = 4 sections per mouse averaged, unpaired t-test). HE, LFB-PAS staining, as well as CNPase, Iba-1 and GFAP immunoreactivity in the corpus callosum. Scale bar, 50 μm.</p

Normal brain morphology and motor skills.

<p>(a) Weight of littermate controls (JunB^f/fc-Jun^f/f) (black bars) and JunB^Δol/c-Jun^Δol double mutants (white bars) at 3 and ≥12 months of age. 22 controls and 30 mutants were analyzed in total. (b, c) Histological analysis (hematoxylin and eosin, HE and luxol fast blue—periodic acid schiff staining, LFB-PAS) of control and mutant brain sections at 6–12 months of age (n≥4 per group; representative pictures are from the corpus callosum brain region of 6 month old mice). Immunostaining for oligodendroglial CNPase, microglial Iba-1, and astrocytic GFAP. Scale bar, 50 μm. For quantifications of the number of Iba-1⁺ cells per visual field see (b) (n = 3–5 mice per group, unpaired t-test, n.s., not significant). (d, e) The grid test (d) assessing limb strenght and subtle motor coordination deficits like slipping at ≥12 months of age (n = 15 controls, and n = 13 double mutants). Motor performance in the rotarod test (e) (3 consecutive trials; after 3 exercise trials the day before). Matched 2way ANOVA, Bonferroni post-test, n.s., not significant; *<i>P</i> < 0.05.</p

Similar clinical and histopathological EAE phenotype after oligodendroglial knock-out of JunB and c-Jun.

<p>(a) Day of onset and maximal clinical EAE score in MOG_35–55-immunized control (JunB^f/fc-Jun^f/f) and JunB^Δol/c-Jun^Δol double mutants (n = 12–19, unpaired t-test). (b) Number of CD45^high leukocytes isolated from cerebellum/spinal cords of controls and mutant mice. Data were pooled from 2 independent experiments (n = 10 MOG peptide-immunized mice per group and n = 4 naïve mutants, unpaired t-test). (c) Semiquantitative histology scores of inflammation and demyelination in spinal cords of MOG peptide-immunized mutant mice (mean clinical EAE score 2.3) and controls (mean EAE score 2.0; n = 4 per group). (d) Representative images of HE, LFB-PAS staining, as well as CNPase, Iba-1 and GFAP immunoreactivity in the spinal cords.</p

Deletion of JunB and c-Jun in oligodendrocytes.

<p>(a) Schematic diagram of MOGiCre x JunB^f/f and c-Jun^f/f (JunB^Δol/Jun-c^Δol) mice. (b) Genotyping PCR using genomic DNA derived from various peripheral organs and CNS regions from a JunB^flox/flox (top) and c-Jun^flox/flox (bottom) MOGiCre/+ mutant (JunB^Δol/c-Jun^Δol) and from a homozygous JunB^flox/flox/c-Jun^flox/flox MOGiCre-negative control (JunB^f/f/c-Jun^f/f). Li, liver; Ki, kidney, Spl, spleen; Hr, heart; Thy, thymus, Lu, lung; Ln, lymph node; Br, brain; Cb, cerebellum; Sc, spinal cord. (c) Western Blot analysis for JunB, c-Jun and vinculin (loading control) in protein lysates obtained from CNS spinal cord tissue of junB^f/fc-Jun^f/f controls and JunB^Δol/c-Jun^Δol double mutants (n = 2 individual animals per group).</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

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